Post on 25-Jun-2020
Characterization of the carbonic anhydrase gene family and other key osmoregulatory
genes in Australian freshwater crayfish (genus Cherax)
Muhammad Yousuf Ali
BSc (Fisheries), MSc (Animal Breeding & Genetics)
Principal Supervisor: Dr. Peter J Prentis (EEBS, QUT)
Associate Supervisors: Dr. Ana Pavasovic (School of Biomedical Sciences, QUT
Professor Peter B Mather (EEBS, QUT)
School of Earth, Environmental and Biological Sciences
Science and Engineering Faculty
Queensland University of Technology
Brisbane, Australia
A thesis submitted in fulfilment of requirements for the degree of
Doctor of Philosophy
2016
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Key Words
Acid-base, carbonic anhydrase (CA), cytoplasmic carbonic anhydrase (CAC), membrane-
associated glycosyl-phosphatidylinositol-linked carbonic anhydrase (CAg), Cherax, Cherax
quadricarinatus (Cq); Cherax destructor (Cd), Cherax cainii (Cc); crayfish, expression,
genomics, gills, glycosyl-phosphatidylinositol-linked (GPI-linked), H+-ATPase (HAT); marron,
Na+/K+-ATPase (NKA); osmoregulation, osmoregulatory genes, pH balance, physiology,
quantitative real-time polymerase chain reaction (qRT-PCR), redclaw, transcriptome, V-type
H+-ATPase, V-type: vacuolar-type, yabby
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Abstract
(Each chapter contains its own abstract. It is a general abstract of the study.)
Cherax quadricarinatus (Redclaw), C. destructor (Yabby) and C. cainii (Marron) are an
abundant group of economically important freshwater crayfish and have been developed
for aquaculture production in many countries of the world including Australia, Mexico,
Ecuador, Uruguay, Argentina and China. They are endemic to Australia, and possess distinct
natural distributions that span a diverse range of aquatic environments. Redclaw crayfish
occurs naturally in rivers across northern Australia with an abrupt environmental pH
gradient of 6.2-8.2; Yabby across southern and central Australia with a natural pH range 6-9;
and Marron in south-west Western Australia with a natural pH range 7-8.25. These species
have the capacity to acclimate to wide fluctuations in environmental parameters including
pH and salinity. The unique physiological characteristics and their economic significance
have made these three species an important group for physiological research on freshwater
crustaceans. Despite this, genetic information for these crayfish is still largely limited to
basic population genetic data on natural variation in wild stocks. To date, so far as we know,
no studies have attempted to identify or characterise specific candidate genes that have a
role in systemic acid-base balance or salinity regulation in freshwater crayfish. Thus,
genomic and molecular studies aimed at characterising the major genes (or variants of the
genes) involved in maintaining systemic pH and salinity balance are essential because it will
provide a first step toward improving our understanding of their response to environmental
changes.
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As crayfish are farmed in a wide range of culture conditions, optimisation of water quality
parameters, particularly pH and salinity, are crucial for their maximum growth performance.
Previous reports have shown that fluctuations in pH and salinity in culture environments can
have a negative impact on growth and physiology of crayfish. It is also evident that
physiological processes involved in osmoregulation and systemic acid-base balance are
undertaken by a range of enzymes encoded by transport-related genes. Having considered
this, we have sequenced, identified and characterised most major candidate genes involved
in systemic pH balance and osmoregulation including: Carbonic anhydrase, Na+/K+-ATPase
(NKA), V-type-H+-ATPase (HAT) and Na+/K+/2Cl- cotransporter (NKCC). We also shed light on
the expression and evolution of some major genes that have roles in pH balance or salinity
regulation.
In our first study (Chapter 2), we deeply sequenced the whole gill transcriptome from C.
quadricarinatus and generated 36,128 assembled contigs with an average length of 800bp.
Approximately 40% of contigs received significant BLAST hits and 22% were assigned gene
ontology terms. Full length sequences were identified and fully annotated for three CA
isoforms (cytoplasmic, membrane-associated and β-CA), Na+/K+-ATPase, V-type H+-ATPase,
sarcoplasmic Ca+-ATPase, Arginine kinase and Calreticulin. In order to understand potential
roles of the candidate genes in response to environmental pH variation, gene expression
analysis for CA, V-type H+-ATPase and Arginine kinase were undertaken at 24 h after
exposure to different pH conditions (pH 6, 7 and 8). The expression results from one
sampling point suggested that cytoplasmic carbonic anhydrase might be involved in pH
balance, as the gene was differentially expressed across the three pH treatments, while the
other genes were not.
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To confirm the results of study 1 and further investigate the role of the genes in systemic pH
balance, further in-depth study was carried out that assessed the effects of pH on
expression for carbonic anhydrase (cytoplasmic form CAc and membrane-associated form
CAg), Na+/K+-ATPase α subunit (NKA-α) and V-type H+-ATPase subunit a (HAT-A) in gills of
Redclaw crayfish. Individuals were challenged to pH 6.2, 7.2 (control) and 8.2; and sampling
was done at 0h, 3h, 6h, 12h and 24h post-exposure. All genes showed significant differences
in expression levels, either across pH treatments or over time. Expression levels for CAc
significantly increased at low pH and decreased at high pH conditions 24 h post-transfer.
Though expression levels for CAg increased at both low and high pH conditions at 6 h post
exposure, but expression remained elevated only at low pH (6.2) at the end of the
experiment. Expression of HAT and NKA also showed a significant increase at low pH
conditions, 6 h post transfer, and their expression remained significantly elevated
throughout the experiment. Overall, these results suggest that CAc, CAg, NKA and HAT may
play a role in response to pH change in freshwater crayfish.
In the third study (Chapter 4), we undertook deep sequencing of gill transcriptomes from
two other important freshwater crayfish, C. cainii and C. destructor, and generated full
length sequences of the candidate genes. The sequences from this study and those obtained
from our previous study (study 1) were compared to examine patterns of molecular
sequence variation; and if the genes were evolving with patterns of nucleotide variation
consistent with the action of natural selection. Over 83 and 100 million high quality reads (Q
≥ 30) were assembled into 147,101 and 136,622 contigs for C. cainii and C. destructor,
respectively. A total of 24630 and 23623 contigs received significant BLASTx hits and
allowed the identification of multiple gill expressed candidate genes associated with pH
balance and osmoregulation. The comparative molecular analysis of the candidate genes
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showed a high level of sequence conservation across the three Cherax sp. All candidate
genes showed patterns of molecular variation consistent with neutral evolution. Overall
these results demonstrate a high degree of conservation in these candidate genes, despite
the ecological and evolutionary divergence of the species studied.
In fourth study (included in Chapter 4), we carried out a tissue-specific expression
(occurrences) analysis of 80 candidate genes that have potential role in pH balance and
ionic/osmotic regulation across different organs (gills, hepatopancreas, heart, kidney, liver,
nerve and testes). Approximately 50% of the candidate genes were expressed in all tissue
types examined. The largest number of genes was observed in nerve tissue (84%) and gills
(78%). The study also revealed that most of the candidate genes that have a role in systemic
acid-base balance and osmoregulation (CA, NKA, HAT, NKCC, NBC, NHE) were expressed in
all tissue types. This indicates the potential for an important physiological role for these
genes outside of osmoregulation in other tissue types.
Overall, we generated, for the first time, full length sequences for major systemic acid-base
balance and osmoregulatory genes in freshwater crayfish. We also provided the first report
on the distribution of the genes across multiple tissue types. Moreover, we present the first
in-depth expression study for major transport-related genes in freshwater crayfish. The
study provides valuable genomic information for future genomic studies in decapod
crustaceans, particularly in crayfish. Taken together this information provides much needed
information to help improve crayfish aquaculture in the future.
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Table of Contents
Table of Contents Key Words ....................................................................................................................................................... ii
Abstract .......................................................................................................................................................... iii
Table of Contents........................................................................................................................................... vii
List of Figures ................................................................................................................................................... x
List of Tables .................................................................................................................................................. xii
List of Abbreviations ..................................................................................................................................... xiii
Note on thesis preparation ........................................................................................................................... xiv
Statement of Original Authorship .................................................................................................................. xv
Works published or submitted for publication by the author incorporated into the thesis ........................... xvi
Acknowledgements .................................................................................................................................... xviii
General Introduction .................................................................................................................. 1 Chapter 1.
1.1 Economic and eco-physiological importance of Crayfish (as a model organism) ................................ 1
1.2 Genes involved in pH balance and osmotic/ionic-regulation.............................................................. 6 1.2.1 Carbonic anhydrase and its role in pH balance and osmoregulation ............................................. 9 1.2.2 Na+/K+-ATPase and its role in pH balance and osmoregulation ................................................... 12 1.2.3 V-type-H+-ATPase and its role in pH balance and osmoregulation ............................................... 18 1.2.4 Arginine kinase and its role in pH balance and osmoregulation .................................................. 21 1.2.5 Ca+2-ATPase and its role in pH balance and osmoregulation ....................................................... 21 1.2.6 Ion channels involved in pH balance and osmoregulation ........................................................... 22 1.2.7 Functions of transporters and exchangers in maintaining pH balance and for osmoregulation .... 23
1.3 Knowledge GAP and Research Questions ........................................................................................ 25
1.4 Aims and objectives ....................................................................................................................... 27
Analysis, characterisation and expression of gill-expressed carbonic anhydrase genes in the Chapter 2.freshwater crayfish Cherax quadricarinatus .................................................................................................. 29
2.1 Abstract ......................................................................................................................................... 31
2.2 Introduction ................................................................................................................................... 32
2.3 Material and Methods ................................................................................................................... 35 2.3.1 Sample Preparation ................................................................................................................... 35 2.3.2 RNA extraction, cDNA library construction and sequencing ........................................................ 36 2.3.3 De novo assembly and functional annotation ............................................................................. 36 2.3.4 Identification of CA and other osmoregulatory genes ................................................................. 37
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2.3.5 CA expression patterns under different pH treatments .............................................................. 38
2.4 Results ........................................................................................................................................... 39 2.4.1 Sequencing and assembly .......................................................................................................... 39 2.4.2 Mapping and functional annotation ........................................................................................... 40 2.4.3 Identification and characterization of carbonic anhydrase (CA) genes ........................................ 41 2.4.4 Phylogenetic analysis of carbonic anhydrase .............................................................................. 48 2.4.5 Identification of additional osmoregulatory genes ..................................................................... 50 2.4.6 CA expression patterns under different pH treatments .............................................................. 51
2.5 Discussion ...................................................................................................................................... 52 2.5.1 Carbonic anhydrase genes in Cherax quadricarinatus ................................................................. 53 2.5.2 Other candidate osmoregulatory genes in Cherax quadricarinatus ............................................. 56 2.5.3 Vacuolar-type H+-ATPase ........................................................................................................... 57 2.5.4 Arginine kinase .......................................................................................................................... 58
2.6 Conclusions .................................................................................................................................... 59
2.7 Acknowledgements ........................................................................................................................ 59
2.8 Data Archive .................................................................................................................................. 59
Expression patterns of two alpha Carbonic anhydrase genes, Na+/K+-ATPase and V-type H+-Chapter 3.ATPase in the freshwater crayfish, Cherax quadricarinatus, under different pH conditions ........................... 60
3.1 Abstract ......................................................................................................................................... 62
3.2 Introduction ................................................................................................................................... 64
3.3 Materials and Methods .................................................................................................................. 67 3.3.1 Sample Preparation ................................................................................................................... 67 3.3.2 RNA extraction and cDNA synthesis ........................................................................................... 68 3.3.3 Quantification of mRNA by quantitative Real-Time-PCR (qRT-PCR) ............................................. 68 3.3.4 Data analysis ............................................................................................................................. 69
3.4 Results ........................................................................................................................................... 69 3.4.1 Carbonic anhydrase (CA) ............................................................................................................ 69 3.4.2 Na+/K+-ATPase (NKA) ................................................................................................................. 72 3.4.3 V-type H+-ATPase (HAT) ............................................................................................................. 73
3.5 Discussion ...................................................................................................................................... 74 3.5.1 Carbonic anhydrase (CA) ............................................................................................................ 74 3.5.2 Na+/K+-ATPase (NKA) ................................................................................................................. 76 3.5.3 Vacuolar-type H+-ATPase ........................................................................................................... 78
3.6 Conclusions .................................................................................................................................... 79
Comparative molecular analyses of select pH- and osmoregulatory genes in three freshwater Chapter 4.crayfish Cherax quadricarinatus, C. destructor and C. cainii ........................................................................... 80
4.1 Abstract ......................................................................................................................................... 82
4.2 Introduction ................................................................................................................................... 83
4.3 Material and Methods ................................................................................................................... 84 4.3.1 Sample collection and preparation............................................................................................. 85 4.3.2 RNA extraction, cDNA synthesis and sequencing ........................................................................ 85 4.3.3 Assembly, functional annotation and gene identification ........................................................... 86
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4.3.4 Molecular analyses .................................................................................................................... 86 4.3.5 Tissue-specific expression .......................................................................................................... 88
4.4 Results ........................................................................................................................................... 88 4.4.1 Transcriptome assembly, annotation and gene identification ..................................................... 88 4.4.2 Sequences alignment, domain analysis and phylogenetic relationships ...................................... 93 4.4.3 Comparative molecular evolutionary analyses ......................................................................... 103 4.4.4 Tissue-specific expression analyses .......................................................................................... 107
4.5 Discussion .................................................................................................................................... 108 4.5.1 Comparative molecular analyses .............................................................................................. 109 4.5.2 Tissue-specific expression analyses .......................................................................................... 112
4.6 Conclusions .................................................................................................................................. 114
4.7 Nucleotide sequence accession number ........................................................................................ 114
4.8 Supplementary data .................................................................................................................... 114
General Discussion and Conclusion ......................................................................................... 115 Chapter 5.
References .............................................................................................................................. 125 Chapter 6.
Appendices ............................................................................................................................. 140 Chapter 7.
7.1 Appendix 1: Data distribution of the assembled contigs for C. quadricarinatus .............................. 140
7.2 Appendix 2: Top-hit Species distribution of the assembled contigs for Redclaw. ............................ 141
7.3 Appendix 3: Gene Ontology distribution of the gill transcripts from Redclaw ................................. 142
7.4 Appendix 4: Multiple alignment of partial C. quadricarinatus CA................................................... 143
7.5 Appendix 5 : Full length nucleotide sequences of C. quadricarinatus CAg ...................................... 144
7.6 Appendix 6: Statistics for contigs from C. cainii (Marron) and C. destructor (Yabby) ...................... 145
7.7 Appendix 7: Gene Ontology classification in C. cainii and C. destructor.......................................... 146
7.8 Appendix 8: List of all possible contigs with their sequence description, length and roles in pH and/or salinity balance ........................................................................................................................................ 147
7.9 Appendix 9: Identity matrix across species based on amino acid compositions (7.9.1-7.9.11) ........ 151
7.10 Appendix 10: Tissue-specific expression of major pH- and osmoregulatory genes in Redclaw crayfish Cherax quadricarinatus ............................................................................................................................ 157
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List of Figures
Figure 1.1 Natural distributions of Cherax quadricarinatus, C. destructor and C. cainii across Australia ................................................................................................................................ 4 Figure 1.2 Generalized ion-transport model in crustacean gills ............................................. 7 Figure 1.3 Mechanism of Na+/K+ transport by Na+/K+ -ATPase (NKA) .................................. 14 Figure 1.4 Schematic diagram of V-ATPase (HAT) showing the arrangement of the different subunits .............................................................................................................................. 20 Figure 2.1 Full length nucleotide sequences of cytoplasmic carbonic anhydrase (ChqCAc) .. 43 Figure 2.2 Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus cytoplasmic carbonic anhydrase isoform (ChqCAc) .................................. 44 Figure 2.3 Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus membrane-associated isoform (ChqCAg) ................................................... 45 Figure 2.4 Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus β-CA isoform (ChqCA-beta) ........................................................................ 47 Figure 2.5 Bootstrapped neighbor-joining tree of deduced amino acid sequences showing relationships between the genes identified from our study................................................. 49 Figure 2.6 Relative mRNA expression levels of Cytoplasmic carbonic anhydrase, GPI-linked carbonic anhydrase, V-type H-ATPase and Arginine kinase.................................................. 52 Figure 3.1 Relative expression of cytoplasmic carbonic anhydrase ...................................... 71 Figure 3.2 Relative expression of membrane-associated carbonic anhydrase ...................... 71 Figure 3.3 Relative expression of Na+/K+-ATPase ................................................................. 73 Figure 3.4 Relative expression of V-type-H+-ATPase ............................................................ 74 Figure 4.1 Multiple alignments of amino acid sequences from three Cherax Carbonic anhydrase (CA) with other species. ..................................................................................... 96 Figure 4.2 Phylogenetic relationships between the three Cherax crayfish with other organisms ........................................................................................................................... 97 Figure 4.3 Multiple alignment of three Cherax Na+/K+-ATPase α-subunit amino acid sequences with other crustaceans ...................................................................................... 99 Figure 4.4 Multiple alignment of three Cherax V-type-H+-ATPase subunit-a amino acid sequences with other species............................................................................................ 102 Figure 4.5 Tissue-specific expression/occurrences of important genes associated with pH balance or ion-regulation in Redclaw crayfish (Cherax quadricarinatus) ............................ 108 S Figure 2.1 Data distribution of the assembled contigs after blasting, mapping and annotation. ....................................................................................................................... 140 S Figure 2.2 Major top-hit species distribution on the basis of BALST search using the assembled contigs from Redclaw (C. quadricarinatus) ....................................................... 141 S Figure 2.3 Gene Ontology distribution of the gill transcripts from Cherax quadricarinatus based on Biological processes, Molecular functions and Cellular components. ................. 142
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S Figure 2.4 Multiple alignment of translated amino acid sequences of two partial C. quadricarinatus CA ............................................................................................................ 143 S Figure 4.5 The proportion and number of contigs assigned to Gene Ontology groups in C. cainii and C. destructor ..................................................................................................... 146
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List of Tables
Table 1.1 Environmental tolerances of Redclaw (C. quadricarinatus), Yabby (C. destructor) and Marron (C. cainii) ............................................................................................................ 5 Table 1.2 List of some important genes responsible for acid-base balance and osmotic/ionic-regulation in crustaceans ...................................................................................................... 8 Table 1.3 Published studies that have identified genes associated with pH balance and osmotic/ionic-regulation in decapod crustacean taxa ......................................................... 15 Table 2.1 Details of primers used in this study for amplification of the target genes at RT-PCR ........................................................................................................................................... 39 Table 2.2 Summary statistics for assembled contigs generated from C. quadricarinatus gill transcriptomes. ................................................................................................................... 41 Table 4.1 Summary statistics for assembled contigs from different organs of Cherax quadricarinatus, C. destructor and C. cainii ......................................................................... 91 Table 4.2 Full length coding sequences of the candidate genes involved in pH balance and osmotic/ionic regulation amplified from the gill transcriptomes of C. quadricarinatus, C. destructor and C. cainii ........................................................................................................ 92 Table 4.3 Molecular evolutionary analyses between three Cherax crayfish (C. quadricarinatus, C. destructor and C. cainii) at amino acid/codon level ............................. 104 Table 4.4 Molecular evolutionary analyses across crustacean species at Amino acid level 106 S Table 4.1 Summary statistics for assembled contigs greater than 200 bp generated from C. cainii (Marron) and C. destructor (Yabby) gill transcriptomes using Trinity de novo assembler and cd-HIT clustering tool. ................................................................................................ 145
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List of Abbreviations
aa= amino acid
AK= arginine kinase
bp= base pair
CA= carbonic anhydrase
CAb= beta (β) carbonic anhydrase
CAc= cytoplasmic carbonic anhydrase
CAg= membrane-associated glycosyl-phosphatidylinositol-linked carbonic anhydrase
Cc= Cherax cainii (Marron)
CD=expressed codons
Cd=Cherax destructor (Yabby)
Cq= Cherax quadricarinatus (Redclaw)
CRT= calreticulin
GPI= glycosyl-phosphatidylinositol
HAT= H+-ATPase or V-type H+-ATPase
HAT-116k=Vacuolar type H+-ATPase 116 kda
NBC=Na+/HCO3- cotransporter
NCX1 = Na+/Ca+2 exchanger 1
NHE3 = Na+/H+ exchanger 3
NKA= Na+/K+-ATPase
NKCC = Na+/K+/2Cl- cotransporter
ORF= open reading frame
qRT-PCR = quantitative real-time polymerase chain reaction
SERCA= sarco/endoplasmic reticulum Ca+2-ATPase
UTR: untranslated region
V-type= vacuolar-type
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Note on thesis preparation
Each chapter contains its own introduction, methodology, results, discussion and conclusion
sections.
Chapter 2 to 4 of this thesis represent individual, independent studies that each have a
discrete set of specific objectives and introduction and discussion sections particular to each
study. As such, there is some necessary repetition of information in the General
Introduction (Chapter 1) and General Discussion and Conclusions sections (Chapter 5) when
compared with the introductions and discussions sections of chapter 2 to 4.
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Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet requirements
for an award at this or any other higher education institution. To the best of my knowledge
and belief, the thesis contains no material previously published or written by another
person except where due reference is made.
Signature: Muhammad Yousuf Ali
Date: April, 2016
QUT Verified Signature
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Works published or submitted for publication by the author incorporated into the thesis
Statement of contribution to jointly authored works in the thesis
1. Ali, M.Y., Pavasovic, A., Mather, P.B. and Prentis, P.J., 2015. Analysis, characterisation and expression of gill-expressed carbonic anhydrase genes in the freshwater crayfish Cherax quadricarinatus. Gene (Elsevier) 564, 176-187
This manuscript is incorporated as Chapter 2 of this thesis. The first author was responsible
for conducting the research, analysis and interpretation of data, and writing this manuscript.
The co-authors were involved in refining the experimental design and provided conceptual,
logistical and editorial support.
2. Ali, M.Y., Pavasovic, A., Mather, P.B. and Prentis, P.J., 2015. Transcriptome analysis and characterisation of gill-expressed carbonic anhydrase and other key osmoregulatory genes in freshwater crayfish Cherax quadricarinatus. Data in Brief (Elsevier) 5, 187-193.
This manuscript is incorporated in Chapter 2 of this thesis. The first author was responsible
for conducting the research, analysis and interpretation of data, and writing this manuscript.
The co-authors were involved in refining the experimental design and provided conceptual,
logistical and editorial support.
3. Ali, M. Y., Pavasovic, A., Mather, P. B., & Prentis, P. J. (2015). Expression patterns of two alpha Carbonic anhydrase genes, Na+/K+-ATPase and V-type H+-ATPase in the freshwater crayfish, Cherax quadricarinatus, under different pH conditions. (manuscript in preparation)
This manuscript is incorporated as Chapter 3 of this thesis. The first author was responsible
for conducting the research, analysis and interpretation of data, and writing this manuscript.
The co-authors were involved in refining the experimental design and provided conceptual,
logistical and editorial support.
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4. Ali, M.Y., Pavasovic, A., Amin, S., Mather, P.B. and Prentis, P.J., 2015. Comparative analysis of gill transcriptomes of two freshwater crayfish, Cherax cainii and C. destructor. Marine Genomics (Elsevier) 22, 11-13.
This manuscript is incorporated in Chapter 4 of this thesis. The first author was responsible
for conducting the research, analysis and interpretation of data, and writing this manuscript.
The co-authors were involved in refining the experimental design and provided conceptual,
logistical and editorial support. In addition, the third co-author was involved in data analysis.
5. Ali, M. Y., Pavasovic, A., Acharige, L. D., Mather, P. B., & Prentis, P. J. (2015). Comparative molecular analyses of select pH- and osmoregulatory genes in three freshwater crayfish Cherax quadricarinatus, C. destructor and C. cainii. (manuscript in preparation)
This manuscript is incorporated in Chapter 4 of this thesis. The first author was responsible
for conducting the research, analysis and interpretation of data, and writing this manuscript.
The co-authors were involved in refining the experimental design and provided conceptual,
logistical and editorial support. In addition, the third co-author was involved in sample
collection, preparation and library preparation for sequencing.
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Acknowledgements
At first I would like to express my heartiest gratitude to all of my PhD supervisors; Dr. Peter J
Prentis (School of Earth, Environmental and Biological Sciences, Science and Engineering
Faculty, Queensland University of Technology), Dr. Ana Pavasovic (School of Biomedical
Sciences, Faculty of Health, Queensland University of Technology) and Professor Peter B
Mather (School of Earth, Environmental and Biological Sciences, Science and Engineering
Faculty, Queensland University of Technology) for their excellent supervision, advice,
guidance and constructive comments throughout the project and writing this thesis.
I would like to thank Australian government for awarding me IPRS and APA scholarships;
and special thanks for the authority of QUT for proving me Vice Chancellor’s Top Up
scholarship and providing me necessary facilities to conduct my PhD project. I also like to
acknowledge the funds provided by QUT (Queensland University of Technology) Higher
Degree Research Support and a QUT ECARD grant awarded to Peter Prentis that facilitated
this research.
Thanks to all in QUT Molecular Genetic Resource Facility (MGRF) and technical staff at
Banyo Pilot Plant Precinct for their cordial help during my project period. Especially, Vincent
Chand who arranged and organised everything nicely for rearing live crayfish at Banyo Pilot
Plant Precinct and arranged all types of facilities at QUT Molecular Genetics Lab. I would
also thank to Ms. Kylie Agnew-Francis and Hamish Kelly for sequencing my samples and to
Shane Russell for providing me some equipment for Banyo Aqualab. I also thank to
administration, technical, academic and research staff within the School of Earth,
Environmental and Biological Sciences for making a friendly environment with a feeling of
belonging to a community of people.
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I greatly acknowledge the support and cooperation given by freshwater crayfish suppliers
(Redclaw: Peter and Ethel Moore – Cherax park, Theebine, Gympie, QLD; Marron: Peter
McGinty – Aquatic Resources Management Pty Ltd., Manjimup west, WA; Yabby: Peter and
Margaret Burns – B and B yabby farm, Mudgee, NSW) by supplying us good quality animals.
Finally, I would like to acknowledge all the members of Physiology Genomics Lab (PGL)
where our lab group met regularly and discussed issues related to our project works, shared
our experiences and learnt new knowledge. Special thanks to Shorash Amin, Joachim Surm
and Jonathon Muller for helping me in RNA extraction and data analysis. I particularly
acknowledge Dr. Lalith Dammannagoda Acharige who helped me in many ways in sample
collection and lab work. I would also like to thank Lifat Rahi (PhD student, EEBS, QUT) for
assisting me in RNA extraction and library preparation for NGS-sequencing.
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General Introduction Chapter 1.
1.1 Economic and eco-physiological importance of Crayfish (as a model organism)
Crustacean aquaculture, a multi-billion dollar industry around the world, has been
expanding more rapidly than finfish and mollusc aquaculture over the last three decades
(FAO, 2011; FAO, 2012; Saoud et al., 2012; Saoud et al., 2013). Commercial crustacean
aquaculture now contributes approximately 9.6% to global aquaculture production (FAO,
2012) and is dominated by penaeid species. Freshwater species now also make a significant
contribution to crustacean production (29.4%). Of the farmed freshwater crustacean
species, crayfish contribute the highest production, followed by Chinese mitten crab and
freshwater prawns (FAO, 2012).
As demand for freshwater crustaceans continues to grow, there is pressure to expand
production levels for this class of organisms, by exploiting desirable traits and optimising
production practices. Selection of desirable traits has traditionally been associated with
traits or genes related to growth performance and/or disease resistance, with less focus on
selection for ecological traits, that would allow species to respond in positive ways where
there are fluctuations in culture conditions, such as tolerance to changes in water pH and
salinity levels.
Abrupt fluctuations in abiotic parameters including pH and salinity, within a production
setting can often have significant impacts on ion transport, endocrine function, physiological
traits and growth rate in many cultured crustaceans (Boron, 1986; Minh Sang and Fotedar,
2004; Pan et al., 2007; Ye et al., 2009). Pan et al., (2007) reported significantly lower growth
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rate in the marine shrimp, Litopenaeus vannamei cultured at 25 and 22 % salinity compared
with at 31 % salinity (a salinity that approximately equals to natural environment). They also
observed significantly lower weight gain at pH 7.1 and 9.1 as compared with that at pH 8.1
(optimal pH). In another study, the euryhaline shrimp (Penaeus latisulcatus) was exposed to
hyper-saline condition (46 ppt), where it showed both reduced hepatosomatic index and tail
muscle index (Minh Sang and Fotedar, 2004). Reduced growth performance in this species
was suggested to result from higher energy requirements diverted from growth to allow
individuals to maintain internal ionic concentration. Ye et al. (2009) also observed poorer
growth performance and lower feed conversion ratios in tiger shrimp (Penaeus monodon)
exposed to both higher and lower salinities outside their optimal salinity level (25 ppt).
Increased energy expenditure on osmoregulation can produce many problems in cultured
organisms including increased moisture content in muscle tissue, increased oxygen
consumption and higher ammonia-N excretion (Silvia et al., 2004). Therefore, a better
understanding of the genes that control osmoregulation and their function is required to
optimise the productivity of commercially cultured crustaceans under variable water quality
parameters.
Cherax quadricarinatus (Redclaw), C. destructor (Yabby) and Cherax cainii (Marron) are the
most abundant and economically important freshwater crayfish species in the genus Cherax.
These species have been developed for aquaculture production in Australia and elsewhere
around the world, most notably in Mexico, Ecuador, Uruguay, Argentina and China
(Macaranas, 1995; FAO, 2010; Saoud et al., 2012; Saoud et al., 2013). All three commercially
important Cherax species are endemic to Australia, and possess different natural
distributions that span a diverse range of aquatic environments (Fig. 1.1 and Table 1.1).
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Redclaw crayfish, C. quadricarinatus occurs naturally in rivers across northern Australia,
from the Kimberley region of north-western Western Australia across the Gulf of
Carpentaria to the tip of Cape York (Fig. 1.1). This species also occurs in the southern half of
Papua New Guinea (PNG). Wild populations of redclaw crayfish from northern Australia
occur naturally in waters with an abrupt environmental (pH) gradient; at low pH conditions
(pH 6.2-6.5 in northern Queensland, Australia) or at higher pH conditions (pH 8-8.2 in
Northern Territory, Australia) (Merrick and Lambert, 1991; Macaranas, 1995; Baker et al.,
2008) (Fig. 1.1). Yabby (C. destructor) is distributed across southern and central Australia,
notably in New South Wales, South Australia (SA) and Victoria with a natural pH range 6-9;
while Marron (C. cainii) occurs in south-west Western Australia with a natural pH range 7-
8.25 (Merrick and Lambert, 1991; YGA, 1992; Lawrence and Morrissy, 2000; Lawrence et al.,
2002) (Fig.1.1 and Table 1.1). However, all the three Cherax species have been translocated
to various places across Australia for commercial culture (Wingfield, 2008).
In general, all three species have the capacity to adapt to, and tolerate, fluctuations in
environmental parameters including pH, salinity, temperature and dissolved oxygen (see
references in Table 1.1). These physiological characteristics, as well as their economic
significance, has made them an important model for physiological research in crustaceans.
Despite this, genetic information for these crayfish species is still largely limited to
population genetic data describing genetic polymorphisms in natural populations (Miller et
al., 2004; Austin et al., 2014; Gan et al., 2014). Thus, genomic and molecular studies directed
at characterising the major genes involved in systematic acid-base balance and
osmoregulation are novel and important because they will allow a better understanding of
Page 4
their response to environmental variables including changes in physico-chemical water
properties.
Figure 1.1 Natural distributions of Cherax quadricarinatus, C. destructor and C. cainii across Australia
Page 5
Table 1.1 Environmental tolerances of Redclaw (C. quadricarinatus), Yabby (C. destructor) and Marron (C. cainii)
pH Salinity (ppm)
Temperature (oC)
Dissolved Oxygen (ppm)
Source Reference
Low
High
Gro
wth
im
paire
d Max
Deat
h
Opt
imal
Min
Max
Min
for
grow
th
Stre
ss
Opt
imal
Dist
ress
Redclaw
18000 20-34 (Jones, 1990) 6.5 8 (Merrick and Lambert, 1991)
23-31 10 36 (Jones, 1998) 12000 18000 23-28 10 35 >5 1 (Mosig, 1998) 17000 28 34 (DPI, 2005)
6.5 8 12000 18000 22-31 10 35.5 34 >5 1 ← Mean
Yabby
>5000 16000 28 15 34 (Mills, 1983) 6000-
8000 14000-15000
22000 15 (Morrissy et al., 1990)
7.5 8.5 >6000 25-28 ≥4 (Merrick and Lambert, 1991) 7 7.5 5-7 2 (YGA, 1992) 6 9 20-26 1 35 (Mosig, 1998)
7.5 10.5 17000 25000 22-28 1 36 15 >4 (DPI, 2005) 7 8.9 6000 15750 21000 22-27.5 1 35.5 15 34 5 2 ← Mean
Marron
24 6.5 2.0 (Shipway, 1951)
8 30 12-13 (Morrissy et al., 1990)
7 8 17000 17-25 ≥6 (Merrick and Lambert, 1991))
7 8.5 >6000-8000
15000 24 30 12.5 6 3 (Lawrence, 1998)
18-24 >6 (Mosig, 1998)
17000 25 31 >6 (DPI, 2006)
18-24 4 26 12 (Sampey, 2007)
7 8.25 7000 16333 17.5-24.5
>4 28.6 12.3 >6 2.5 ← Mean
Page 6
1.2 Genes involved in pH balance and osmotic/ionic-regulation
Many enzymes and transporters contribute to ion transport in crustacean taxa including;
Na+/K+-ATPase (NKA), V-type-H+-ATPase (HAT), carbonic anhydrase (CA), Na+/K+/2Cl-
cotransporter (NKCC), (Na+)Cl− /HCO3− (NCB) and Na+/H+ exchangers (NHE) (Zhang et
al., 2000a; Weihrauch et al., 2001a; Gao and Wheatly, 2004a; Serrano et al., 2007; Tsai
and Lin, 2007; Freire et al., 2008; Pongsomboon et al., 2009; McNamara and Faria,
2012a; Havird et al., 2013; Tongsaikling, 2013; Havird et al., 2014). Table 1.2 summarises
some of the major genes that have already been identified and shown to be involved in
maintenance of systemic acid-base balance and ion-regulation/osmoregulation in
decapod crustaceans. The inferred transport model and hypothetical schematic
pathways for how these genes act are shown in Fig. 1.2 Briefly, CA produces H+ and
HCO3- ions through a reversible reaction, CO2+H2O ←→H++HCO3
-, thereafter the H+ and
HCO3- serve as anti-porters for Na+/H+(NH4
+) exchangers and Cl-/HCO3- cotransporter
(see comprehensive reviews by: Freire et al., 2008; Henry et al., 2012; Romano and
Zeng, 2012). NKA pumps Na+ ions out of the cell and draws K+ ions in, and thus
establishes an electrochemical gradient that acts as a driving force for transport of Na+
and K+ ions by other transporters including NKCC (Lucu and Towle, 2003; Jayasundara et
al., 2007; Leone et al., 2015; Li et al., 2015). Basolateral NKCC absorb Na+, K+ and Cl-
ions at low salinities, and apical NKCC extrude Na+, K+ and Cl- ions at high salinities to the
external environment, through the gradient driven by NKA (Onken and Riestenpatt,
1998; Weihrauch et al., 2004a). HAT (H+-ATPase) pumps protons (H+) and acidifies
intracellular organelles that help to maintain pH balance in crustacean taxa (Faleiros et
Page 7
al., 2010; Lee et al., 2011; Towle et al., 2011; Boudour-Boucheker et al., 2014; Lucena et
al., 2015).
Figure 1.2 Generalized ion-transport model in crustacean gills
Generalized ion-transport model in crustacean gills (after Onken and Riestenpatt, 1998; Weihrauch et al., 2002; Weihrauch et al., 2004a; Weihrauch et al., 2004b). (1) Carbonic anhydrase (CA); (2) Cl−/ HCO3
- exchanger; (3) Na+/K+-ATPase (NKA); (4) V-type-H+-ATPase (HAT); (5) Na+/H+ exchanger (NHE); (6) Na+/K+/2Cl− co-transporter (NKCC); (7) K+ channel; (8) Cl− channels; (9) paracellular diffusion of NH4
+ via vesicles acidified by V-ATPase that protonate NH3. Note: At high salinities, apical NKCC extrude Na+, K+ and Cl- ions to the external environment, through a gradient driven by NKA, while at low salinities, basolateral NKCC absorb these Na+, K+ and Cl- ions. CA catalyzes the reversible reaction: H++HCO3
-←→CO2+H2O, where H+ and HCO3
- serve as anti-porters for (2) and (5). Thus, CA plays a vital role in pH balance, ion transport and gas exchange.
Page 8
Table 1.2 List of some important genes responsible for acid-base balance and osmotic/ionic-regulation in crustaceans
Gene Enzymes Functions Reference CAc Cytoplasmic carbonic anhydrase (CAc) pH balance and osmoregulation (Serrano et al., 2007; Pongsomboon et al., 2009; Liu et al., 2015)
CAg GPI-linked carbonic anhydrase (CAg) pH balance and osmoregulation (Serrano et al., 2007; Serrano and Henry, 2008; Liu et al., 2015)
NKA Na+/ K+-ATPase alpha subunit pH balance and osmoregulation (Chaudhari et al., 2015; Han et al., 2015; Leone et al., 2015)
HAT V-type-H+-ATPase catalytic subunit a pH balance and osmoregulation (Pan et al., 2007; Wang et al., 2012; Havird et al., 2014)
NKCC Na+/K+/2Cl- cotransporter pH balance and osmoregulation (Towle et al., 2001; Havird et al., 2013; Havird et al., 2014)
NBC Na+/HCO3- cotransporter pH balance and osmoregulation (Pan et al., 2007)
NCBE Na+-driven Cl−/HCO3 –exchanger pH balance and osmoregulation (Freire et al., 2008)
NHE
Na+/H+ Exchanger
pH balance and osmoregulation (Towle and Weihrauch, 2001; Weihrauch et al., 2004a; Ren et al., 2015)
NCX Na+/Ca+2 Exchanger Osmoregulation (Freire et al., 2008)
AK arginine kinase pH balance and osmoregulation (Serrano et al., 2007; Havird et al., 2014)
ALP alkaline phosphatase pH balance (Tongsaikling, 2013)
CRT calreticulin Osmoregulation (Luana et al., 2007; Visudtiphole et al., 2010; Watthanasurorot et al., 2013; Duan et al., 2014; Lv et al., 2015; Xu et al., 2015)
SERCA sarcoplasmic endoplasmic reticulum calcium-transporting ATPase
Ion regulation (Zhang et al., 2000b; Wheatly et al., 2002; Ahearn et al., 2004; Mandal et al., 2009)
K+ Channel K+ Channel Osmoregulation (Ren et al., 2015)
Aquaporin integral membrane protein Aquaporins Osmotic signal transduction Cell volume regulation
(Chung et al., 2012)
MAPK P38 mitogen-activated protein kinases Osmotic signal transduction response to osmotic shock
(Sun et al., 2015)
ITG Integrin Response to salinity stress and controls homeostasis
(Huang et al., 2015)
Page 9
1.2.1 Carbonic anhydrase and its role in pH balance and osmoregulation
Carbonic anhydrase (CA) is a Zn-metalloenzyme that catalyses the inter-conversion of CO2
and HCO3− through the reversible reaction: CO2 + H2O ↔ HCO3
− + H+; where H+ and HCO3-
serve as anti-porters for Cl−/ HCO3− exchanger and Na+/H+ exchanger. Consequently,
members of this gene family have been shown to play an important and large role in pH
balance, ion transport, shell formation, bone deposition and gas exchange in eumetazoans
(Freire et al., 2008; Henry et al., 2012; Romano and Zeng, 2012). CA exists in three distinct
forms in arthropods; cytoplasmic CA, membrane-associated CA (GPI-linked) and beta CA (β-
CA) (Serrano et al., 2007; Serrano and Henry, 2008; Zimin et al., 2008; Syrjanen et al., 2010;
Werren et al., 2010; Colbourne et al., 2011; Jillette et al., 2011; Nygaard et al., 2011). While
cytoplasmic CA and GPI-linked CA are both members of the alpha carbonic anhydrase gene
family they have an independent origin to β-CA, which is a member of the beta carbonic
anhydrase gene family.
Carbonic anhydrases are widely distributed ion transport-related enzymes and members of
the alpha CA family that have been well-studied in crustaceans (for examples; (Serrano et
al., 2007; Serrano and Henry, 2008; Pongsomboon et al., 2009; Liu et al., 2015) and also see
the reviews by: (Freire et al., 2008; Henry et al., 2012; Romano and Zeng, 2012). A
substantial number of studies have been undertaken on its distribution among decapod
species, biochemical- and molecular characterisation of its function, expression levels and
its physiological role(s) in decapod crustaceans (Henry and Cameron, 1982; Henry and
Kormanik, 1985; Wheatly and Henry, 1987; Böttcher et al., 1990; Böttcher et al., 1991;
Onken and Riestenpatt, 1998; Henry et al., 2002; Henry et al., 2003; Henry and Campoverde,
Page 10
2006; Roy et al., 2007; Serrano et al., 2007; Serrano and Henry, 2008). Alpha CA genes,
cytoplasmic CA and GPI-linked CA, play a vital role in ion-regulation/osmoregulation
because they provide H+ and HCO3− ions which are used as counterions for exchange of
cations and anions, in particular Na+ and Cl- (Henry and Cameron, 1983; Henry, 2001; Freire
et al., 2008; Henry et al., 2012; Havird et al., 2013). Its role in systematic acid-base balance
has also been established in a wide range of marine crustacean taxa including; prawns (Gao
and Wheatly, 2004a; Pongsomboon et al., 2009; Liu et al., 2010; Pan et al., 2011; Hu et al.,
2013); and crabs (Serrano et al., 2007; Serrano and Henry, 2008; Liu et al., 2012), but
information for freshwater species is limited.
The main CA enzyme that has been shown to respond to pH changes and plays a significant
role in systematic acid-balance in decapod crustaceans is cytoplasmic CA. The expression of
cytoplasmic CA has been demonstrated to be strongly induced in crustaceans by changes in
water salinity (Serrano and Henry, 2008), where the induction of CAc expression was more
pronounced when individuals were exposed to low salinity conditions (Henry et al., 2012). In
fact, previous studies have reported a 10-fold increase in CAc expression in the posterior
gills of two marine crab species C. sapidus and C. maenas after 6 h exposure to low salinity
levels, and the CAc expression levels increased to approximately 100-fold 24 h post-transfer
(Serrano et al., 2007; Serrano and Henry, 2008). It has been suggested that the change in
expression levels occurs because cytoplasmic CA provides counter ions (H+ and HCO3−) to
the Na+/H+ and Cl−/HCO3− exchanger that drive ion uptake in crab gills when they are
exposed to low salinity levels or low pH environments (Henry and Cameron, 1983; Henry,
1988; Henry, 2001).
Page 11
The role and activity of membrane-associated CA (CAg) and β-CA (CAb) are not clearly
resolved in systematic acid-balance and osmoregulation in decapod crustaceans (Havird et
al., 2013). For example, some studies have not found clear evidence that these two genes
are differentially expressed in response to changes in water pH and/or salinity (Henry, 1988;
Serrano et al., 2007). In fact, in studies that do find changes in the expression of the
membrane bound CA form, this transcript shows much lower induction than the cytoplasmic
form when exposed to low pH and low salinity conditions (Serrano et al., 2007; Serrano and
Henry, 2008; Liu et al., 2015). CAg is mainly located on the outer membrane of cells and is
believed to facilitate mobilization of hemolymph HCO3− to molecular CO2 and assists with
CO2 excretion via the gills (Henry, 1987). Thus, this gene (CAg) may play a less significant
role in maintenance of acid-base balance in freshwater crayfish than cytoplasmic CA.
It remains unknown if CA-β is expressed in the gills of decapod crustaceans or if this enzyme
plays a role in either systematic acid-balance and osmoregulation. Recent studies have
shown that the homologous β CA protein in Drosophila is a highly active mitochondrial
enzyme (Syrjanen et al., 2010). This mitochondrial CA may have a functional role in the
CO2/HCO3− interaction by providing counterions for the uptake of energy-linked
mitochondrial Ca2+ (Dodgson et al., 1980). However, CA-β was not identified in any of the
complete mitochondrial genomes of Cherax quadricarinatus (Gan et al., 2014), C. destructor
(Miller et al., 2004) and C. cainii (Austin et al., 2014) in Cherax crayfish. Thus, the role and
activity of CA-β in crayfish remains unclear.
Given that some CA enzymes play an important role in acid-base balance and
osmoregulation (particularly cytoplasmic CA), CA in general has received only limited
attention in crayfish. In this regard, freshwater crayfish may provide an interesting model
Page 12
for identification and characterisation of the various CA forms because these taxa
experience in nature hypo-osmotic environments that can vary widely in pH (Baker et al.,
2008).
1.2.2 Na+/K+-ATPase and its role in pH balance and osmoregulation
Na+/K+-ATPase (NKA), often referred to as the Na+/K+ pump or sodium pump, is an
electrogenic transmembrane enzyme located in the plasma membrane of cells that pumps
sodium ions out of cells and potassium ions into the cell. Ion uptake in many crustaceans is
largely controlled by the activity of this enzyme (Havird et al., 2013; Chaudhari et al., 2015;
Han et al., 2015; Leone et al., 2015; Li et al., 2015). The NKA protein is normally located in
the gill basolateral membrane and provides the major driving force for transport of Na+ and
K+ ions by establishing an electrochemical gradient between the inside and outside of a cell
(Towle and Kays, 1986). In addition to active transport of Na+ and K+, NKA also helps to;
maintain resting potential, regulate cell volume and affects transport of other ions as well.
Moreover, NKA functions as a signal transducer to regulate the ERK/MAPK pathway
(extracellular signal-regulated kinases or mitogen-activated protein kinases) and
intracellular calcium. In most animal cells, NKA is responsible for the major component of
energy expenditure (Skou and Esmann, 1992; Scheiner-Bobis, 2002). Of several major ion-
regulating enzymes in crustaceans (Na+/K+-ATPase, V-H+-ATPase, HCO3--ATPase and
Carbonic anhydrase), NKA accounts for about 60-70% of the total ATPase activity (Skou and
Esmann, 1992; Morris, 2001). For example, in the freshwater shrimp, Macrobrachium
olfersii, NKA function contributed approximately 70% of total ATPase activity; with the
remaining 30% largely attributed to V-type and F0F1-ATPase activity (Furriel et al., 2000). In
Page 13
post larvae Litopenaeus vannamei, exposed to different salinity levels, Pan et al. (2007)
reported that NKA accounted for appriximately 62–78% of the total ATP activity; which
varied across different pH levels.
NKA consists of three different subunits; α, β and γ (Skou and Esmann, 1992; Pressley,
1995; Blanco and Mercer, 1998). The alpha subunit, the largest one (molecular mass ≈ 112-
115 kda), contains binding sites for cations and ATP, and thus responsible for the catalytic
function and ion regulation. The β-subunit, the second largest unit (molecular mass ≈ 40-60
kda), is usually involved in protein maturation and assists in anchoring the enzyme complex
in membranes (Blanco and Mercer, 1998). This process includes several steps (Fig. 1.3
illustrates the process) and after a single cycle, it pumps 3 Na+ ions out of the cell and draws
2 K+ ions into the cell (Skou, 1988; Glynn, 1993; Scheiner-Bobis, 2002) .
NKA-α subunit has been shown to be important for osmoregulation/ion-regulation but
results vary across euryhaline organisms when they are challenged with changes in salinity
and pH levels in water (Towle et al., 2001; Parrie and Towle, 2002; Pan et al., 2007). Relative
activity levels and gene expression of α-NKA are highly induced under salinity-stressed
conditions in some species (Henry et al., 2002; Luquet et al., 2005; Jayasundara et al., 2007;
Li et al., 2015). In contrast, other studies have reported the down-regulation of NKA-α
expression following salinity challenges in the swimming crab Portunus trituberculatus (Xu
and Liu, 2011) and in the shore crab Carcinus maenas (Jillette et al., 2011). For example, Han
et al. (2015) reported a significant decrease in NKA-α expression in the first 6 h after
individuals were transferred from 35‰ (ppt) salinity to 15, 13, 11, and 9‰ salinity in P.
trituberculatus. The diverse activities of NKA-α under changes in water salinity are also
Page 14
demonstrated as some other studies have not observed any significant changes in
expression levels in decapod crustaceans (Havird et al., 2014).
Tissue-specific gene expression patterns and enzyme activity studies have demonstrated
that gill is the tissue with highest levels of NKA expression in decapod crustaceans: A.
anguilla (Cutler et al., 1995), C .sapidus (Towle et al., 2001) F. heteroclitus (Semple et al.,
2002), S. sarba (Deane and Woo, 2005), P. marmoratus (Jayasundara et al., 2007), A.
schlegeli (Choi and An, 2008) and P. trituberculatus (Han et al., 2015). For instance, Han et
al. (2015) observed approximately 40 fold increase in NKA mRNA expression in the gills of
swimming crab P. trituberculatus as compared to that in hemocytes, and almost double
when compared with Hepatopancreas.
Figure 1.3 Mechanism of Na+/K+ transport by Na+/K+ -ATPase (NKA) Complete reaction cycle of NKA. Step1: NKA binds 3 Na+ and ATP in the E1 conformational state. Step 2: NKA is phosphorylated at an aspartate residue. This leads to the occlusion of three Na+ ions. Step 3: Na+ are released to the extracellular side. Step 4: New conformational state (E2-P) binds K+ Step 5: Binding of K+ leads to dephosphorylation of the enzyme and to the occlusion of two K+ cations. Step 6: K+ is then released to the cytosol after ATP binds to the enzyme with low affinity. (based on (Skou, 1988; Glynn, 1993; Scheiner-Bobis, 2002)).
Page 15
Table 1.3 Published studies that have identified genes associated with pH balance and osmotic/ionic-regulation in decapod crustacean taxa
Species Organ Stressor Level of Stress
Dur
atio
n
Genes Expression Reference
Callinectes sapidus (blue crab)
Gills salinity * 35→5 ‰ NKA No significant differences in expression (Towle et al., 2001)
Penaeus chinensis Gills pH 6←7.5→8.5 14d NKA Down-regulated significantly below and after pH 7.5 (Wang et al., 2002) Carcinus maenas GIlls salinity 32→10‰ CAc
AK CAc: Upregulated (5 fold at 24 h post-transfer) AK: did not change
(Henry et al., 2003)
Chasmagnathus granulatus ( euryhaline crab)
Gills salinity 2←30→45‰
NKA- α HAT-B NKCC AK (Ct)
NKA- α: upregulated (35 to 55 fold by 24 h at 2‰; 28 fold after 96h at 45‰) HAT-B: slightly upregulated (5-8 fold by 24h at 2‰; 10 fold after 96 h at 45‰) NKCC: upregulated (10-22 fold by 24 h at 2‰; 60 fold after 96h at 45‰) AK: slightly upregulated ( 3-4-fold by 24 h; 2-3 fold after 96 h at 45‰)
(Luquet et al., 2005)
Carcinus maenas Gills salinity 32→10‰ 32→25→20→15→10‰
7d CA 32→10‰: upregulated (10 fold at 24 h when transferred 32→10‰) 32→25→20→15→10‰: upregulated (after 7 d, 10 fold at 15‰; 2-2.5 fold at 20/25‰; 5 fold at 10‰) (CA activity also increased in similar pattern)
(Henry et al., 2006)
Litopenaeus vannamei ( white shrimp)
Gills pH salinity
7.1←8.1→9.1 22‰←31‰
4d
NKA HAT HCO3
--ATPase
pH: NKA upregulated below and above pH 8.1 at 24h (overall, NS) HAT and HCO3
-ATPase showed negative correlation with pH (S) Salinity: NKA showed negative correlation with salinity HAT and HCO3
-ATPase not significantly changed
(Pan et al., 2007)
Callinectus sapidus (blue crab)
Gills salinity 35→15‰ 28d CAc CAg NKA-a AK
CAc: highly up-regulated (100 fold) CAg: non-significantly up-regulated (3 fold) NKA: significantly up-regulated (3-4 fold) AK: not significantly changed
(Serrano et al., 2007)
Carcinus maenas (crab)
Gills salinity 32→15‰ 14d CAc CAg NKA AK
CAc: upregulated (100 fold by 24 h) CAg: significantly up-regulated (2 fold at 24h and 3 fold at 7 days) [CA activity 16 fold increase at 14 days] NKA: upregulated (10-fold at 96 h and 20 fold after 14 days) AK: increased 1.5 fold at 7 d, but unchanged over 14 days (housekeeping
(Serrano and Henry, 2008)
Page 16
gene) Pachygrapsus marmoratus (euryhaline shore crab)
Gills salinity 10←36→45‰
NKA Up-regulated at lower salinity (10‰) No change at higher salinity (45‰)
(Jayasundara et al., 2007)
Penaeus monodon (shrimp)
Gills Epipodites Antennal gland
salinity 25→3‰ 14d CA Gills: Significantly up-regulated after 24h Epipodites and antennal gland: down-regulated
(Pongsomboon et al., 2009)
Litopenaeus vannamei (Pacific shrimp)
Gills Hepatopancreas
pH salinity
5.6←7.4→9.3 5←10←20‰
24h NKA-b HAT-a
pH 5.6: NKA and HAT up-regulated (17 and 50 fold respectively) after 6h pH 9.3: NKA and HAT up-regulated (4 and 10 fold respectively) after 3h NKA and HAT upregulated (17 and 45 fold respectively) after 12h at salinity 5‰
(Wang et al., 2012)
Euryhaline sps
Gills (plus others)
salinity meta-analysis of 59 published qPCR works
NKA CA HAT NKCC CFTR
NKA, CA, HAT and NKCC up-regulated CFTR unchanged
(Havird et al., 2013)
Halocaridina rubra ( anchialine shrimp)
Gills salinity 2,15←32→45‰
7d NKA CAc CAg HAT-B NKCC AK
Overall, all unchanged after 7 days NKA down-regulated after 24 h at 15‰ and 45‰ (by 26% and 35% respectively) HAT down-regulated in all transfers between 3-48 h (50-fold lower at 45‰ after 24 h) NKCC up-regulated slightly after 24 h at 2‰ (2.6-fold) CAs down-regulated after 48 h at 15‰ (by 76%)
(Havird et al., 2014)
Callinectes sapidus and Carcinus maenas
Gills salinity 32→25→22.5‰
7d CA Upregulated ( 90-fold in C. sapidus and 2-fold in C. maenas within 6 h) (300% and 100% increased CA activity at 4 days respectively)
(Mitchell and Henry, 2014)
Macrobrachium amazonicum (river shrimp)
Gills (adult and juvenile)
pH 6.5→7→7.5→8→8.5
same day
HAT (activity)
Increased when pH increased from 6.5 t0 7.5, but decreased when pH increased up to 8.5 Same pattern in adult and juvenile.
(Lucena et al., 2015)
Exopalaemon carinicauda (ridgetail white prawn)
Gills Hepatopancreas Hemocytes Muscle
salinity 4←32→39‰
24h NKA-α Up-regulated both in low and high salinities in gills (12 fold in 4‰ at 32h and 19.5 fold in 39‰ at 24 h) Highest expression in gill
(Li et al., 2015)
Callinectes ornatus (ornate blue crab)
Gills salinity 33→21‰ 10d NKA Expression up-regulated within 1-2-h ( ≈ 8-fold after 24-h) Activity increased (≈ 2.5-fold after 10d).
(Leone et al., 2015)
Penaeus monodon Gills salinity 5←25→35‰
20d NKA Expression up-regulated (1.5 fold at 5‰) Activity increased at 5‰ (3 folds) Activity decreased at 35‰.(0.48 folds)
(Chaudhari et al., 2015)
Page 17
Note: NKA=Na+/K+-ATPase, CAc=Cytoplasmic carbonic anhydrase, CAg=GPI-linked carbonic anhydrase, HAT-A=Vacuolar type H+-ATPase, NKCC = Na+/K+/2Cl- cotransporter, AK=Arginine kinase, SERCA=Sarco/endoplasmic reticulum Ca+2-ATPase, CALR=Calreticulin, * The root of the arrow indicates the control and the head of the arrow points towards the treatment where the samples were transferred to
Portunus trituberculatus (swimming crab)
Gills Hepatopancreas Hemocytes Muscle
salinity 9,11,13,15←35‰
72h NKA Down-regulated in all transfers after 6 h Up-regulated significantly in all transfers after 24 h
(Han et al., 2015)
Litopenaeus vannamei (the pacific shrimp)
Gill pH salinity
7.4←8.2→9 20←30←35‰
72h CAc CAg
pH: CAc up-regulated within 12h (5 & 4.5 fold after 48h at pH7.4 and 9 respectively) CAg up-regulated within 12h (2.5 & 3 fold after 12h at pH7.4 and 9 respectively ) Salinity: CAc up-regulated within 24h (5.5 & 3.5 fold after 24h at salinity 20 and 35‰ respectively) CAg up-regulated within 24h (2.5 & 1.5 fold after 24h at salinity 20 and 35‰ respectively )
Liu et al., 2015
Page 18
1.2.3 V-type-H+-ATPase and its role in pH balance and osmoregulation
Vacuolar-type H+-ATPase (HAT) or V-ATPase are a group of highly conserved membrane-
associated enzymes that acidify a wide range of intracellular organelles and pump protons
across the plasma membranes in multiple tissue-types. HAT are ATP-dependent proton
pumps that couple the energy for ATP hydrolysis to actively transport protons into different
compartments of the cell or outside of the cell via the plasma membrane (Forgac, 2007).
HATs are organized into two major multi-subunit functional domains, V0 and V1 that are
interconnected to each other. The integral membrane associated V0 domain has six different
subunits (a, d, c, c', c", and e) and the peripherally associated V1 domain consists of eight
different subunits (A–H) arranged in different ways to undertake the entire function of this
protein (Kitagawa et al., 2008; Muench et al., 2011; Marshansky et al., 2014; Rawson et al.,
2015) (Fig. 1.4). The A and B subunits of V1 domain possess the main catalytic sites that are
involved in ATP hydrolysis (Saroussi and Nelson, 2009; Nakanishi-Matsui et al., 2010; Toei et
al., 2010). ATP hydrolysis in the V1 domain drives the central rotary mechanism that HATs
use to perform their main function, the unidirectional transport of H+ across the cell
membrane (Imamura et al., 2003). The major responsibility of the Vo domain is to
translocate protons, where two protons are translocated every time ATP is hydrolysed
(Johnson et al., 1982). The majority of HAT subunits are expressed as multiple isoforms that
have been shown to have tissue-specific expression patterns (Toei et al., 2010). Factors
affecting the regulation of HAT activity include; dissociation of the V1 and V0 domains,
selective targeting of specific HAT units and the strength of coupling between proton
transport and ATP hydrolysis (Kane, 2006; Toei et al., 2010).
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HAT has been shown to be one of the master enzymes that regulate acid-base balance and
osmoregulation in many organisms (Forgac, 1998; Saliba and Kirk, 1999; Beyenbach, 2001;
Kirschner, 2004; Covi and Hand, 2005). Its involvement in osmoregulation in freshwater taxa
has also been demonstrated by a number of gene expression experiments and protein
activity studies (Faleiros et al., 2010; Lee et al., 2011; Towle et al., 2011). This enzyme
pumps protons to acidify intracellular organelles, thus influencing Na+ uptake and Cl−/HCO3−
exchange (Weihrauch et al., 2004a; Martin Tresguerres et al., 2006). It also plays a pivotal
role in branchial NH3 excretion (Weihrauch et al., 2002; Weihrauch et al., 2009; Martin et
al., 2011; Weihrauch et al., 2012). Although, expression and activity levels of V(H+)-ATPase
may be regulated by extracellular pH, studies on the effects of pH on this enzyme in
crustaceans to date are rare (Pan et al., 2007; Lucena et al., 2015). In contrast, much more
work has been directed to attempting to understand the effects of salinity on V(H+)-ATPase
activity (Table 1.2).
HAT expression has been found to be commonly up-regulated when individuals are exposed
to low pH or low salinities (Pan et al. , 2007,(Havird et al., 2013). Pan et al. (2007) reported
a negative correlation between expression of HAT and pH level, where this enzyme is highly
expressed under low pH conditions but lowly expressed at higher pH. Expression of HCO3-
ATPase was also modified in a pattern similar to HAT, showing peak changes up to 24 h in all
treatment groups (pH 7.1, 7.6, 8.6, 9.1 as compared with a control group (pH 8.1). A few
studies have also detected the down-regulation of HAT following transfer of individuals
between different salinity environments (Havird et al., 2014). Havird et al. (2014) showed
significant decreases in the expression level of HAT when individuals were transferred from
32‰ to 2, 15 and 45‰ between 3 and 48 h post transfer. The lowest decline 24 h post-
Page 20
transfer was at 45‰, where the expression level was 50-fold lower than in control salinity
condition. The study, however, did not find any significant changes in HAT expression after
48 h to 7 days (Havird et al., 2014). Lucena et al. (2015) reported significant down-regulation
of gill V(H+)-ATPase activity at pH conditions both below and above neutral conditions (pH of
7.5), where they reported a 4-fold decrease at pH 6.5 and 2.5 fold decrease at pH 8.5. This
pattern was observed in both juvenile and adult individuals of the river shrimp, M.
amazonicum. Overall, there is a general pattern that HAT expression is upregulated at low
pH or low salinities and down-regulated at high pH or high salinity levels, but with some
variation among species. It remains to be tested whether HAT expression in freshwater
crayfish responds in similar way.
Figure 1.4 Schematic diagram of V-ATPase (HAT) showing the arrangement of the different subunits
(Beyenbach and Wieczorek, 2006)
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1.2.4 Arginine kinase and its role in pH balance and osmoregulation
Arginine kinase is an enzyme that plays an important role in maintaining ATP levels in the
cells of invertebrates (Song et al., 2012) and has been suggested to play a role in
osmoregulation and acid-base balance in some crustaceans. In fact, this enzyme has been
shown to be differentially expressed in certain crustacean species, when transferred to
different ionic conditions (Kinsey and Lee, 2003; Pan et al., 2004; Yao et al., 2005; Liu et al.,
2006; Silvestre et al., 2006; Abe et al., 2007; Song et al., 2012). This pattern does not hold
for all decapod crustaceans, as some species show no significant difference in arginine
kinase expression when transferred among different ionic conditions. For example, the
relative abundance of AK mRNA was not different in the crab species, C. maenus, when
individuals were transferred to low salinity conditions (Kotlyar et al., 2000; Towle and
Weihrauch, 2001). While the gene was not differentially expressed, arginine kinase protein
levels did increase in low pH treatments where V-type H+-ATPase and cytoplasmic CA
protein abundance also increased. Weihrauch et al. (2001b) studied osmoregulation in
crustaceans, and suggested that AK levels increased in some species, to maintain ATP levels,
that are required for active ion transport and other cellular processes that require ATP. This
hypothesis would explain the increased levels of AK in lower pH treatments when active
transport enzymes are also upregulated. Whether this pattern of increased AK gene
expression at low pH occurs in freshwater crayfish species remains untested.
1.2.5 Ca+2-ATPase and its role in pH balance and osmoregulation
Ca+2-ATPase or the calcium pump is an important enzyme that is required for transbranchial
Ca2+ absorption and secretion (Wheatly et al., 2002; Ahearn et al., 2004). Ca2+-ATPase has
Page 22
been identified from branchial tissues in crustaceans including crayfish and shrimp (Chen et
al., 2002; Gao and Wheatly, 2004b; Gao and Wheatly, 2007; Wang et al., 2013b). It is
commonly located in the basolateral membrane of epithelial cells.
Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase has been identified and characterised in a
number of crustaceans (Zhang et al., 2000b; Mandal et al., 2009). This enzyme has also been
implicated in the moulting process as it plays an active role in Ca2+ absorption during post-
moult period in crustacea. Molecular data on, and mRNA expression profiles for this
enzyme, however, are still very limited, even though the kinetic characteristics of the Ca2+
pump have been widely studied. The role of Ca2+-ATPase in systematic pH balance need to
be further explored in freshwater crustaceans.
1.2.6 Ion channels involved in pH balance and osmoregulation
A number of ion channels; including the Na+ ion channel, K+ ion channel, Cl- ion channel and
Ca+2 ion channel are involved in direct transport of some key osmoregulatory ions both into
and out of cells in crustaceans. The function of these ion channels can be seen in
hyperosmoregulating crustacean taxa, where active NaCl absorption via the epithelium
membrane compensates for passive loss of NaCl as a result of an external dilute medium
(Kirschner, 2004). Basolaterally located membrane transporters, including the Na+, K+ and Cl-
channels are ubiquitous in all NaCl absorbing epithelia (Onken et al., 1991; Zeiske et al.,
1992; Onken and Riestenpatt, 1998; Onken et al., 2003).
Epithelial Na+ channels have been identified in gills of the Chinese mitten crab (E. sinensis)
where the majority of osmoregulation and pH balance take place (Riestenpatt et al., 1994).
Basolateral, as well as an apical K+ channel have been identified across split gill lamellae in a
Page 23
number of weak hyperosmoregulators, including C. maenas and C. granulata, using Ba2+ or
Cs+ and short-circuit current analysis (Onken and Graszynski, 1989; Onken et al., 1991;
Riestenpatt et al., 1996; Onken and Riestenpatt, 1998). Onken et al. (1991) also presented
additional evidence for a K+ channels when they examined the effects of hemolymph-side
Ba2+ or high K+ on the membrane voltage. Basolateral Cl− channels have also been identified
via transbranchial voltage and short-circuit current analysis in isolated gills of crustacean
taxa (Gilles and Pequeux, 1986; Siebers et al., 1990; Onken et al., 1991; Riestenpatt et al.,
1996). While these ion channels have been identified in the gills of various decapod
crustaceans, little is known about whether these genes are expressed in the gills of
freshwater crayfish.
1.2.7 Functions of transporters and exchangers in maintaining pH balance and for
osmoregulation
Transporters and exchangers have been identified in crustaceans that play fundamental
roles in maintenance of pH balance and osmotic/ionic-regulation. They are associated with
active or secondary ion transport in and out of cells. Of these, some of the most important
include; the Na+/K+/2Cl- cotransporter, Na+/HCO3- cotransporter, Cl−/HCO3
– exchanger,
Na+/H+ Exchanger, Na+/Ca+2 exchanger and the Na+/NH4+ exchanger (Table 1.2 and 1.3).
1.2.7.1 Na+/K+/2Cl- cotransporter (NKCC)
The Na+/K+/2Cl- cotransporter (NKCC) is a membrane transport protein that assists in active
transport of Na+, K+ and Cl- across cell membranes (Haas, 1994). As NKCC moves each solute
in the same direction, sometimes it is termed as a symporter. NKCC that has a 1Na+:1K+:2Cl-
Page 24
stoichiometry, maintains electroneutrality via transport of two positively charged ions
(Na+and K+) in parallel with two negatively charged ions (2Cl-).
Two forms of this protein have been identified, NKCC1 and NKCC2, and are encoded by two
different genes referred to as SLC12A2 and SLC12A1, respectively. NKCC1 is usually
distributed throughout the body and plays a vital role in exocrine glands (organs that
secrete fluids); while NKCC2 is limited to the kidney in invertebrates where it plays an active
role in reabsorption of Na+, K+ and Cl- ions into hemolymph (Haas and Forbush III, 2000).
NKCC1 is commonly located in the basolateral membrane of cells. This location in the
basolateral membrane, the closest part of the cell from the hemolymph vessels, facilitates
NKCC1 to transport Na+, K+ and Cl- from the hemolymph into the cell (Delpire et al., 1999).
The energy required to move the Na+ ion (along with K+ and Cl- ) across the cell membrane is
supplied by the electrochemical gradient of Na+, and this sodium gradient is established by
the ATP-dependent enzyme Na+/K+-ATPase (NKA).
NKCC has been identified in the crabs, C. maenas and C. sapidus, where strong expression
was found in the posterior gills of C. sapidus (Towle et al., 2001). NKCC has been suggested
to play a vital role in apical NaCl absorption in weak hyper-osmoregulators (see review: Haas
and Forbush III, 2000). Gene expression and relative activities of NKCC have been
investigated in a number of decapod crustaceans (Havird et al., 2013; see details in Table
1.3). In fact, a meta-analysis of 59 published qPCR studies showed that expression and/or
activity levels of NKCC were up-regulated after salinity transfer (Havird et al., 2013). Havird
et al. (2014), however, did not observe any change in NKCC expression levels in an
anchialine shrimp, Halocaridina rubra, after being transferred to 15, or 45‰ salinity from
32‰.; but NKCC was up-regulated slightly after 24 h at 2‰ (2.6 fold).
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1.2.7.2 Bicarbonate (HCO3-) transporters
Bicarbonate transporters are a group of proteins, involved in the transport of HCO3- ions,
that drive HCO3- out of the cell while drawing in other important ions (Na+ and Cl- ). This
enzyme is usually located in the apical region of an epithelial cell. The most important
bicarbonate transporters in crustaceans are Na+/HCO3- and Cl-/HCO3
-. Bicarbonate transport
is one the principal pH regulators and has been well studied euryhaline crustacean species
(Henry et al., 2003; Weihrauch et al., 2004a; Tsai and Lin, 2007; Freire et al., 2008; Henry et
al., 2012; Romano and Zeng, 2012). This process also plays an important role in movement
of acids and bases in many parts of the body, notably in gills, kidney, hepatopancreas and
intestine. The presence of Cl-/HCO3 exchangers in crustacean gills has been proposed based
on the equimolar exchange of Cl- for HCO3 or because of experiments that have used
different anion transport inhibitors to infer their function (Ehrenfeld, 1974; Gilles and
Péqueux, 1985; Gilles and Pequeux, 1986; Péqueux and Gilles, 1988; Lucu, 1989; Onken and
Graszynski, 1989; Onken et al., 1991). Molecular identification and characterization of such
exchangers in crustacean gill tissue, however, is still lacking. Like Na+/H+ exchangers, apical
Na+/HCO3- and Cl-/HCO3
- may be involved in transbranchial NaCl absorption and in
hemolymph acid–base regulation, but information on this is still lacking. They may also
regulate cell pH and volume when located in the basolateral gill cell membrane. This
enzyme has been well studied in vertebrate animals, but has received much less attention in
crustaceans (Pan et al., 2007).
1.3 Knowledge GAP and Research Questions
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The genus Cherax, an important group of freshwater crayfish in the family Parastacidae, is
the largest and most widespread group of freshwater crayfish in the southern hemisphere.
It represents an important ecological and evolutionary component of biodiversity in fresh
waters in this region (Riek, 1969; Crandall et al., 1999). Cherax spp. are confined largely to
lakes, rivers and freshwater streams in most regions in Australia and New Guinea
(Munasinghe et al., 2004) with Australia possessing the highest diversity (Riek, 1969;
Crandall et al., 1999). A number of species of Cherax crayfish have natural distributions that
are limited by strict pH ranges and consequently adaptation to these environmental pH may
have occurred in this group. Consequently, the identification of genes involved in systemic
acid-base balance and osmoregulation may be good candidates to examine for evidence of
selection across Cherax species.
Crayfish of the genus Cherax span a diverse range of aquatic environments and can tolerate
fluctuations in a range of environmental parameters, such as pH and salinity (Bryant and
Papas, 2007; McCormack, 2014). Due to the unique physiological characteristics of these
animals and relative economic importance of certain species, they provide models for
physiological and genomic research in crustaceans. Genetic information about Cherax spp.,
however, remains largely limited to mitochondrial and population genetic data (Miller et al.,
2004; Baker et al., 2008; Austin et al., 2014). There is a lack of basic information available on
genes that contribute to pH balance and osmoregulation under different acidic and basic
water conditions, in Cherax, and in freshwater crayfish in general. To date, no studies we are
aware of have attempted to identify or characterise specific candidate genes that are
involved in systemic acid-base balance in freshwater crayfish. This is a large knowledge gap
Page 27
in this economically important group that this study aims to fill by studying these genes in
Cherax crayfish.
Recently a few studies have been conducted on genomics of Cherax species, and only two
stress-related genes, HSP70 (heat-shock protein) and hemocyanin have been characterised
in these species (Fang et al., 2012; Wang et al., 2013a). No studies, to the best of our
knowledge, have focused on either CA gene family or other key osmoregulatory genes such
as Na+/K+-ATPase and H+-ATPase in this group of freshwater crayfish. The focus here,
therefore, will be on the characterization of functional genes and identifying the functional
mutations in carbonic anhydrase genes in addition to other osmoregulation genes or genes
involved in acid-base balance using selected Australian freshwater crayfish species in the
genus Cherax. Thus, this project will try to address the following specific questions:
• Which genes are the major genes responsible for pH balance and osmoregulation in
Cherax crayfish?
• Are there any functional mutations at the nucleotide and protein levels between the
same genes (particularly CA gene family, Na+/K+-ATPase and H+-ATPase) in
congeneric Cherax species across Australia?
• Do the expression patterns of these genes vary under natural pH range conditions or
between different tissue types?
1.4 Aims and objectives
The overall aim of the project was to use a functional and comparative genomics approach
to identify a number of potentially important putative or candidate genes and specific
functional mutations in crayfish that affect adaptation to natural environmental pH and
Page 28
salinity ranges where species occur in natural ecosystems. The specific objectives of the
project include;
• To sequence and annotate the gill transcriptomes from three economically and
ecologically important Australian endemic freshwater crayfish species; Cherax
quadricarinatus (Redclaw), C. destructor (Yabby) and C. cainii (Marron) and to
identify candidate genes that have potential roles in maintaining pH balance and in
osmoregulation (particularly CA, NKA, and HAT) .
• To document the expression patterns of key genes across a range of sub-lethal pH
treatments in order to determine their roles in maintaining pH balance.
• To generate full-length sequences for specific candidate genes and to compare
patterns of nucleotide and functional variation in the same genes in congeneric
Cherax species that are exposed to a wide diversity of different natural pH
environments.
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Analysis, characterisation and Chapter 2.expression of gill-expressed carbonic anhydrase genes in the freshwater crayfish Cherax quadricarinatus
Published as:
1. Ali, M.Y., Pavasovic, A., Mather, P.B. and Prentis, P.J., 2015. Analysis, characterisation and expression of gill-expressed carbonic anhydrase genes in the freshwater crayfish Cherax quadricarinatus. Gene (Elsevier) 564, 176-187
(http://dx.doi.org/10.1016/j.gene.2015.03.074)
2. Ali, M.Y., Pavasovic, A., Mather, P.B. and Prentis, P.J., 2015. Transcriptome analysis and characterisation of gill-expressed carbonic anhydrase and other key osmoregulatory genes in freshwater crayfish Cherax quadricarinatus. Data in Brief (Elsevier) 5, 187-193.
(http://dx.doi.org/10.1016/j.dib.2015.08.018)
Page 30
Analysis, characterisation and expression of gill-expressed
carbonic anhydrase genes in the freshwater crayfish Cherax
quadricarinatus
Muhammad Yousuf Ali1, Ana Pavasovic2, Peter B Mather1 and Peter J Prentis1*
1School of Earth, Environmental and Biological Sciences, Queensland University of Technology, Brisbane, QLD
4001, Australia
2School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4001, Australia
* Corresponding author: Peter J Prentis, p.prentis@qut.edu.au
Email addresses: MYA: my.ali@qut.edu.au AP: a.pavasovic@qut.edu.au PBM: p.mather@qut.edu.au PJP: p.prentis@qut.edu.au
Page 31
2.1 Abstract
Changes in water quality parameters such as pH and salinity can have a significant effect on
productivity of aquaculture species. Similarly, relative osmotic pressure influences various
physiological processes and regulates expression of a number of osmoregulatory genes.
Among those, carbonic anhydrase (CA) plays a key role in systemic acid–base balance and
ion regulation. Redclaw crayfish (Cherax quadricarinatus) are unique in their ability to thrive
in environments with naturally varied pH levels, suggesting unique adaptation to pH stress.
To date, however, no studies have focused on identification and characterization of CA or
other osmoregulatory genes in C. quadricarinatus. Here, we analysed the redclaw gill
transcriptome and characterized CA genes along with a number of other key
osmoregulatory genes that were identified in the transcriptome. We also examined patterns
of gene expression of these CA genes when exposed to three pH treatments.
In total, 72,382,710 paired end Illumina reads were assembled into 36,128 contigs with an
average length of 800bp. Approximately 37% of contigs received significant BLAST hits and
22% were assigned gene ontology terms. Three full length CA isoforms; cytoplasmic CA
(ChqCAc), glycosyl-phosphatidylinositol-linked CA (ChqCAg), and β-CA (ChqCA-beta) as well
as two partial CA gene sequences were identified. Both partial CA genes showed high
similarity to ChqCAg and appeared to be duplicated from the ChqCAg. Full length coding
sequences of Na+/K+-ATPase, V-type H+-ATPase, sarcoplasmic Ca+-ATPase, Arginine kinase,
calreticulin and Cl- channel protein 2 were also identified. Only the ChqCAc gene showed
significant differences in expression across the three pH treatments.
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These data provide valuable information on the gill expressed CA genes and their expression
patterns in freshwater crayfish. Overall our data suggest an important role for the ChqCAc
gene in response to changes in pH and in systemic acid–base balance in freshwater crayfish.
Key words: Redclaw, Cherax quadricarinatus, gills, osmoregulatory genes, carbonic
anhydrase
2.2 Introduction
Crustacean aquaculture is a multi-billion dollar industry, which contributes approximately
9.6% to the global aquaculture production (FAO, 2012). Among the variety of species
commercially cultured, culture of crustacean species has grown more rapidly than finfish
and molluscan production over the last three decades (FAO, 2011). Currently, crustacean
production is dominated by penaeid species, while a significant contribution now comes
from production of freshwater species (29.4%). Of the freshwater crustacean species,
crayfish contributes the highest production, followed by Chinese mitten crab and freshwater
prawns (FAO, 2012). Redclaw crayfish (Cherax quadricarinatus von Martens 1968) are
among the most important commercial species developed for aquaculture production in
Australia and elsewhere around the world, most notably Mexico, Ecuador, Uruguay,
Argentina and China (Macaranas, 1995; FAO, 2010).
As demand for freshwater crustaceans continues to grow, there is pressure to further
develop production for this class of organisms, by exploiting desirable traits and optimising
production practices. Selection of desirable traits has traditionally been associated with
traits or genes relating to growth performance and disease resistance, with less focus on
Page 33
selection of traits, which would deem the culture species ‘hardy’ in light of significant
fluctuations in culture conditions, such as tolerance of changes in pH and salinity.
Fluctuations in abiotic parameters, such as water chemistry, within a production setting
frequently have significant impact on the physiology and growth performance of many
cultured crustaceans. For example, after the euryhaline shrimp (Penaeus latisulcatus) was
exposed to a high salinity (46 ppt), it showed a reduced hepatosomatic index and tail muscle
index (Minh Sang and Fotedar, 2004). Reduced growth performance in this species was
suggested to result from higher energy requirements used to maintain internal ionic
concentration. (Ye et al., 2009) also observed reduced growth performance and poor feed
conversion ratios in tiger shrimp (Penaeus monodon) exposed to either low or high salinities
outside the optimal salinity level of 25 ppt. This resulted from increased energetic
expenditure on molting and excretion in this species. Increased energy expenditure on
osmoregulation can produce many problems in culture including increased moisture
content in muscle tissue, increased oxygen consumption and ammonia-N excretion (Silvia et
al., 2004). Therefore, a better understanding of the genes that control osmoregulation and
their function is required to optimise the production of commercially cultured crustaceans
under different water quality parameters.
Ion-regulatory genes have been reported in a number of decapod crustaceans (e.g. Na+/K+-
ATPase (Tsai and Lin, 2007); V-type H+-ATPase (Weihrauch et al., 2001b); Carbonic
anhydrase (CA) and Alkaline Phosphate (Tongsaikling, 2013); Ca2+ATPase (Zhang et al.,
2000a; Gao and Wheatly, 2004a; Tongsaikling, 2013). Among these, CA is one of the master
genes that has been reported to be involved in acid-base regulation in a wide variety of
marine crustacean taxa: e.g. shrimp (Gao and Wheatly, 2004a; Pongsomboon et al., 2009;
Page 34
Liu et al., 2010; Pan et al., 2011; Hu et al., 2013); crab (Serrano et al., 2007; Serrano and
Henry, 2008; Liu et al., 2012). Carbonic anhydrases also plays vital role in osmoregulation
because they provide H+ and HCO3- ions which are used as counterions for exchanging
cations and anions, especially Na+ and Cl- (Henry and Cameron, 1983; Henry, 2001; Freire et
al., 2008; Henry et al., 2012). Despite its role in acid-balance and osmoregulation, CA has
received limited attention in freshwater crustacean taxa (but see (Colbourne et al., 2011)).
In this regard, freshwater crayfish may provide an interesting model for identification and
characterising the functions of CA isoforms because these taxa experience hypo-osmotic
environments that can vary widely in pH (Baker et al., 2008). The redclaw crayfish, Cherax
quadricarinatus, is an excellent candidate to examine CA and other osmoregulatory genes in
freshwater crustaceans. This species has a natural range that spans an abrupt
environmental (pH) gradient, with populations occurring in waters with low pH (6) (northern
Queensland, Australia) and higher pH (8) (Northern Territory, Australia). Previous research
(Macaranas, 1995) on this species reported a fixed allozyme difference at a carbonic
anhydrase (CA) allozyme locus between C. quadricarinatus populations collected from either
side of this pH gradient. This locus was the only fixed difference observed across this
gradient and the authors of the paper suggest that this CA isoform may play an important
role in maintaining systemic acid–base balance and ion regulation under different water
chemistries. Consequently, identifying and functionally characterising allelic variants of CA
genes in this species will help improve our understanding of how this important freshwater
species copes with fluctuations in water quality, in particular pH stress.
To better understand osmoregulation in C. quadricarinatus we sequenced the gill
transcriptome of this species to identify endogenous CA genes expressed in gill tissues from
Page 35
individuals exposed to different pH conditions. Gill tissue was chosen for sequencing as it is
the key organ for ion-exchange and acid-base regulation in freshwater crayfish. This
transcriptome sequence was also used to identify other major osmoregulatory genes
including Na+/K+-ATPase, V-type H+-ATPase, sarcoplasmic Ca+-ATPase, arginine kinase,
calreticulin, Cl- channel protein, Na+/K+/2Cl− co-transporters, Na+/H+ exchanger and alkaline
phosphatase. Gene expression patterns of the identified CA genes as well as some of the
osmoregulatory genes were examined when exposed to three pH treatments.
2.3 Material and Methods
2.3.1 Sample Preparation
Live redclaw crayfish (Cherax quadricarinatus) were obtained from Cherax Park Aquaculture,
Theebine, Queensland, Australia. Animals were housed in rectangular glass tanks (capacity
27 L each) and acclimated at 270C, pH 8 and conductivity 500 (μS/cm) for 2 weeks before
the start of the experiment. During the acclimation period all animals were fed regularly
with formulated feed pellets. Water quality was maintained at temperature 27-280C, pH 7.5
and conductivity 450-550 μS/cm with a computer-controlled filtration system (Technoplant).
Feeding was stopped 24 h before the treatment. Animals were distributed into three
separate glass tanks (25×18×15 cm) with water temperature set at 25.5±0.90C and
conductivity 521.8±29 (μS/cm). A total of nine individuals (average length 131.9±6 mm and
body weight 56.3±6 g) were used for the experiment. These animals were placed in three
different pH levels (6, 7 and 8), a treatment within the tolerance range of this species
(Macaranas, 1995). Gill tissue was extracted at 3, 6 and 12 h. Continuous aeration was
provided to tanks using aquarium air pumps.
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2.3.2 RNA extraction, cDNA library construction and sequencing
Prior to tissue extraction, animals were euthanized in crushed ice for 5-10 minutes. Gill
tissue were dissected out and immediately stored in RNAlater® solution. Tissues preserved
in RNAlater® were stored at -800C prior to RNA extraction.
Total RNA was extracted from the pooled gill tissues, which contained equal amounts of
sample from each of the animals from all treatments using a TRIZOL/Chloroform extraction
(Chomczynski and Mackey, 1995) and then purified further using a RNeasy Midi Kit (cat #
75144, QIAGEN). RNA yield and quality were checked using agarose gel electrophoresis,
spectrophotometer absorbance and a bioanalyzer using a 2100 RNA nanochip. RNA was
sequenced at the Beijing genomics institute and prepared using the same protocol as
(Prentis and Pavasovic, 2014).
2.3.3 De novo assembly and functional annotation
Illumina paired end sequences were assembled into contigs using CLC Genomics Workbench
6.0.2 with multiple kmers of 15, 20, 25, 30, 35, 40 and 45bp. It was found that a kmer of
25bp provided an assembly with the best quality metrics with the fewest chimeric
sequences based on CEGMA and CD-Hit (Knudsen T and Knudsen B, 2013). The assembled
data were blasted, mapped and annotated using Blast2Go Pro software (Camacho et al.,
2009), while contigs longer than 8000bp were manually blasted. Sequence annotation
information was retrieved for sequences that had BLASTx queries that exceeded a
stringency of e-value<10-5. For contigs that received significant BLAST hits with protein
function information, Gene Ontology (GO) terms were assigned. Gene ontologies from
identified ESTs were categorized into GO categories from Biological Process, Molecular
Function and Cellular Component using web-based batch analyzer software ‘CateGOrizer’
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(Hu et al., 2008). The distribution of the GO categories was mapped using WEGO (Ye et al.,
2006). To predict the metabolic pathways for each contig Enzyme Commission numbers
were assigned and the relevant maps from the Kyoto Encyclopedia of Genes and Genomes
were downloaded (Kanehisa and Goto, 2000).
2.3.4 Identification of CA and other osmoregulatory genes
The transcriptome data set was examined to identify transcripts that matched CA genes and
other major osmoregulatory genes. The identified gene sequences were aligned with the
non-redundant protein database at NCBI (National Centre for Biotechnology Information) in
order to compare the similarity to previously identified and annotated genes. Following this
validation step, three prime and five prime untranslated regions (UTR) as well as open
reading frames were determined using ORF Finder. Open reading frames were translated
into proteins. The potential cleavage site of the signal peptide was predicted using PrediSi
(PrediSI, 2014) and N-linked glycosylation sites were predicted with N-GlycoSite (Zhang et
al., 2004) using NXS/T model (where N=Aspargine, S=Serine, T=Theronine and X=any amino
acid). The translated amino acids sequencs were used as BLASTp queries against the NCBI
database. Up to 20 top BLAST hits were downloaded for each gene and aligned with the C.
quadricarinatus candidate gene in BioEdit (version 7.2.5; Hall, 1999) using a ClustalW
alignment platform (Larkin et al., 2007). For the full-length CA genes Neighbour Joining trees
were generated in Geneious (version 8.0.4) ) using the Jukes Cantor method with 1000
bootstraps (Kearse et al., 2012). The putative protein domains of the open reading frames
was analysed using SMART (Simple Modular Architecture Research Tool) database (Schultz
et al., 1998).
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2.3.5 CA expression patterns under different pH treatments
Live C. quadricarinatus individuals were obtained from Cherax Park Aquaculture, and
acclimated to lab conditions for two weeks under the same conditions as animals used for
RNA sequencing. Following acclimation, animals were transferred to water containing three
pH treatments, pH 6, 7 and 8, which are in a similar range as the two pH environments in
natural habitats. Animals were harvested after 24 hours and gills were extracted
immediately after animals were euthanized in crushed ice for 5-10 minutes. Three replicates
animals were used in all experiments. Specific quantitative real-time PCR primers were
designed for transcripts that identified as the three CA genes, V-type H+-ATPase, Arginine
kinase and 18s rRNA based on the transcripts obtained in our study (Table 2.1). Realtime
PCR conditions included a pre-incubation of 950C for 5 minutes, followed by a total 45 cycles
of three-step amplification of 950C for 10 seconds; 600C for 10 seconds and 720C for 10
seconds using a LightCycler 96 RT-PCR machine and reagents (Roche, Version 04, Cat.
No.06924204001). Ribosomal 18S was used as an internal control gene to normalize sample-
to-sample variations. The relative expression of the target genes were measured as a ratio
(Ratio=concentration of target gene/concentration of 18S gene) using Relative Quant
analysis tool described in the Light Cycler 96 system operator’s guide, version 2.0. An one-
way ANOVA was used to determine if any of the genes showed significant expression
differences across the three pH treatments.
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Table 2.1 Details of primers used in this study for amplification of the target genes at RT-PCR
Target genes Primers Primer Sequence (5’→3’) Tm (0C) Product Size (bp)
Cytoplasmic CA (ChqCAc)
CAcF CTTGCTGTCCTGGGAATGTT 64.0 196 CAcR CATAGCATGGTGGAGTGGTG 64.1
GPI-linked CA (ChqCAg)
CAgF GGCACTAGGCTCTGAACACA 57.0 149
CAgR CTGACACCTCCAGCATCACT 56.8
Beta CA (ChqCAb)
CAbF CAGTTTGAGGTTGTGCGAGA 64.1 168 CAbR CTCCTCATGCCCACAAAGAT 64.0
Na+K+ATPase alpha
NaKF TGGTGTTGAGGAGGGAAGAC 64.2 130 NaKR ACCCAATGGTAGAGGCACAG 63.9
V-typeH+ATPase alpha
VHaF ATCGAGTATTGGCTGCTGCT 63.8 142 VHaR ACTGGGATCCAACATTCAGC 63.9
Calreticulin
CalF TACCAGATCCTGATGCCACA 64.2 106 CalR GGCTTCCATTCACCCTTGTA 63.8
Ca+2 ATPase
Ca+2ATPF TGGCTCTTCTTCCGCTACAT 63.8 107 Ca+2ATPR TAGCTCAAGTGAGGGCCAGT 63.9
Arginine kinase
AKF GGAGGATCATCTCCGCATTA 63.9 103 AKR GGGAACACGCTTTTCAATGT 63.8
Chloride channel protein
Cl-ChF CTCCGAAAAGCGACGTAAAG 63.6 196 Cl-ChR ACGGGAGAGTGAGCGACTAA 63.9
2.4 Results
2.4.1 Sequencing and assembly
Transcriptome sequencing of the C. quadricarinatus gill library resulted in over 72 million
(72,382,710) high quality paired end sequence reads after low quality reads were removed.
Over 36 thousand (36,128) contigs with a length greater than 300 bp were assembled. The
average contig length was 800 bp, the N50 was 936 bp and the longest contig was 14,972 bp
(Table 2.2).
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2.4.2 Mapping and functional annotation
Almost 37% of contigs (13,355) received significant BLASTx hits (S Fig. 2.1 in Appendix 1).
Overall there was a negative relationship between BLAST success and contig length, with
contigs shorter than 800 bp in length having low BLASTx success. Of contigs receiving
significant BLASTx hits, the most common top hit species was Daphnia pulex, a distantly
related microcrustacean species, but currently the only crustacean with a complete genome
sequence. Arthropod species represented the majority of top BLAST hits (17% of the total
Top-BLAST Hits; and 76% of the top 30 Species) (S Fig. 2.2 in Appendix 2). Twenty two
percent of contigs (7,975) were assigned Gene Ontology (GO) terms (S Fig. 2.1 in Appendix
1). In total 48,684 GO terms were assigned to contigs, based on GO classification.
Approximately 48.5%, 29.2% and 22.3% of the GO terms were assigned to Biological
Processes, Molecular Function and Cellular Component GO categories, respectively (S Fig.
2.3 in Appendix 3). Sequences that had valid enzymes codes were assigned to 118 different
KEGG pathways. Contigs were most commonly assigned to purine metabolism (201
sequences, 42 enzymes), oxidative phosphorylation (94 sequences, 8 enzymes) and nitrogen
metabolism (85 sequences, 13 enzymes).
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Table 2.2 Summary statistics for assembled contigs generated from C. quadricarinatus gill transcriptomes.
2.4.3 Identification and characterization of carbonic anhydrase (CA) genes
From BLASTx searches we were able to identify five transcripts belonging to the CA gene
family. The CA transcripts included three complete CA isoforms; a cytoplasmic CA (ChqCAc,
KM538165), a glycosyl-phosphatidylinositol-linked CA (ChqCAg, KM538166), and a β-CA
(ChqCA-beta, KM538167); as well as two partial CA cds, ChqCA-p1 (KM610228) and ChqCA-
p2 (KM880150). The open reading frames of the three full length transcripts: ChqCAc,
ChqCAg and ChqCAb were validated using RT-PCR and matched the transcript sequences
with 100% nucleotide identity.
The cytoplasmic CA transcript (ChqCAc, KM538165) was 1527 bp in length and contained a
78 bp 5′-UTR, 816 bp ORF (including the stop codon), and a 633 bp 3′-UTR (Fig. 2.1). The
deduced protein encoded by ChqCAc contained 271 amino acids, no signal peptide and
contained two putative N-glycosylation motifs: NKS (aa. position 122) and NGS (aa. position
187). A possible polyadenylation cleavage signal (ATTTA) was detected 75bp downstream
from the stop codon. A eukaryotic-type carbonic anhydrase domain was detected within the
deduced polypeptide (aa. residues: 4-265 and e-value 7.07×e-102). These characteristics
suggest that ChqCAc is a cytoplasmic carbonic anhydrase gene.
Statistics bp/number Total number of contigs: 36 128 N50 936 bp N75 535 bp N25 1835 bp Mean contig length 800 bp Minimum contig length 283 bp Length of the longest contig 14 972 bp Number of contigs longer than 500 bp 20 554 Number of contigs longer than 1500 bp 3807 GC content 43%
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A BLASTp search determined that the ChqCAc was highly conserved in decapod crustaceans
and had greatest similarity with cytoplasmic CA isoforms from P. monodon (76% identity),
Litopenaeus vannamei (76% identity), and Callinectes sapidus (73.8% identity). Multiple
alignment of the ChqCAc with the homologous genes in arthropods, fish and humans
showed that three histidine residues involved in the zinc binding network (Histidine-94,
Histidine-96 and Histidine-119 using CA-II of Homo sapiens as a reference) were conserved
in all the aligned sequences (Fig. 2.2). Amino acid residues associated with the hydrogen
bond around the active site were also conserved.
The transcript identified as the glycosyl-phosphatidylinositol (GPI)-linked CA isoform of C.
quadricarinatus (ChqCAg, KM538166) contained a total 3352 bp of nucleotide sequence
consisting of 83 bp 5′-UTR, 933 bp ORF (including the stop codon), and a 2336 bp 3′-UTR (S
Fig. 2.5 in Appendix 5). The open reading frame was translated into a protein sequence and
the predicted protein was 310 amino acids and identified as a membrane-associated GPI-
anchored protein with an N-terminal signal (Met1 to Gly22). A single N-glycosylation motif,
NIK, was identified at position 189. Five possible polyadenylylation cleavage signals (mRNA
instability motif ATTTA) were predicted in the 3’ UTR region (S figure 2.5 in Appendix 5). A
eukaryotic-type carbonic anhydrase domain was identified within the translated
polypeptide (amino acid residues: 28-281 and e-value 6.9×e-119).
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1 gtg ctc ttc cga tct cct taa acg tgc aag tga agg tac gct ctt 45 46 tcc tcc gct cct cta ctc ttc tac caa ttt gaa ATG GTC GGC TGG 90 M V G W 91 GGA TAC GCA AAG GCA AAC GGA CCC GCC ACT TGG CCA AAC CTG TTT 135 5 G Y A K A N G P A T W P N L F 19 136 CCC ATC GCA GGT GGA AGG CGG CAG TCC CCT ATT GAT ATC AAG TGC 180 20 P I A G G R R Q S P I D I K C 34 181 GAG GCT TGC CAG AAA GAT TCC AGT CTT TCC AAG ATC CAG GCT GTG 225 35 E A C Q K D S S L S K I Q A V 49 226 TAC AGC GGC ATC AAG GTC TCA GAG TTG TCT AAC ACG GGA CAC GCC 270 50 Y S G I K V S E L S N T G H A 64 271 TGG AAG GCA CAA GTC TCC GGA GGC AGT TCA AGC CTG AAG GGC GGT 315 65 W K A Q V S G G S S S L K G G 79 316 CCA CTG GGC ACT GAC GAG TAC GTG CTA GAA CAG TTC CAC TGT CAC 360 80 P L G T D E Y V L E Q F H C H 94 361 TGG GGC AAG ACC AAC GAC AAA GGT TCA GAA CAC ACT GTC AAT GGC 405 95 W G K T N D K G S E H T V N G 109 406 AAG TGC TAC GCT GCT GAG CTA CAC TTG GTA CAC TGG AAT AAG AGC 450 110 K C Y A A E L H L V H W N K S 124 451 AAA TTC TCA AGC TTT GCT GAA GCA GCA TCC TCT GAC GGC GGT CTT 495 125 K F S S F A E A A S S D G G L 139 496 GCT GTC CTG GGA ATG TTT TTA GAG GTT GGC AGT GAA CAC GAG GAA 540 140 A V L G M F L E V G S E H E E 154 541 CTG AAC AAG GTG TGC AAG CTA CTA CCC TTC GTG CAA CAC AAG GGT 585 155 L N K V C K L L P F V Q H K G 169 586 CAG GCT ATT ACT GTC ACT GAA CCA ATC AGT CCA GCT GCG TTC ATC 630 170 Q A I T V T E P I S P A A F I 184 631 CCC AAA AAT GGC TCC TAC TGG ACA TAT GCT GGC TCC CTC ACC ACT 675 185 P K N G S Y W T Y A G S L T T 199 676 CCA CCA TGC TAT GAG TCT GTC ACC TGG ATC GTA TAC GAA CAT CCC 720 200 P P C Y E S V T W I V Y E H P 214 721 ATC CAG GTG TCT CAG ACA CAG ATG GAC GCT TTC AGG TCA ATG AAG 765 215 I Q V S Q T Q M D A F R S M K 229 766 TCT TAT CAT CCT TGC GAG ACT TGT CCT GAG GAT GAG CTA GCA GGA 810 230 S Y H P C E T C P E D E L A G 244 811 GCA CTG GTT GAA AAC TAT CGA CCC CCG TGT CCT CTC TGT GAC CGA 855 245 A L V E N Y R P P C P L C D R 259 856 GTT GTC AGA GTT TAC AAG GAT TCA GTA CAA GAG GAA TAA tca gtc 900 260 V V R V Y K D S V Q E E * 272 901 tac aga aat cgc tat act gga ctg cat ata caa aaa tag tgt ctg 945 946 aac atg ata aaa att ggc acg tat att tac att ctg att atg cag 990 991 aaa ctg aag tga cga ttg tat ttc agg gca ttg tat tat tgt gaa 1035 1036 tgg cac aag aaa cat tta aaa aga atg aag gtg tgt atc tca taa 1080 1081 atg aaa act gac ttg aag ctt tcg gag gct aaa att ata cag tac 1125 1126 cat atg cag aaa tca att tta aaa gca aga taa ata att gtt gaa 1170 1171 agg gac ttt att aag cag cag cga tag tta taa ata aaa cca ata 1215 1216 cag gac caa ata gca aga gga gca gga cat tgt cga gaa cca acg 1260 1261 aga gga aca cag tac taa gaa ccc agc aac agc acc tcg ata cca 1305 1306 aga acc aac aag agg atc aca gta cca aga acc aac aag agg agc 1350 1351 aca gtg cca aga acc aaa agg agg aac aca gta cca aga acc atc 1395 1396 aag aac agc aca gtg cca aga acc aaa aga agg aac aca gta cca 1440 1441 agt acc aac aag agc agc agg gta cca aaa tct aac aac aat aca 1485 1486 aac aat cat aac tca gtc ctg tat tgt gct ttg caa cac aca 1527
Figure 2.1 Full length nucleotide sequences of cytoplasmic carbonic anhydrase (ChqCAc)
Full length nucleotide sequences along with corresponding deduced amino acid sequences of a CAc (cytoplasmic carbonic anhydrase, ChqCAc: KM538165) cDNA amplified from the gills Redclaw crayfish, C. quadricarinatus. Sequences are numbered at both sides of each line. The start codon and stop codon are in bold. N-linked glycosylation sites (NXS/T) are shaded and in bold. One putative AU-rich mRNA instability motif (polyadenylylation cleavage signal) (ATTTA) is in bold and underlined. 3’ and 5’ untranslated regions (UTR) are in small letters and expressed codons (CDS) are in capital letters.
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Figure 2.2 Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus cytoplasmic carbonic anhydrase isoform (ChqCAc)
Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus CAc isoform (cytoplasmic carbonic anhydrase, ChqCAc: KM538165) with some representative cytoplasmic CA protein sequences from crustaceans: tiger shrimp Penaeus monodon (PmCA: ABV65904), white shrimp Litopenaeus vannamei CAc (LvCAc: ADM16544), blue crab Callinectes sapidus (CasCAc: ABN51213) and horse crab Portunus trituberculatus (PtCAc: AFV46144); the insects: mosquito Aedes aegypti CA (AaCA: XP_001650043), Anopheles gambiae CA (AgCA: ABF66618) and fruit fly Drosophila melanogaster CA-I (DmCA: NP_523561); sea lamprey Petromyzon marinus CA (PmaCA: AAZ83742); winter flounder Pseudopleuronectes americanus CA (PaCA: AAV97962); and human Homo sapiens CA-I (HsCA-I: NP_001729), CA-II (HsCA-II: NP_000058), CA-III (HsCA-III: NP_005172). Colours indicate the similarity. The symbols under the sequences: + signs indicate all putative
Page 45
active site residues; z represents the predicted Zinc-binding histidine residues; black dots localize the active-sites involved in hydrogen bond network.
Figure 2.3 Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus membrane-associated isoform (ChqCAg)
Page 46
Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus CAg isoform (Glycosyl-phosphatidylinositol-linked carbonic anhydrase, ChqCAg: KM538166) with some representative GPI-linked CA isoform sequences from the crustaceans such as the blue crab Callinectes sapidus (CasCAg: EF375491), the horse crab Portunus trituberculatus (PtCAg: AFV46145), white shrimp Litopenaeus vannamei (LvCAg: AGC70493) and the littoral crab Carcinus maenas (CmCAg: ABX71209); from the insect Anopheles gambiae (AngCA: ABF66618); from the vertebrates such as the batrachian Xenopus laevis (XelCA: AAH42287), the agnatha Petromyzon marinus (PmCA: AAZ83742), the teleostei Tribolodon hakonensis CA-II (TrhCA: BAB83090) and the mammal Homo sapiens (HsCA-I: NP_001729; HsCA-II: NP_000058; HsCA-III: NP_005172). Colours indicate the similarity. The symbols under the sequences: + signs indicate all putative active site residues; z represents the predicted Zinc-binding histidine residues; black dots localize the active-sites involved in hydrogen bond network.
The ChqCAg predicted protein shared strong similarity with GPI-linked CA isoforms
identified in other crustacean taxa (C. sapidus: ABN51214, 74% identity; L. vannamei :
AGC70493, 73% identity; P. trituberculatus: AFV46145, 73% identity). Multiple alignment of
the ChqCAg with the homologous genes in arthropods and vertebrates revealed that three
histidine residues involved in the zinc binding were conserved as well as amino acid residues
involved in the hydrogen bond around the active site (Fig. 2.3).
The β-CA transcript (ChqCA-beta, KM538167) was 1021 bp in length and consisted of a 194
bp 5′-UTR, 768 bp ORF (including the stop codon), and a 59 bp 3′-UTR. The coding sequence
produced a predicted protein of 255 amino acids with no signal peptide sequence and no N-
glycosylation motifs. No polyadenylylation cleavage signal was identified in the 3’ UTR
region. The predicted ChqCA-beta protein contained a conserved β-CA-super family domain
(residues: 27-229 and e-value 6.84×e-36).
BLASTp analysis of the ChqCA-beta protein determined that it had high similarity to other β-
CA proteins identified in arthropods including D. pulex (EFX79480, 63% identity);
Acyrthosiphon pisum (XP_001943693, 65% identit) and Nasonia vitripennis (XP_001606972,
64% identity). Multiple alignment of the ChqCA-beta protein with homologous proteins in
arthropod species identified that four amino acid residues involved in zinc binding, C-39, D-
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41, H-102 and C-105, were conserved (Fig. 2.4) as were amino acid residues associated with
the hydrogen bond around the active site.
Figure 2.4 Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus β-CA isoform (ChqCA-beta)
Multiple alignment of deduced amino acid sequences of a complete C. quadricarinatus β-CA isoform (Beta carbonic anhydrase, ChqCA-beta: KM538167) with some representative β-CA isoform sequences from the microcrustacean Daphnia pulex (DapCA-beta: EFX79480); from the small arthropods such as the insects, pea aphid Acyrthosiphon pisum (AcpCA-beta: XP_001943693), small fly Nasonia vitripennis (NavCA-beta: XP_001606972), the ant Camponotus floridanus (CafCA-beta: EFN64927), the jumping ant Harpegnathos saltator (HasCA-beta: EFN90033), the ant Acromyrmex echinatior (AceCA-beta: EGI68846), the red dwarf honey bee Apis florea (ApfCA-beta: XP_003692734), the alfalfa leafcutter bee Megachile rotundata (MerCA-
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beta: XP_003699501), the southern house mosquito Culex quinquefasciatus (CuqCA-beta: XP_001849341),the fruit fly Drosophila mojavensis (DrmCA-beta: XP_001999442) and the silkworm Bombyx mori (BomCA-beta: XP_004922999). Colours indicate the similarity. The symbols under the sequences: z represents the predicted active-site Zinc-binding residues.
The partial coding sequence of a different CA gene, which we named ChqCA-p1 (KM610228)
was 390bp in length and consisted of 386 bp of coding sequence (128 amino acids) and a
4bp 5’ UTR region. One putative N-glycosylation motif: NRT (amino acid position 30) was
identified in the ChqCA-p1 predicted protein sequence. Homology searches determined that
the ChqCA-p1 protein showed some similarity with GPI-linked CA isoforms identified in
other crustacean taxa including; L. vannamei (AGC70493, 40% identity), C. maenas
(ABX71209, 42% identity) and P. trituberculatus (AFV46145, 42% identity). A second partial
transcript, which we named ChqCA-p2 (KM880150) was 495 bp in length and translated into
a partial protein of 165 amino acids. No start codon; stop codon or N-glycosylation site
could be identified in the partial transcript. ChqCA-p2 was similar with the ChqCAg
transcript in both the nucleotide and protein sequence. BLASTp analysis determined that
this sequence was similar to GPI-linked CA isoforms from other crustacean taxa including; P.
trituberculatus (AFV46145, 52% identity), C. maenas (ABX71209, 52% identity) and C.
sapidus (ABN51214, 51% identity).
2.4.4 Phylogenetic analysis of carbonic anhydrase
A Neighbour Joining tree of the translated CA transcripts identified in this study showed that
the ChqCAc transcript clustered cytoplasmic CA forms from arthropods, fish and humans
(Fig. 2.5). The ChqCAg transcript was resolved in well supported clade that contained GPI-
anchored CA from arthropods. The partial transcript, ChqCA-p2 was sister to the GPI-linked
CA clade indicating that it shared a close relationship with the GPI-linked CA proteins from
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arthropods. The ChqCA-beta transcript and one partial CD (ChqCA-p1) could not be aligned
with other CA proteins and were excluded from this analysis.
Figure 2.5 Bootstrapped neighbor-joining tree of deduced amino acid sequences showing relationships between the genes identified from our study
Bootstrapped neighbor-joining tree of deduced amino acid sequences showing relationships between the genes identified from our study Cherax cytoplasmic carbonic anhydrase (ChqCAc:KM538165), Cherax Glycosyl-phosphatidylinositol-linked carbonic anhydrase (ChqCAg:KM538166, Cherax carbonic anhydrase partial cds (ChqCAp2: KM880150) and other CA isoforms: horse crab Portunus trituberculatus CAc (AFV46144), Portunus trituberculatus CAg (AFV46145), tiger shrimp Penaeous monodon (ABV65904); the blue crab C. sapidus CAc (ABN51213); C. sapidus CAg (ABN51214); white leg shrimp Litopenaeus vannamei CAc (ADM16544), Litopenaeus vannamei CAg (AGC70493); littoral crab Carcinus maenas (ABX71209), mosquito Aedes aegypti (XP_001650043), Anopheles gambiae (ABF66618), fruit fly Drosophila melanogaster (NP_523561), honey bee Apis mellifera (XP_392359), silk worm Bombyx mori (NP_001040186), flour beetle Tribolium castaneum (XP_974322), lamprey Petromyzon marinus (AAZ83742) and winter flounder Pseudopleuronectes americanus (AAV97962), and Homo sapiens CA-I (NP_001729), CA-II (NP_000058), CA-III (NP_005172), CA-IV (NP_000708), CA-V (NP_001730), CA-VI (NP_001206), CA-VII (NP_005173) CA-RP-VIII (NP_004047), CA-IX (NP_001207), CA-RP-X (NP_001076002), CA-RP-XI (NP_001208), CA-XII (NP_001209), CA-XIII (NP_940986) and CA-XIV (NP_036245). In the figure c, g and p after CA indicate cytoplasmic, GPI-linked (Glycosyl-phosphatidylinositol-linked) and partial cds respectively.
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2.4.5 Identification of additional osmoregulatory genes
We identified over a hundred different transcripts from C. quadricarinatus gills that had
been previously reported to be associated with osmoregulation, acid-base balance and ion
transport in other (Appendix 8). Of these transcripts, major osmoregulatory genes
previously identified in crustaceans included Na+/K+-ATPase, V-type H+-ATPase,
sarco/endoplasmic reticulum Ca+-ATPase (SERCA), alkaline phosphatase, arginine kinase,
calrecticulin, Na+/K+/2Cl− cotransporter (NKCC), Na+/H+ exchanger, Na+/HCO3− cotransporter
and Cl- channel protein (Table 2.3). For example, we identified multiple subunits of V-type
H+-ATPase including the alpha, B, C, D and H subunits. A number of other solute carrier gene
families were also identified.
Table 2.3 List of top candidate genes involved in pH and/or salinity balance identified from the gill transcriptomes of C. quadricarinatus.
Function Gene name Contig number
Contig length (bp)
ORF (aa)
ORF position
CD type
GenBank Accession #
pH balance Cytoplasmic carbonic anhydrase 720 1527 271 79-894 Full KM538165
pH balance GPI-linked carbonic anhydrase 458 3352 310 84-1016 Full KM538166
pH balance β-Carbonic anhydrase 19805 1021 255 195-962 Full KM538167
pH balance Carbonic anhydrase alpha 2595 549 165 1-495 Partial KM880150
pH balance GPI-linked CA 2596 390 128 4-389 Partial KM610228
salinity regulation
Na+/K+-ATPase alpha subunit 246 2391 673 19-2040 Full KM538168
pH/salinity regulation
V-type H ATPase alpha subunit 791 2959 820 129-2591 Full KM538169
pH/salinity regulation
V-type H ATPase 116 kda subunit a isoform 1
15404 1898 593 86-1867 Full KM610229
pH/salinity regulation
V-type H ATPase Subunit B 395 2008 489 83-1522 Full KM880151
pH/salinity regulation
v-type H ATPase Subunit C 2517 1348 386 101-1261 Full KP063232
pH/salinity regulation
V-type-H-ATpase Subunit H 2008 2070 478 52-1488 Full KP063233
salinity regulation
Sarco/endoplasmic reticulum Ca2+-ATPase
2145 4631 1020 375-3437 Full KM538171
salinity regulation
Arginine kinase 30 1197 210 242-874 Full KM610226
salinity regulation
Calreticulin 929 1722 402 124-1332 Full KM538170
salinity regulation
Cl- channel protein 2 3994 2408 686 41-2101 Full KM610227
salinity regulation
Alkaline phosphatase 13232 1115 361 28-1113 Partial KM610230
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pH/salinity regulation
Na+/HCO3 -cotransporter 4984 2023 614 180-2022 Partial KM659402
salinity regulation
Cl-/HCO3 exchanger 5045 1471 463 81-1470 Partial KM659403
salinity regulation
K+/Cl- symporter 28739 445 128 28-444 Partial KM880152
pH/salinity regulation
Na+/H+ exchanger 3 5168 2826 935 21-2825 Partial KM880153
salinity regulation
Na+/Ca2+ exchanger 1-like 2402 2687 835 178-2685 Partial KM880154
2.4.6 CA expression patterns under different pH treatments
The only transcript to show significant changes in expression levels across the three pH
treatments was ChqCAc (F2 = 14.6064; P < 0.005). This transcript was most highly expressed
in the pH 6 treatment and had decreased expression at both the pH 7 and 8 treatments. The
mRNA expression of CAs showed about 2-fold increase in pH 6 after 24 h, as compared to
that of pH 7 and it was approximately 6-fold when compared to pH 8 (Fig. 2.6). The V-type
H+-ATPase subunit A transcript showed a trend in its expression pattern similar to that of
ChqCAc, but did not show differential expression (F2 = 5.0104; P = 0.053). The ChqCA-beta
transcript was very lowly expressed and we could not determine reliable expression results
for this transcript. All other transcripts were not differentially expressed across the pH
treatments.
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Figure 2.6 Relative mRNA expression levels of Cytoplasmic carbonic anhydrase, GPI-linked carbonic anhydrase, V-type H-ATPase and Arginine kinase.
Relative mRNA expression levels of Cytoplasmic carbonic anhydrase (ChqCAc), GPI-linked carbonic anhydrase (ChqCAg), V-type H-ATPase and Arginine kinase observed after 24hrs. Relative expression levels were calculated as a ratio (conc. of target gene/conc. of 18S gene), using 18S as a reference gene.
2.5 Discussion
The data generated in this study contributes to our understanding of the genes involved in
acid-base balance in freshwater crayfish, while also providing genomic resources for a non-
model crayfish for which limited transcriptome data currently exists (Yudkovski et al., 2007;
Glazer et al., 2013). We identified 21 different genes, which have been shown to play an
important role in osmoregulation or acid-base balance in other crustacean species. In
addition, this study demonstrated that the expression of cytoplasmic carbonic anhydrase is
significantly affected by changes in water pH, but not the GPI-linked or beta carbonic
anhydrase genes.
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2.5.1 Carbonic anhydrase genes in Cherax quadricarinatus
Carbonic anhydrase is a universal and well-studied transport-related enzyme in crustaceans.
A substantial number of studies have been undertaken on its occurrence, biochemical- and
molecular characteristics, expression levels and physiological role in decapod crustaceans
(Henry and Cameron, 1982; Henry and Kormanik, 1985; Wheatly and Henry, 1987; Böttcher
et al., 1990; Böttcher et al., 1991; Onken and Riestenpatt, 1998; Henry et al., 2002; Henry et
al., 2003; Henry and Campoverde, 2006; Roy et al., 2007; Serrano et al., 2007; Serrano and
Henry, 2008). In this study we present the full coding sequence of three distinct gill-
expressed CA isoforms (cytoplasmic, GPI-linked and β-class CA) in redclaw crayfish as well as
information on their expression patterns in response to changes in water pH. The existence
of multiple alpha CA genes in the gills of freshwater crayfish supports previous biochemical
and physiological research which demonstrated the existence of two different CA types in
crustacean gills; a cytoplasmic and a membrane-associated CA (Serrano et al., 2007; Serrano
and Henry, 2008; Jillette et al., 2011). More recently we have also identified these CA
isoforms in Cherax cainii and C. destructor, which are similar in length and sequence identity
(Ali et al., 2015a).
The cytoplasmic CA (ChqCAc) identified here was similar both in length and amino acid
composition to cytoplasmic CA from other crustaceans (Serrano et al., 2007; Pongsomboon
et al., 2009; Liu et al., 2012; Hu et al., 2013). This cytoplasmic CA was the only gene
differentially expressed among pH treatments indicating that it may play an important role
in acid-base balance in this species. Previous research has shown that the expression of
cytoplasmic CA is strongly induced in crustaceans by changes in water salinity (Serrano and
Henry, 2008). The expression of CAc is more pronounced when exposed to low salinity,
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rather than in high salinity (Henry et al., 2012). In our study, we observed about 2-fold and
6-fold increase in mRNA expression of CAc after 24 h exposure to low pH (pH 6), as
compared to that of pH 7 and pH8 respectively (Fig. 2.6). Some previous studies have
reported approximately 10-fold increase of CAc expression in the posterior gills of both C.
sapidus and C. maenas after 6 h of low salinity transfer, and the expression level increased
to approximately 100-fold on 24 h post-transfer and remained static onwards (Serrano et
al., 2007; Serrano and Henry, 2008). It is suggested that this change in expression occurs
because cytoplasmic CA provides the counter ions H+ and HCO3− ions to the Na+/H+ and
Cl−/HCO3− exchanger to drive ion uptake in the gills of crustaceans when exposed to low
salinity or pH environments (Henry and Cameron, 1983; Henry, 1988; Henry, 2001).
Consequently, we hypothesise that the C. quadricarinatus cytoplasmic CA isoform may play
an important role in maintaining internal pH conditions in high and low pH streams across
northern Australia.
The membrane-associated GPI-linked CA from redclaw (ChqCAg) was similar to homologous
proteins from other decapod crustaceans. We found no evidence that this enzyme was
differentially expressed in response to changes in pH. This result is similar to previous
studies on osmoregulation in crustaceans (Henry, 1988; Serrano et al., 2007), which have
found that the change in expression of membrane bound isoform was much less than for
the cytoplasmic form when exposed to low salinity conditions. The difference in expression
levels of ChqCAc and ChqCAc can be attributed to isoform-specific physiological functions.
The presence of CAg is mainly on the outer membrane of the cells that is believed to
facilitate the mobilization of hemolymph HCO3- to molecular CO2 and assist in CO2 excretion
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through the gills (Henry, 1987). Thus, we hypothesise that this gene may play a less
important role in acid-base balance in freshwater crayfish than cytoplasmic CA.
Here we identified and characterized a novel β-CA gene (ChqCA-beta) which represents the
first full sequence of a beta CA gene from a crayfish (C. quadricarinatus). β-CAs have been
observed and well documented in archaea, bacteria, algae and fungi; in most plants; and in
some invertebrates (Hewett-Emmett, 2000; Zimmerman and Ferry, 2008), but their activity
are still not clear in the animal kingdom. Previously the presence of β-CAs in the animal
kingdom was controversial and sometimes ignored as they seemed to be uncommon or
result from poor quality sequences (Zimmerman and Ferry, 2008). However, β-CA genes
have recently been reported in the microcrustacean D. pulex (Colbourne et al., 2011) and
also in other arthropods (Zimin et al., 2008; Syrjanen et al., 2010; Werren et al., 2010;
Nygaard et al., 2011). More recently, we have also characterized β-CA in other freshwater
crayfish Including C. destructor and C. cainii .These results suggest that β-CA is widespread in
arthropods. The β isoform of CA has not previously been identified in the genomes of
vertebrate species and the loss of β-CA gene in the chordate lineage might have occurred
either in the last common ancestor of all chordates (Syrjanen et al., 2010). Our rt-PCR
results show that CA-β gene was expressed in the gills of C. quadricarinatus at very low
expression levels compared the other CA genes. This indicates that the β isoform of CA
identified here does not play an important role in acid-base balance in freshwater crayfish.
In fact, recent studies have shown that the homologous β CA protein from Drosophila is a
highly active mitochondrial enzyme (Syrjanen et al., 2010). Previous papers show that
mitochondrial CAs have a functional role in the CO2/HCO3− interaction by providing
counterions for the uptake of energy-linked mitochondrial Ca2+ (Dodgson et al., 1980).
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Although β-CAs catalyse a similar reaction as other CA isoforms there are some important
structural differences between these classes. In α-, γ- and δ-CAs the active Zn-binding site is
coordinated by three histidines (Fig. 2.2 and 2.3), whereas in β-CAs it is linked with one
histidine and two cysteine residues (Cox et al., 2000). In spite of this structural difference, β-
CAs basically follow the same molecular mechanism for reversible hydration of CO2 into H+
and HCO3- like α-CAs (Strop et al., 2001). Our limited gene expression data suggest no major
function of β-CAs in pH balance in freshwater crayfish. However, to confirm the activities of
β-CAs in crayfish and other decapod crustaceans, more detailed physiological and
biochemical investigations are needed.
The two partial CA transcripts identified, ChqCA-p1 and ChqCA-p2 showed greatest protein
similarity with the GPI-linked CA isoform (S Fig. 2.4 in Appendix 4; Ali et al., 2015c). As these
genes show greatest similarity with the GPI-linked CA isoform we hypothesise that these
transcripts may represent duplicated copies of ChqCAg. GPI-linked carbonic anhydrase
genes show evidence of duplication in a range of species including fish and other
vertebrates (Tolvanen et al., 2013). In fact, it has been hypothesised that the extensive
duplication of GPI-linked carbonic anhydrase genes in fish has resulted in different isoforms
undertaking slightly different roles in breathing and acid excretion (Tolvanen, 2013). As
crayfish also inhabit an aquatic environment the duplication of GPI-linked carbonic
anhydrase genes in C. quadricarinatus may also have different functions in similar
physiological processes in these species.
2.5.2 Other candidate osmoregulatory genes in Cherax quadricarinatus
We have found a number of candidate genes that have previously been identified as playing
a role in osmoregulation and pH balance in crustaceans. Many of these genes have been
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shown to be important for adaptation in response to salinity gradients and also in stress
response to abrupt changes in pH or water chemistry (Henry et al., 2002; Serrano and
Henry, 2008; Liu et al., 2010; Havird et al., 2013). One group of candidate genes of particular
interest are those involved in ion transport and systemic acid–base balance. These genes
included sodium-potassium ATPase, sarco/endoplasmic reticulum Ca2+-ATPase, arginine
kinase, calreticulin, Na+/H+ exchanger, Na+/HCO3− cotransporter, alkaline phosphatase,
multiple subunits of vacuolar proton pump and a number of different genes from the solute
carrier gene family. In this section, we will discuss two of these genes for which we had gene
expression data, V-type H+-ATPase and arginine kinase, and whether they may play an
important role in acid-base balance in freshwater crayfish.
2.5.3 Vacuolar-type H+-ATPase
The V-type H+-ATPase is a key enzyme that regulates osmoregulation and pH balance in
many organisms (Forgac, 1998; Saliba and Kirk, 1999; Beyenbach, 2001; Kirschner, 2004;
Covi and Hand, 2005). Its involvement in freshwater osmoregulation has also been shown by
a number of studies of gene expression and protein activity (Faleiros et al., 2010; Lee et al.,
2011; Towle et al., 2011). This protein pumps protons to acidify intracellular organelles and
in this process influence HCO3− and Cl− exchange and Na+ uptake (Weihrauch et al., 2004a;
Martin Tresguerres et al., 2006) and also plays a vital role in branchial NH3 excretion
(Weihrauch et al., 2002; Weihrauch et al., 2009; Martin et al., 2011; Weihrauch et al., 2012).
The large number of V-type H+-ATPase subunits expressed in the gills of C. quadricarinatus
suggests that it may play an important role in this tissue. The gene expression data for V-
type H+-ATPase subunit A was close to significant; so it may play a role in acid-base balance
in crustaceans. Further gene expression studies on this gene with greater replication than
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used in the current study may elucidate whether V-type H+-ATPase subunit A is involved in
acid-base balance in C. quadricarinatus. Examining the expression of other V-type H+-ATPase
subunits may also shed further light on other members of this gene family in acid-base
balance in freshwater crayfish.
2.5.4 Arginine kinase
Arginine kinase is a kinase enzyme that plays an important role in maintaining ATP levels in
invertebrates (Song et al., 2012)and has been suggested to play a role in osmoregulation
and acid-base balance in crustaceans. In fact, this enzyme was shown to be differentially
expressed in some crustacean species, but not in others, when transferred to different ionic
conditions (Kinsey and Lee, 2003; Pan et al., 2004; Yao et al., 2005; Liu et al., 2006; Silvestre
et al., 2006; Abe et al., 2007; Song et al., 2012). In our study we found no evidence for
arginine kinase playing a role in acid-base balance in C. quadricarinatus as this gene was not
differentially expressed across pH treatments. The enzyme activity and mRNA abundance of
arginine kinase also did not change when C. maenus was transferred to low salinity (Kotlyar
et al., 2000; Towle and Weihrauch, 2001). While the gene was not differentially expressed
arginine kinase levels did increase in low pH treatments where V-type H+-ATPase and
cytoplasmic CA were also increased. Weihrauch (Weihrauch et al., 2001b) in a study on
osmoregulation in crustaceans suggested that while arginine kinase levels are increased in
some species it is to maintain ATP levels, which are required for active ion transport and
other cellular processes that require ATP. This hypothesis would explain the increased levels
of arginine kinase in the lower pH treatment when active transport enzymes are also
upregulated.
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2.6 Conclusions
From the gene expression data obtained from this study, we suggest that cytoplasmic
carbonic may play an important role in systemic acid-base balance in freshwater crayfish
while other CA genes probably play only a limited role in this process. This data is important
because we still know remarkably little about which genes are important for acid-base
balance in freshwater crayfish.
2.7 Acknowledgements
This work was funded by the QUT (Queensland University of Technology) Higher Degree
Research Support and a QUT ECARD grant awarded to PP. We are grateful to the valuable
guidance and support of all the group members of Physiological Genomics Lab at QUT.
2.8 Data Archive
Raw sequence data was deposited in the SRA (Short Read Archive) repository of NCBI under
the accession number PRJNA275170. Data can be downloaded through the link:
http://www.ncbi.nlm.nih.gov/sra/?term=PRJNA275170
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Expression patterns of two alpha Chapter 3.Carbonic anhydrase genes, Na+/K+-ATPase and V-type H+-ATPase in the freshwater crayfish, Cherax quadricarinatus, under different pH conditions
(Manuscript is ready to be submitted very soon.)
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Expression patterns of two alpha Carbonic anhydrase genes,
Na+/K+-ATPase and V-type H+-ATPase in the freshwater
crayfish, Cherax quadricarinatus, under different pH
conditions
Muhammad Yousuf Ali1, Ana Pavasovic2, Peter B Mather1 and Peter J Prentis1*
1School of Earth, Environmental and Biological Sciences, Queensland University of Technology, Brisbane, QLD
4001, Australia
2School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4001, Australia
* Corresponding author: Peter J Prentis (p.prentis@qut.edu.au)
Email addresses: MYA: my.ali@qut.edu.au AP: a.pavasovic@qut.edu.au PBM: p.mather@qut.edu.au PJP: p.prentis@qut.edu.au
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3.1 Abstract
Osmoregulation and systemic acid-base balance in decapod crustaceans are largely
controlled by a set of transport-related enzymes including carbonic anhydrase (CA), Na+/K+-
ATPase (NKA) and V-type-H+-ATPase (HAT). Variable pH levels and changes in osmotic
pressure can have a significant impact on the physiology and behaviour of crustaceans.
Therefore, it is crucial to understand the mechanisms via which an animal can maintain its
internal pH balance and regulate the movement of ions into and out of its cells. Here, we
examined expression patterns of the cytoplasmic (CAc) and membrane-associated form
(CAg) of CA, NKA α subunit and HAT subunit a in gills of the freshwater crayfish Cherax
quadricarinatus. Expression levels of the genes were measured at three pH levels, pH 6.2,
7.2 (control) and 8.2 over a 24 hour period. All genes showed significant differences in
expression levels, either among pH treatments or over time. Expression levels of CAc were
significantly increased at low pH and decreased at high pH conditions 24 h after transfer to
these treatments. Expression increased in low pH after 12 h, and reached their maximum
level by 24 h. The membrane-associated form CAg showed changes in expression levels
more quickly than CAc. Expression increased for CAg at 6 h post transfer at both low and
high pH conditions, but expression remained elevated only at low pH (6.2) at the end of the
experiment. Expression of CqNKA significantly increased at 6 h after transfer to pH 6.2 and
remained elevated up to 24 h. Expression for HAT and NKA showed similar patterns, where
expression significantly increased 6 h post transfer to the low pH conditions and remained
significantly elevated throughout the experiment. The only difference in expression
between the two genes was that HAT expression decreased significantly 24 h post transfer
to high pH conditions. Overall, our data suggest that CAc, CAg, NKA and HAT gene
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expression is induced at low pH conditions in freshwater crayfish. Further research should
examine the physiological underpinnings of these changes in expression to better
understand systemic acid/base balance in freshwater crayfish.
Key words: osmoregulation, acid-base, pH balance, gills, expression, crayfish, Redclaw
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3.2 Introduction
Of all currently available farmed freshwater crayfish, Redclaw (Cherax quadricarinatus) is
the most important commercial species developed for aquaculture production in Australia.
It is also an important commercial species in other areas of the world, most notably Mexico,
Ecuador, Uruguay, Argentina and China (FAO, 2010; Saoud et al., 2012). Redclaw occurs
naturally across Northern Australia as well as Southern Papua New Guinea. Wild Australian
population of Redclaw are distributed over a distinct pH gradient; one area has low pH
(≈6.2) in north Queensland and the other area higher pH (≈8.2) on the western side of
Northern Territory (Macaranas, 1995; Bryant and Papas, 2007; Baker et al., 2008). Previous
research on this species reported a fixed allozyme difference at a carbonic anhydrase (CA)
allozyme locus between C. quadricarinatus populations collected from either side of this pH
gradient (Macaranas, 1995). The authors suggested that this CA isoform may play an
important role in maintaining systemic acid–base balance and ion regulation under different
water chemistry (Macaranas, 1995). This indicates that CA genes and potentially other
genes involved in systemic acid-base balance or ion transport probably play an important
role in the response to changes in water chemistry.
In the areas Cherax species occur and are cultured, pH Levels fluctuate not only among
natural water bodies, but vary widely within water-bodies over time (Boyd, 1990). For
example, many aquaculture ponds are built in areas with acid sulphate soils or areas with
acid precipitation which can lead to decreased pH levels within water-bodies (Haines,
1981). Regardless of the causes, fluctuations in pH have been demonstrated to have a great
impact on the distribution, growth, behaviour and physiology in many crustaceans including
Cherax crayfish (Chen and Chen, 2003; Pavasovic et al., 2004; Pan et al., 2007; Yue et al.,
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2009; Haddaway et al., 2013; Kawamura et al., 2015; Kim et al., 2015). It is also evident that
environmental pH has a great impact on acid-base balance and electrolyte concentrations in
the haemolymph of freshwater crayfish (Morgan and McMahon, 1982; Wood and Rogano,
1986; Zanotto and Wheatly, 1993; Wheatly et al., 1996). For example, water with a low pH
level has been shown to cause acid–base imbalance (decreased pH in haemolymph) and
disturbed ion regulation (decreased Na+ and Cl- concentrations and increased K+) in
freshwater crayfish (Morgan and McMahon, 1982; Wood and Rogano, 1986; Wheatly et al.,
1996). Therefore, a better understanding of the effect of external pH on gene expression in
pH induced and ion transport-related genes in gills of freshwater crayfish is needed to
elucidate what role these important genes are playing in response to changes in water pH.
Systemic acid-base balance and ion-regulation in crustaceans are largely controlled by a set
of transport-related enzymes including, carbonic anhydrase (CA), Na+/K+-ATPase (NKA) and
V-type-H+-ATPase (HAT); and gills are the main organs where these functions take place
(Freire et al., 2008). CA produce H+ and HCO3- ions through a reversible reaction, CO2+H2O
←→H++HCO3-, thereafter the H+ and HCO3
- serve as anti-porters for Na+/H+(NH4+)
exchangers and Cl-/HCO3- cotransporter (Freire et al., 2008; Henry et al., 2012; Romano
and Zeng, 2012). NKA pumps Na+ ions out of the cell and draws K+ ions in, and thus
establishes an electrochemical gradient that acts as a driving force for transport of Na+ and
K+ ions by other transporters including Na+/K+/2Cl- cotransporter (Lucu and Towle, 2003;
Jayasundara et al., 2007; Leone et al., 2015; Li et al., 2015). H+-ATPase pumps protons (H+)
and acidifies intracellular organelles that help to maintain pH balance in crustacean taxa
(Faleiros et al., 2010; Lee et al., 2011; Towle et al., 2011; Boudour-Boucheker et al., 2014;
Lucena et al., 2015).
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Recently, two distinct forms of CA as well as key systemic acid-base balance genes Na+/K+-
ATPase (NKA) and V-type-H+-ATPase (HAT) were identified through transcriptome
sequencing in multiple species of the genus Cherax; Cherax quadricarinatus, C. destructor
and C. cainii (Ali et al., 2015a; Ali et al., 2015b). More recently, comparative molecular
analysis between these species showed that there were very few non-synonymous
mutations in CA or other key osmoregulatory genes that are likely to lead to large
differences in protein function within and between these species (Our unpublished results).
Therefore, we hypothesize that differential expression of CA genes and other important ion
transport and systemic acid-base balance genes may enable Redclaw crayfish to survive in
both acidic and alkaline water conditions. However, this hypothesis has to be thoroughly
investigated through a gene expression study at varied pH conditions.
Despite the fact that expression and activity levels of the ion-transport enzymes are
probably regulated by extracellular pH, studies on the effects of pH on the expression of
these genes in crustaceans is limited (Pan et al., 2007; Wang et al., 2012; Liu et al., 2015;
Lucena et al., 2015). Most of the previous work has investigated the effect of salinity on
expression of ion-transport genes in decapod crustaceans (for example; CA (Serrano et al.,
2007; Serrano and Henry, 2008; Pongsomboon et al., 2009); NKA (Mitchell and Henry, 2014;
Chaudhari et al., 2015; Han et al., 2015; Leone et al., 2015; Li et al., 2015); HAT (Luquet et
al., 2005; Havird et al., 2014). However, to the best of our knowledge, all the studies have
been undertaken in euryhaline species, and none have investigated freshwater crayfish.
Recently, we have reported gene expression of CA and HAT in C. quadricarinatus, but the
study was limited to one sampling point and mainly comprised the description of a
transcriptome dataset (Ali et al., 2015b). Thus, in this study we have undertaken a time
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course of expression patterns for the key genes involved in pH-balance and osmoregulation
under varied pH conditions in C. quadricarinatus. The study reports the expression patterns
of two forms of alpha carbonic anhydrase (a cytoplasmic form, referred to as CqCAc and a
membrane-associated form, referred to as CqCAg); sodium-potassium pump Na+/K+-ATPase
α subunit (CqNKA); and proton (H+) pump V-type-H+-ATPase subunit a (CqHAT) at three
different pH levels, pH 6.2, 7.2 (control) and 8.2, in the gills of C. quadricarinatus over a time
course of 24 hours.
3.3 Materials and Methods
3.3.1 Sample Preparation
Live inter-moult C. quadricarinatus were obtained from Theebine, Queensland, Australia.
Animals were housed in rectangular glass tanks (size: 25×18×15 cm, capacity: 27 L each) at
QUT (Queensland University of Technology) Aquaculture facility and acclimated at pH 7.2 ±
0.14 for three weeks before the experiment. Other water quality parameters were as
follows: temperature 20.9±0.90C, conductivity 405±42 μS/cm. Water quality was maintained
with a computer-controlled filtration system (Technoplant). During the acclimation period
all animals were fed regularly with formulated feed pellets.
Feeding was stopped 24 h before the pH treatments were undertaken. A total of 45 animals
(weight 41±4 g and length 11.8±0.6) were distributed into separate tanks. The animals were
stressed by two pH treatments, pH 6.2 and pH 8.2, a treatment within the tolerance range
of this species (Macaranas, 1995; Bryant and Papas, 2007); and pH 7.2 was used as a no
change control. Gills were extracted from three individuals as biological replicates at 0 h, 3
h, 6 h, 12 h and 24 h post-exposure for each treatment.
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3.3.2 RNA extraction and cDNA synthesis
Prior to tissue extraction, animals were euthanized in crushed ice for 5-10 minutes. Gill
tissue were dissected and immediately frozen in liquid nitrogen. Total RNA was extracted
from individual gill tissue, from 0.1 g of tissue from each animal, using a TRIZOL/Chloroform
extraction (Chomczynski and Mackey, 1995) and then purified using a RNeasy Midi Kit (cat #
75144, QIAGEN) using an existing protocol (Prentis and Pavasovic, 2014). Genomic DNA was
digested with Turbo DNA-free kit (REF-AM1907, Ambion RNA, Life Technologies, USA) and
RNA quality and concentration were checked using a Bioanalyzer 2100 RNA nanochip
(Agilent Technologies).
Complementary DNA (cDNA) was synthesized by reverse transcription from 1 μg of total
RNA using SensiFAST cDNA synthesis protocol (Bioline, Australia, Cat # BIO-65054). The
reaction was made in a final volume of 20 μl with 1 μg RNA template, 1x TransAmp Buffer, 1
μl Reverse Transcriptase and of DNase/RNase free water as required. The resulting cDNA
samples were stored at −20°C until used as templates for real-time quantitative PCR.
3.3.3 Quantification of mRNA by quantitative Real-Time-PCR (qRT-PCR)
The relative abundance of mRNA levels was measured using the quantitative real-time PCR
machine LightCycler 96 (Roche, Version 04) using FastStart Essential DNA Green Master
(Roche, Germany, Cat. No.06924204001). Three replicate animals were used for all sampling
points in all experimental treatments and all qRT-PCR amplifications were carried out in
triplicate. Gene-specific quantitative real-time PCR primers were designed in primer3 using
the settings from Amin et al. (2014) for transcripts that were identified as: two forms from
the alpha CA gene family (CqCAc and CqCAg), CqNKA, CqHAT and 18s rRNA (Cq18s) (Ali et
al., 2015b). The RT-PCR reaction contained 1 μl cDNA template, 1 μl Green Master, 2 μl
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primers (1 μl forward and 1 μl reverse, concentration 10 μmole) and DNase free water
required to make the final volume up to 20 μl. Real-time PCR conditions included a pre-
incubation of 950C for 5 minutes, followed by a total 45 cycles of three-step amplification of
950C for 10 seconds; 600C for 10 seconds and 720C for 10 seconds. Ribosomal 18S was used
as an internal control gene (whose expression levels did not change under different
treatments) to normalize sample-to-sample variation. Negative controls (without cDNA
template) were also used. The relative expression of the target genes were measured as a
ratio (concentration of target gene/concentration of 18S gene) according to Pfaffl (2001).
3.3.4 Data analysis
The relative expression values for the genes were obtained using the Relative Quant analysis
tool described in the Light Cycler 96 system operator’s guide, version 2.0. Statistical
analyses were performed using Minitab software (version 17). A one-way ANOVA was
undertaken with a Fisher’s tests to determine if any of the genes showed significant
expression differences across the three pH treatments or time points at p<0.05.
3.4 Results
3.4.1 Carbonic anhydrase (CA)
We analysed expression patterns of two forms of carbonic anhydrase, CqCAc and CqCAg.
The results showed that CqCAc was only differentially expressed at 24 hour time point,
across all three treatments, low pH (6.2), high pH (8.2) and the control pH treatment (7.2)
(Fig. 3.1). At 24 h, the expression levels of CqCAc were significantly upregulated with an
approximately 3 fold increase at pH 6.2 (p=0.007, F-value 22.14) whereas, at pH 8.2, the
expression levels were significantly down-regulated (p=0.023, F-value 18.60). CqCAc
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expression levels were consistent in control group (pH 7.2) over the experiment. For pH 6.2
treatment, CqCAc showed consistent expression up to 6 h mark; after this expression of
CqCAc started increasing dramatically with the initial increase of ≈2 fold at 12 h, and 3 fold
at 24 h (p=0.007, F-value 22.14). At pH 8.2, CqCAc expression did not change significantly up
to the 12 h mark, but there was a significant decrease in expression 24 h post transfer
(p=0.023, F-value 18.60).
The pattern of CqCAg expression was similar to CqCAc, but CqCAg was induced more quickly
than CqCAc (Fig. 3.2). The expression of CqCAg initially increased at 6 h post transfer at both
pH 6.2 and pH 8.2; although these increases were not statistically significant (p>0.05, F value
1.79). The expression levels remained elevated at pH 6.2, but non-significantly, for any time
points post-transfer. Expression levels of CqCAg in the pH 8.2 treatment gradually increased
until 6 h but started to decrease 12 h post transfer and expression decreased significantly by
≈2.5 fold at 24 h as compared to the initial expression level (p=0.01, F-value 33.82).
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Figure 3.1 Relative expression of cytoplasmic carbonic anhydrase
Relative mRNA expression of CqCAc (cytoplasmic carbonic anhydrase) in the gills of Redclaw crayfish (Cherax quadricarinatus) acclimated to pH 7.2 (control) and after being transferred to pH 6.2 and pH 8.2 at various times for up to 24 hours. Vertical bars represent the mean±s.e.m (n=3). Different letters above the bars denote significant differences from the control group in the same time of sampling at the 0.05 level ( one-way ANOVA, Tukey’s and Fisher post-hoc tests). Asterisk (*) indicate the significant differences (at 0.05 level) in expression levels over the course of exposure time compared with the initial level (0 h) of mRNA-expression within the same treatment group. Expression levels were normalized with respect to reference gene 18S (internal control gene) for the same sample, and calculated as a ratio (conc. of target gene/conc. of 18S gene) according to Pfaffl (2001).
Figure 3.2 Relative expression of membrane-associated carbonic anhydrase
Relative mRNA expression of CqCAg (membrane-associated carbonic anhydrase) in the gills of Redclaw crayfish (C. quadricarinatus) acclimated to pH 7.2 (control) and after being transferred to pH 6.2 and pH 8.2 at various times for up to 24 hours. Vertical bars represent the mean±s.e.m (n=3). Different letters above the
Page 72
bars denote significant differences from the control group in the same time of sampling at the 0.05 level ( one-way ANOVA, Tukey’s and Fisher post-hoc tests). Asterisk (*) indicate the significant differences (at 0.05 level) in expression levels over the course of exposure time compared with the initial level (0 h) of mRNA-expression within the same treatment group.
3.4.2 Na+/K+-ATPase (NKA)
Expression of CqNKA was significantly different between treatments (p= 0.021, F-value 9.19)
from 6 h onwards. At pH 6.2 expression of CqNKA increased ≈4.5 fold at 6 h and remained
differentially expressed up to 24 h post transfer (≈3 fold expression increase compared to
time 0)(Fig. 3.3, p= 0.016 and F-value 16.37). In contrast, the magnitude of increase in
expression of CqNKA at pH 8.2 was less than that at pH 6.2. At pH 8.2, the highest level of
expression was reached at 12 h post-transfer with an increase of ≈2.5 fold, but the change
was not statistically significant (p=0.36, F-value 1.02). At 24 h post-exposure to high pH (pH
8.2) the expression levels sharply decreased almost to its initial level of expression (0 h) (Fig.
3.3).
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Figure 3.3 Relative expression of Na+/K+-ATPase
Relative mRNA expression of CqNKA (Na+/K+-ATPase) in the gills of Redclaw crayfish (C. quadricarinatus) acclimated to pH 7.2 (control) and after being transferred to pH 6.2 and pH 8.2 at various times for up to 24 hours. Vertical bars represent the mean±s.e.m (n=3). Different letters above the bars denote significant differences from the control group in the same time of sampling at the 0.05 level ( one-way ANOVA, Tukey’s and Fisher post-hoc tests). Asterisk (*) indicate the significant differences (at 0.05 level) in expression levels over the course of exposure time compared with the initial level (0 h) of mRNA-expression within the same treatment group.
3.4.3 V-type H+-ATPase (HAT)
The expression of CqHAT was similar to that of CqNKA, and demonstrated significantly
higher levels of expression 6 h after transfer to pH 6.2 (p=0.023 and F-value 18.74 at 6h;
p=0.005 and F-value 53.30 at 24 h ). This pattern of increased expression remained fairly
constant approximately 2-3 fold higher than initial expression until the experiment ceased
(Fig. 3.4). For pH 8.2 the expression of CqHAT was not significantly different from that of the
control group until 24 h post-transfer. At 24 h post-exposure the expression level dropped
sharply to less than the initial level of expression (≈35% of the control level, p=0.012, F-value
29.62).
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Figure 3.4 Relative expression of V-type-H+-ATPase
Relative mRNA expression of CqHAT (V-type-H+-ATPase) in the gills of Redclaw crayfish (C. quadricarinatus) acclimated to pH 7.2 (control) and after being transferred to pH 6.2 and pH 8.2 at various times for up to 24 hours. Vertical bars represent the mean±s.e.m (n=3). Different letters above the bars denote significant differences from the control group in the same time of sampling at the 0.05 level ( one-way ANOVA, Tukey’s and Fisher post-hoc tests). Asterisk (*) indicate the significant differences (at 0.05 level) in expression levels over the course of exposure time compared with the initial level (0 h) of mRNA-expression within the same treatment group.
3.5 Discussion
The expression of all candidate genes was induced by transfer to low pH. The timing of
induction varied between the candidate genes as did the fold increase of expression. The
role that these candidate genes may play in systemic acid-base balance is discussed in the
following subsections.
3.5.1 Carbonic anhydrase (CA)
In our study, expression levels of CqCAc increased upon exposure to low pH conditions (≈3
fold at 24 hour post-exposure) (Fig. 3.1). Other studies have reported increased levels of CA
expression in crustaceans after being exposed to low pH conditions (Liu et al., 2015) or low
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salinity levels (Jayasundara et al., 2007; Serrano et al., 2007; Serrano and Henry, 2008;
Pongsomboon et al., 2009; Mitchell and Henry, 2014). For instance, Liu et al. (2015)
reported a 5 fold increase in expression of CAc in the euryhaline shore crab Pachygrapsus
marmoratus 12h after transfer to a pH 7.4 treatment (control pH 8.2). A large induction of
CAc was also reported by Serrano et al. (Serrano et al., 2007) (100 fold increase in blue crab
Callinectus sapidus), Serrano and Henry (Serrano and Henry, 2008) (100 fold increase in
green crab Carcinus maenas), Mitchell and Henry (Mitchell and Henry, 2014) (90 fold
increase in C. sapidus), and Henry et al. (2006) (10 fold increase in C. maenas) following
transfer to low salinity water. The pattern of CAc expression we found in our study was
similar to that seen in other studies where CAc expression was more pronounced at low
salinity or low pH conditions (Jayasundara et al., 2007; Serrano et al., 2007; Serrano and
Henry, 2008). It is suggested that this change in expression occurs due to the fact that
cytoplasmic CA provides the counter ions H+ and HCO3− ions to the Na+/H+ and Cl−/HCO3
−
exchanger to drive ion uptake in the gills of crustaceans when exposed to low pH or low
salinity environments (Henry and Cameron, 1983; Henry, 1988; Henry, 2001).
Previous studies have shown decreased level of Cl- concentrations in freshwater crayfish
including Procambarus clarkii, Orconectes propinguus and Orconectes rusticus after
exposure to low pH conditions (Wood and Rogano, 1986; Zanotto and Wheatly, 1993).
Therefore, it is possible that in C. quadricarinatus, also a freshwater crayfish species, that
internal Cl- concentrations may decrease in haemolymph under low pH conditions;
increasing the activity of Cl−/HCO3− exchanger to compensate for the loss of Cl-. The activity
of CAc should increase under low-pH stress because HCO3− are supplied by CAc through
hydration of CO2. From these findings, we can infer that the cytoplasmic CA form from C.
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quadricarinatus may play an important role in maintaining internal pH conditions in low
streams across northern Australia where they occur naturally.
Our study showed that the timing for initial induction of CqCAg was faster than that of
CqCAc in both pH conditions. For CqCAc, the initial increase in expression was observed only
after 12 h post-exposure, and only at the lower pH (pH 6.2); while for CqCAg, the initial
increase started at 6 h post-transfer, in both low pH and high pH conditions (Fig. 3.2). This
finding suggests that CqCAg is more sensitive to pH changes, and responds quicker. Liu et al.
(2015) also reported similar pattern of expression for the same two forms of CA in
Litopenaeus vannamei, where CAg was induced more quickly than the CAc. This indicates
that CAg might be more sensitive to changes in pH than CAc at both low and high pH levels.
We also observed that the cytoplasmic CA (CqCAc) was induced more at low pH than high
pH (Fig. 3.1); and had a greater magnitude of ‘inductive scope’ (degree of differences in
expression between the maximal level of expression and baseline expression). In contrast,
CAg showed increased induction at higher pH levels. Liu et al. (2015) also observed similar
patterns of CA gene expression, where they found that CAc was induced more under low pH
conditions, and that CqCAc was induced at a higher level compared to CAg. Very
interestingly, the patterns of sensitivity and induction at different pH conditions are very
similar to expression patterns of Cag and CAc under different salinity conditions in other
decapod crustaceans (Serrano et al., 2007; Serrano and Henry, 2008).
3.5.2 Na+/K+-ATPase (NKA)
In our study, expression of CqNKA was significantly upregulated at low pH conditions but
non-significantly increased at high pH (pH 8.2) (Fig. 3.3). Previous studies have reported
similar patterns of NKA expression, where they found increased levels of either NKA
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transcription or NKA enzyme activity under both low and/or high pH conditions (Pan et al.,
2007; Wang et al., 2012). In an experiment with L. vannamei, Pan et al. (2007) reported 3-4
fold increases in NKA activity 24 h after exposure to low pH (pH 7.1) and high pH conditions
(pH 9.1). In the present study, we report a maximum increase of 4.5 fold in expression at
low pH conditions and 2.5 fold increase at high pH conditions. This indicates that Na+/K+-
ATPase from C. quadricarinatus is induced more strongly to low pH, rather than the high-pH
conditions. Similarly, Wang et al. (Wang et al., 2012) in L. vannamei documented a 17 fold
and 4 fold increase in NKA expression after exposure to low pH (at 6 h post-exposure to pH
5.6) and high pH conditions (at 3h post-exposure to pH 9.3). The main reason for the
differences reported in the level of NKA expression among our study and the two L.
vannamei studies is probably related to differences in the pH treatments (transfer from pH
7.4 to pH 5.6 and 9.3).
In crustacean species, a large number of previous studies have examined the effect of
salinity on expression of transport-related genes, but few studies have focused on the
effects of external pH (Wang et al., 2002; Pan et al., 2007; Wang et al., 2012; Li et al., 2015).
Changes in expression patterns of CqNKA induced by pH in our study are quite similar with
that induced by salinity in other crustacean species, i.e. higher induction levels at low
salinity exposure (Luquet et al., 2005; Jayasundara et al., 2007; Pan et al., 2007; Serrano et
al., 2007; Wang et al., 2012; Havird et al., 2013; Han et al., 2015; Leone et al., 2015; Li et al.,
2015). As published reports show that Na+ concentration decreases and Na+ concentration
increases in the haemolymph of the freshwater crayfish Procambarus clarkia and
Orconectes rusticus upon exposure to low-pH conditions (Morgan and McMahon, 1982;
Wood and Rogano, 1986; Wheatly et al., 1996), it is logical that the NKA expression level
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should also increase in C. quadricarinatus under similar conditions. The main reason of
increased expression levels for CqNKA in C. quadricarinatus may be attributed to increased
level of NKA activity in the gills. Because, Na+/K+-ATPase is a key enzyme that pumps Na+
into haemolymph and draws K+ in the cell, it establishes electrochemical gradients that act
as a driving force for trans-epithelial movement of many monovalent ions including Na+, K+,
H+, Cl- across the gills in crustacean taxa (Havird et al., 2013; Chaudhari et al., 2015; Han et
al., 2015; Leone et al., 2015; Li et al., 2015). Overall, these findings suggest that NKA plays a
role in maintaining systemic acid-base balance in Cherax crayfish.
3.5.3 Vacuolar-type H+-ATPase
In the present study, expression of Vacuolar-type H+-ATPase in C. quadricarinatus was
upregulated at low-pH and down-regulated at high-pH conditions (Fig. 3.4). Previous studies
in crustacean species have also reported that expression levels of Vacuolar-type H+-ATPase
are changed under low and high pH conditions in a similar pattern, but this pattern can vary
among species (Pan et al., 2007; Wang et al., 2012; Havird et al., 2013). For example, in a
study of HAT enzyme activity in L. vannamei, Pan et al. (2007) reported a negative
correlation between activity of HAT and pH levels. HAT expression is probably affected by
changes in external pH, because it is one of the key enzymes that acidifies intracellular
organelles which in turn influence the activity of other ion-transport enzymes including
Na+/H+(NH4+) and Cl−/HCO3
− exchanger (Weihrauch et al., 2002; Weihrauch et al., 2004a;
Luquet et al., 2005; Martin Tresguerres et al., 2006; Pan et al., 2007; Weihrauch et al., 2009;
Faleiros et al., 2010; Martin et al., 2011; Wang et al., 2012; Weihrauch et al., 2012; Havird et
al., 2013).
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In the present study, we report a maximum increase of 2-3 fold in mRNA expression of
CqHAT between 6-12 h after exposure to low pH (6.2) and a 3 fold decrease at high pH (pH
8.2) conditions (Fig. 3.4). Our results are similar to a recent study that reported a significant
down-regulation of 2.5 fold in gill V(H+)-ATPase activity of river shrimp M. amazonicum at
pH 8.5, as compared to that at pH 7.5 (Lucena et al., 2015). Wang et al. (2012), however,
reported increased levels of HAT expression in L. vannamei at both low- and high-pH
conditions, but expression levels were higher at low pH. This variation in the expression of
HAT transcripts or HAT activity are probably attributed to species-specific physiological
differences in species with distinct osmoregulatory capabilities (Anger and Hayd, 2010;
Faleiros et al., 2010; Charmantier and Anger, 2011; Lucena et al., 2015). These findings
suggest that V(H+)-ATPase likely plays an important role in systemic acid-base balance in
freshwater crayfish.
3.6 Conclusions
From the gene expression data, we suggest that cytoplasmic carbonic anhydrase, Na+/K+-
ATPase and V-type-H+-ATPase may play important role in the maintenance of pH balance in
freshwater crayfish, while the membrane-associated CA probably plays a more limited or
indirect role in this process. We can also infer that cytoplasmic CA, Na+/K+-ATPase and V-
type-H+-ATPase are induced more strongly at low pH conditions compared with high pH
conditions. These data can help to provide a better understanding of which genes are
involved in systemic pH balance in freshwater crayfish.
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Comparative molecular analyses of Chapter 4.select pH- and osmoregulatory genes in three freshwater crayfish Cherax quadricarinatus, C. destructor and C. cainii
Part of this chapter published as:
Ali, M.Y., Pavasovic, A., Amin, S., Mather, P.B. and Prentis, P.J., 2015. Comparative analysis of gill transcriptomes of two freshwater crayfish, Cherax cainii and C. destructor. Marine Genomics (Elsevier) 22, 11-13.
(http://dx.doi.org/10.1016/j.margen.2015.03.004)
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Comparative molecular analyses of select pH- and
osmoregulatory genes in three freshwater crayfish Cherax
quadricarinatus, C. destructor and C. cainii
Muhammad Yousuf Ali1, Ana Pavasovic2, Lalith Dammannagoda Acharige1, Peter B Mather1 and Peter J Prentis1*
1School of Earth, Environmental and Biological Sciences, Queensland University of Technology, Brisbane, QLD
4001, Australia
2School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4001, Australia
* Corresponding author: Peter J Prentis (p.prentis@qut.edu.au)
Email addresses: MYA: my.ali@qut.edu.au AP: a.pavasovic@qut.edu.au LDA: l.dammannagodaacharige@qut.edu.au PBM: p.mather@qut.edu.au PJP: p.prentis@qut.edu.au
Page 82
4.1 Abstract
Systemic acid-base balance and osmotic/ionic regulation in decapod crustaceans are in part
maintained by a set of transport-related enzymes such as carbonic anhydrase (CA), Na+/K+-
ATPase (NKA), H+-ATPase (HAT), Na+/K+/2Cl− cotransporter (NKCC), Na+/Cl-/HCO3-
cotransporter (NBC), Na+/H+ exchanger (NHE), Arginine kinase (AK), Sarcoplasmic Ca+2-
ATPase (SERCA) and Calreticulin (CRT). We carried out a comparative molecular analysis of
these genes in three commercially important yet eco-physiologically distinct freshwater
crayfish, C. quadricarinatus, C. destructor and C. cainii, with the aim to identify mutations in
these genes and determine if observed patterns of mutations were consistent with the
action of natural selection. We also conducted a tissue-specific expression analysis of these
genes across seven different organs, including gills, hepatopancreas, heart, kidney, liver,
nerve and testes using NGS transcriptome data. A total of 87290, 136622 and 147101
assembled contigs were obtained from more than 72 million, 83 million and 100 million high
quality reads from the gills of C. quadricarinatus, C. cainii and C. destructor, with a BLAST
success rate of 25.3%, 17% and 18% respectively. The molecular analysis of the candidate
genes revealed a high level of sequence conservation across the three Cherax sp. Hyphy
analysis revealed that all candidate genes showed patterns of molecular variation consistent
with neutral evolution. The tissue-specific expression analysis showed that half of the genes
were expressed in all tissue types examined, while approximately 10% of candidate genes
were only expressed in a single tissue type. The largest number of genes was observed in
nerve (84%) and gills (78%) and the lowest in testes (66%). The tissue-specific expression
analysis also revealed that most of the master genes regulating pH and osmoregulation (CA,
NKA, HAT, NKCC, NBC, NHE) were expressed in all tissue types indicating an important
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physiological role for these genes outside of osmoregulation in other tissue types. The high
level of sequence conservation observed in the candidate genes may be explained by the
important role of these genes as well as potentially having a number of other basic
physiological functions in different tissue types.
Key words: Redclaw, Yabby, Marron, osmoregulatory genes, carbonic anhydrase, Na+/K+-
ATPase, V-type H+-ATPase
4.2 Introduction
In decapod crustaceans, systematic acid-base balance and ion-regulation are processes that
are largely controlled by a set of transport-related enzymes including carbonic anhydrase
(CA), Na+/K+-ATPase (NKA), Vacuolar-type H+-ATPase (HAT), Na+/K+/2Cl− cotransporter
(NKCC), Na+/HCO3- cotransporter (NBC), Arginine kinase (AK), Calreticulin (CRT),
Sarco/endoplasmic reticulum Ca2+ATPase (SERCA), Na+/H+ exchanger (NHE) and Na+/Ca+2
exchanger (NCX) (Pan et al., 2007; Serrano et al., 2007; Freire et al., 2008; Henry et al., 2012;
McNamara and Faria, 2012a; Romano and Zeng, 2012; Havird et al., 2013). The distribution,
expression patterns and activities of these genes have been reported in a number of
decapod crustaceans (for example; CA (Serrano et al., 2007; Pongsomboon et al., 2009; Ali
et al., 2015b; Liu et al., 2015); NKA (Chaudhari et al., 2015; Han et al., 2015; Leone et al.,
2015); HAT (Pan et al., 2007; Wang et al., 2012; Havird et al., 2014; Ali et al., 2015b); NKCC
(Towle et al., 1997; Havird et al., 2013; Havird et al., 2014); NHE (Towle and Weihrauch,
2001; Weihrauch et al., 2004a; Ren et al., 2015); NCX (Flik et al., 1994; Lucu and Flik, 1999;
Flik and Haond, 2000); AK (Serrano et al., 2007; Havird et al., 2014; Ali et al., 2015b); CRT
(Luana et al., 2007; Visudtiphole et al., 2010; Watthanasurorot et al., 2013; Duan et al.,
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2014). Despite this, we know little about the molecular evolution of these key gene classes
or their tissue specific expression in decapod crustaceans.
Cherax quadricarinatus (Redclaw), Cherax destructor (Yabby) and Cherax cainii (Marron) are
the most abundant and commercially important freshwater crayfish, endemic to Australia.
These species occur in a diverse range of aquatic environments and can tolerate wide
fluctuations in a range of water parameters such as pH, salinity, dissolved oxygen and
temperature (Bryant and Papas, 2007; McCormack, 2014). This indicates that these crayfish
species have a great capacity to cope with highly variable aquatic environments.
Consequently, because of the physiological robustness of these animals and their economic
significance, it has led to their use in physiological genomic research in crustaceans.
In previous studies we have identified a number of the above mentioned ion-transport
related genes in C. quadricarinatus, C. destructor and C. cainii (Ali et al., 2015a; Ali et al.,
2015b). In the present study, we undertook an in-depth molecular analysis of candidate
systemic acid-base and ion-transport related genes across these three freshwater crayfish
species. In addition, we have performed comparative and evolutionary genomic analysis of
these genes across other related arthropod species. The main focus of this study was to
identify synonymous and non-synonymous mutations in these genes and to determine if the
observed patterns of mutations were consistent with the action of natural selection or
neutral evolution. This study will also investigate the tissue-specific expression of the pH
and ion/osmoregulatory genes in the gills, hepatopancreas, heart, kidney, liver, nervous
system and testes using publically available transcriptome metadata.
4.3 Material and Methods
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4.3.1 Sample collection and preparation
Live redclaw crayfish (C. quadricarinatus) were obtained from Theebine, in Queensland,
Australia; yabby (C. destructor) were sourced from New South Wales, Australia; and Marron
(C. cainii) from Mudgee, Western Australia, Australia. Culture conditions for C.
quadricarinatus has already been described in (Ali et al., 2015b). Cherax destructor and C.
cainii were housed at QUT aquaculture facility under standard culture conditions. Animals
were acclimated to lab conditions for three weeks (210C, pH 7 and conductivity 1700
μS/cm). Before tissue collection, three C. cainii (body length 11 ± 0.9 cm and wet body
weight 57 ± 5g) and three C. destructor (9 ± 0.7 cm and 45 ± 3g) were exposed to pH 6, 7 and
8; a natural pH tolerance range of Cherax species (Macaranas, 1995; Bryant and Papas,
2007).
4.3.2 RNA extraction, cDNA synthesis and sequencing
Gills were excised from the samples, immediately following euthanisation in ice for 5-10
minutes. Tissues were extracted after six hours at each treatment, following euthanisation
in ice-water. Tissue samples were pooled for each species and crushed in liquid nitrogen
before total RNA was extracted using an existing protocol (Prentis and Pavasovic, 2014).
Genomic DNA was digested with Turbo DNA-free kit (Life Technologies) and RNA quality and
concentration was determined using a Bioanalyzer 2100 (Agilent Technologies). RNASeq
library preparation and paired-end sequencing were undertaken on an Illumina Next Seq
500 (NextSeq 150 bp pair-ends) according to the manufacturer's protocol for stranded
library preparation.
Six individual hepatopancreas samples were dissected and homogenised in liquid N2 before
total RNA was extracted using the protocol of Prentis and Pavasovic (2014). Genomic DNA
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was digested with Turbo DNA-free kit (REF-AM1907, Ambion RNA, Life Technologies, USA)
and RNA quality and concentration were checked using the Bioanalyzer 2100 (Agilent
Technologies). RNASeq library preparation and sequencing were undertaken according to
the Ion Proton 200 bp library preparation and sequencing protocol (Thermo Fisher, see
Amin et al., 2014). Raw Illumina sequence data for heart, kidney, liver, nerve and testes
were downloaded from NCBI SRA database as FastQ files (Bio-project No. PRJEB5112).
4.3.3 Assembly, functional annotation and gene identification
Reads which did not meet our quality criteria (Q>30, N bases <1%) were removed before
assembly using Trimmomatic (Version 0.32; Bolger et al., 2014). These filtered reads were
assembled with the Trinity short read de novo assembler using the stranded option for
Illumina data (Version 2014-04-13p1; Haas et al., 2013) and the unstranded option for Ion
Torrent data. Contigs with > 95% sequence similarity were clustered into gene families using
CD-HIT (Version 4.6.1; Huang et al., 2010). Coding sequences representing open reading
frames (ORFs) from the transcripts were determined using Transdecoder (Version
r20140704; Haas et al., 2013).
Assembled data were used as BLASTx queries against the NCBI NR protein database using
BLAST+ (Version 2.2.29; Camacho et al., 2009) with a stringency of 10 x e-5. Gene ontology
(GO) terms were assigned to annotated transcripts using Blast2Go Pro (Version 3.0; Conesa
et al., 2005). Candidate genes were identified based on literature searches, PFAM domains,
and GO terms (Henry et al., 2012; Eissa and Wang, 2014; Ali et al., 2015b).
4.3.4 Molecular analyses
Comparative molecular analyses were carried out on the candidate genes previously
reported to have important role in pH balance and/or ion-/osmo-regulation. The translated
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amino acids sequences from translated ORFs were used as BLASTp queries against the NCBI
NR database. The top BLAST hits were downloaded for each gene and aligned in BioEdit
(version 7.2.5; Hall, 1999) using a ClustalW alignment (Larkin et al., 2007). Neighbour Joining
trees based on amino acid data were generated in Geneious (version 8.0.4)) using the Jukes
Cantor method with 10000 bootstraps (Kearse et al., 2012). Important functional residues
and PFAM domains in the predicted amino acid sequences for each candidate gene were
determined using SMART (Schultz et al., 1998), PROSITE (Sigrist et al., 2013) and NCBI’s
Conserved Domain Database (Marchler-Bauer et al., 2015). Putative signal peptides,
functional motifs and potential cleavage sites of signal peptides were predicted using PrediSi
(PrediSI, 2014) and SignalP 4.0 (Petersen et al., 2011). N-linked glycosylation sites were
predicted with N-GlycoSite (Zhang et al., 2004) using the NXS/T model (where N=Aspargine,
S=Serine, T=Theronine and X=any amino acid).
Molecular evolutionary analyses for each of the gene sequences were generated using
MEGA (version 6; Tamura et al., 2013). Estimates of natural selection for each codon
(codon-by-codon) were determined using HyPHy analysis (hypothesis testing using
phylogenies) according to (Sergei and Muse, 2005). In this analysis, estimates of the
numbers of synonymous (s) and nonsynonymous (n) substitutions for each codon, and their
respective potential numbers of synonymous (S) and nonsyonymous (N) sites were also
determined. These estimates were calculated using joint Maximum Likelihood
reconstructions of ancestral states under a Muse-Gaut model (Muse and Gaut, 1994) of
codon substitution and a General Time Reversible model (Nei and Kumar, 2000). The
differences between the nonsynonymous substitutions rate (per site) (dN = n/N) and
synonymous substitutions rate (dS= s/S) were used for detecting the codons that had
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undergone positive selection. The null hypothesis of neutral evolution was rejected at
p<0.05 (Suzuki and Gojobori, 1999; Kosakovsky and Frost, 2005). All positions in the aligned
sequences containing gaps and missing data were eliminated from the final analysis.
Estimates of evolutionary divergence between the sequences were conducted in MEGA6
using the Poisson correction model (Zuckerkandl and Pauling, 1965). The equality of
evolutionary rate between two sequences from two different species was conducted
according to Tajima's relative rate test using amino acid substitutions model (Tajima, 1993).
4.3.5 Tissue-specific expression
Transcriptomes were assembled separately for seven different C. quadricarinatus organs
including gills, hepatopancreas, heart, kidney, liver, nerve and testes. This analysis examined
the presence or absence of 80 important genes that are directly or indirectly involved in
either acid-base balance or osmotic/ionic regulation across the seven different tissues. Two
data sets (from gills and hepatopancreas) were generated in our lab and the remaining five
were sourced from publicly available NGS-sequenced data archived in NCBI SRA data bank
(Small Reads Archive).
4.4 Results
4.4.1 Transcriptome assembly, annotation and gene identification
4.4.1.1 Transcriptomes of gills from Yabby and Marron
RNA libraries yielded more than 83 million (83,984,583) and 100 million (100,712,892) high
quality (Q ≥ 30) 150 bp paired-end reads for C. cainii and C. destructor, respectively.
Assemblies resulted in 147,101 contigs (C. cainii) and 136,622 contigs (C. destructor) ≥ 200
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bp. Contigs with >70% protein sequence similarity were clustered into 18,325 and 18,113
gene families in C. cainii and C. destructor (Table 4.1). Coding sequences (CDS) representing
open reading frames using Transdecoder are given in supplementary data 1 and 2. Average
contig length (747 and 740) and the N50 statistic (1380 and 1326), which is a weighted
median statistic where half of the entire assembly length is contained in contigs equal to or
larger than this value, were similar in both assemblies. Average contig lengths from our
assemblies were longer than that from other assemblies in crustaceans: e.g. 323 bp in
Macrobrachium rosenbergii (Mohd-Shamsudin et al., 2013) and 492 bp in Euphausia
superba (Clark et al., 2011).
Assembled data were used as BLASTx queries against the NR NCBI protein database using
BLAST+ (Version 2.2.29). More than 23,000 contigs for each species received significant
BLASTx hits against the NR database with a stringency of e-5. The percentage BLAST success
attained for the two transcriptomes (17-18 %) were lower compared to other freshwater
crayfish; Procambarus clarkii (36 %; Shen et al., 2014) and C. quadricarinatus (37 %; Ali et al.,
2015b).
4.4.1.2 Transcriptomes of different organs from Redclaw (C. quadricarinatus)
The seven transcriptome libraries, when combined, constituted a data set of 608 million
high quality (Q ≥ 30) reads. RNA libraries for gills yielded more than 72 million (72,382,710),
83 million (83,984,583) and 100 million (100,712,892) high quality reads for C.
quadricarinatus, C. cainii and C. destructor, respectively. The hepatopancreas transcriptome
from Redclaw yielded ≈65 million reads and 67401 transcripts (Table 4.1). The detailed
statistics of raw reads, assembled contigs and BLAST success are presented in Table 4.1. Our
genes of interest were identified and characterised for the three species and full length
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cDNA of these genes were submitted to NCBI gene-bank database with the accession
numbers (Table 4.2). Most transcripts of interest from the three Cherax species were highly
conserved at the nucleotide level and had predicted proteins of similar size (Table 4.2).
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Table 4.1 Summary statistics for assembled contigs from different organs of Cherax quadricarinatus, C.
destructor and C. cainii
Summary statistics for assembled contigs greater than 200 bp generated from different organs of Cherax quadricarinatus (Redclaw), C. destructor (Yabby) and C. cainii (Marron) using Trinity de novo assembler and cd-HIT clustering tool.
Summary statistics Redclaw Yabby Marron
Gills Hepato- pancreas
Heart Kidney Liver Nerve Testes
Gills Gills
Total reads (million) 72.3 65 47 63 51 64 62 100 83.9
Number of total contigs 87290 67401 38938 66308 47166 66564 63924 136 622 147 101
N50 725 398 720 1033 705 1190 995 1326 1380
Mean contig length 563 386 554 663 548 716 653 740 747
Length of the longest contig 15028 9532 16343 15021 17690 20121 16889 17725 22 324
Number of contigs longer than 500 bp
27973 12055 10129 22622 13665 23290 19642 49678 52 459
Number of contigs longer than 1500 bp
5274 0 2048 6264 2867 6670 5050 16 178 17 486
Number of clusters 24123 21732 23127 19816 22963 22837 22989 18 113 18 325
Blast Success rate (%) 25.3 35 31.2 25.9 27.7 27.1 27.4 17 18
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Table 4.2 Full length coding sequences of the candidate genes involved in pH balance and osmotic/ionic regulation amplified from the gill transcriptomes of C. quadricarinatus, C. destructor and C. cainii
Redclaw Yabby Marron
Genes
Enzymes
GeneID Contig length
Bp
Protein length
(aa)
Accession #
GeneID
Cont
ig le
ngth
Bp
Protein length
(aa)
Accession #
GeneID Contig length
Bp
Protein length
(aa)
Accession #
CAc Carbonic anhydrase alpha CqCAc 1527 271 KM538165
CdCAc 1589
271
KP299962
CcCAc 2795 271 KP221715
CAg Carbonic anhydrase GPI-linked CqCAg 3352 310 KM538166
CdCAg 2465
289 KP299963
CcCAg 4774 310 KP221716
CAb Beta carbonic anhydrase CqCAb 927 257 - CdCAb 2459 257 KP299965
CcCAb 4163 257 KP221717
NKA-α Sodium/potassium ATPase alpha subunit
CqNKA-α 4351 1038 - CdNKA-α 5500
1038 KP299966
CcNKA-α 3888 1038 KP221718
NKA-β Sodium/potassium ATPase beta subunit
CqNKA-β 1662 316 - CdNKA-β 1601
316 KP299967
CcNKA-β 1551 316 KP221719
HAT-c V-type H+-ATPase Catalytic subunit A
CqHATc 2626 622 - CdHATc 2760
622 KP299968
CcHATc 2678 622 KP221720
HAT V-type H+-ATPase 116 kda subunit A
CqHAT 3184 831 - CdHAT 2810 838 KP299969
CcHAT 2908 838 KP221721
AK Arginine kinase CqAK 1432 357 - CdAK 1477
357 KP299970
CcAK 1421 357 KP221722
CRT Calreticulin CqCRT 1722 402 KM538170 CdCRT 1708 403 KP299971 CcCRT 1739 402 KP221723
SERCA Sarco/endoplasmic reticulum Ca+2 ATPase
CqSERCA 4631 1020 KM538171
CdSERCA 4344
1002
KP299972 CcSERCA 6255 1020 KP221724
SEPHS Selenophosphate synthetase CqSEPHS 2142 326 - CdSEPHS 2160
326
KP299975 CcSEPHS 2186 326 KP221726
NKCC Sodium/chloride cotransporter CqNKCC 4643 1061 - CdNKCC 4524
909
KP299986 CcNKCC 4728 1074 KP221733
NHE3 Sodium/hydrogen exchanger 3 CqNHE3 3578 943 CdNHE3 3308
943 KP299982 CcNHE3 4271 986 KP221730
NBC Sodium/bicarbonate cotransporter isoform 4
CqNBCi4 5217 1133 - CdNBCi4 5023
1134 KP299979 CcNBCi4 4986 1133 KP299993
NBC Sodium/bicarbonate cotransporter isoform 5
CqNBCi5 4855 1114 - CdNBCi5 5188
1115 KP299980 CcNBCi5 4915 1114 KP299994
NCX1 Sodium/calcium exchanger 1 CqNCX1 4895 846 - CdNCX1 4148 849 KP299983 CcNCX1 4289 848 KP221731
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4.4.2 Sequences alignment, domain analysis and phylogenetic relationships
Multiple protein alignments were performed for CA, NKA and HAT, as these genes are
considered the most important genes responsible for pH balance and osmoregulation in
crustaceans.
4.4.2.1 Carbonic anhydrase
Cytoplasmic CA from C. quadricarinatus, C. destructor and C. cainii (CqCAc, CdCAc and
CcCAc) encoded a predicted protein of 271 aa (Table 6.2). None of the sequences contained
a signal peptide; while all predicted proteins had two putative N-glycosylation motifs: (NKS
at 122 aa. and NGS at 187 aa. position). BLASTp analysis (homologous protein analysis)
showed that the cytoplasmic CAs from C. quadricarinatus, C. destructor and C. cainii were
highly conserved to other decapod crustaceans and had greatest similarity with cytoplasmic
CA forms from Penaeus monodon (75-76% identity, EF672697), Litopenaeus vannamei (74-
76% identity, HM991703), and Callinectes sapidus (72-74% identity, EF375490). Protein
analysis revealed that cytoplasmic CAs obtained from C. quadricarinatus, C. destructor and
C. cainii had greatest similarity to one another and shared 97-98% identity at the amino acid
level (Appendix 9).
Glycosyl-phosphatidylinositol (GPI)-linked CA forms; CqCAg and CcCAg contained a
predicted protein with 310 aa (CdCAg had a partial CD of 289 aa). The sequence analysis
showed that all the CAg (CqCAg, CdCAg and CcCAg) contained a N-terminal signal peptide of
22 amino acids (Met1 to Gly22). The membrane-associated CA (CAg) obtained from all Cherax
species shared strong similarity with the CAg forms identified in other crustacean species
(for example: 73-74% identity with C. sapidus, EF375491; 71-73% identity with L. vannamei,
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JX975725; 72-73% identity with P. trituberculatus, JX524150); and the similarity among the
three Cherax species ranged between 97-99% protein identity ( Appendix 9).
The β-CA had moderate to high sequence similarity to β-CA proteins obtained from other
arthropod species (for example; 64-65% identity with Acyrthosiphon pisum, XP_001943693;
61-63% identity with D. pulex, EFX79480 (Colbourne et al., 2011) and 62-64% identity with
Nasonia vitripennis, XP_001606972). Cherax quadricarinatus, C. destructor and C. cainii
shared between 98-99% amino acid sequence identity with each other (Appendix 9).
Multiple alignment of all three forms of CAs with the homologous proteins in arthropods
revealed that all active sites, including the zinc binding sites and the hydrogen bonding sites
around the active sites are conserved in all CA proteins (Fig. 4.1). A Neighbour Joining
phylogenetic tree showed that the β-CA proteins formed a separate and well resolved clade
from the alpha CA proteins (CAc and CAg). The three Cherax species formed a separate
group in the β-CA clade that was sister to the same protein from insects. Within the large
alpha CA clade, both the CAg and CAc proteins formed distinct well resolved clades. For the
CAg clade, the three Cherax species formed a monophyletic group that was sister to a group
comprised of crabs Carcinus maenas (ABX71209, Serrano and Henry, 2008), Callinectes
sapidus (ABN51214, Serrano et al., 2007) and Portunus trituberculatus (AFV46145, Xu and
Liu, 2011); with shrimps being more distantly related Litopenaeus vannamei (AGC70493, Liu
et al., 2015) and Halocaridina rubra (AIM43573, Havird et al., 2014). The CAc clade clustered
according to taxonomic affinity where vertebrate species formed a separate group from the
arthropod species. Within the arthropod clade, insects species Drosophila melanogaster
(NP_523561, Hoskins et al., 2007), Apis florea (XM_003696244) and Bombus terrestris
(XP_003395501)) formed a separate and well supported group from decapod crustacean
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species including Callinectes sapidus, (ABN51213, Serrano et al., 2007) and Portunus
trituberculatus (AFV46144, Xu and Liu, 2011), and shrimp species Halocaridina rubra
(AIM43574, Havird et al., 2014), Litopenaeus vannamei (ADM16544, Liu et al., 2015) ,
Penaeus monodon (ABV65904, Pongsomboon et al., 2009) (Fig 4.2). The three Cherax
species clustered together in a discrete group which was sister to the other decapod
species.
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Figure 4.1 Multiple alignments of amino acid sequences from three Cherax Carbonic anhydrase (CA) with other species.
B. CAg
C. β-CA
A. CAc
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Multiple alignments of amino acid sequences from three Cherax Carbonic anhydrase (CA) with other species. Colours indicate the similarity. The black-boxed indicate the predicted active-sites, Zinc-binding residues. A. Multiple alignment of cytoplasmic CA B. Multiple alignment of membrane associated CA, and C. Multiple alignment of β-CA
A. CA
B. NKA
C. HAT-A
Figure 4.2 Phylogenetic relationships between the three Cherax crayfish with other organisms Bootstrapped Neighbour-joining tree showing the phylogenetic relationships between the three Cherax
crayfish with other organisms on the basis of the amino acid sequences. Accession numbers are placed in brackets.
A. Phylogenetic tree of Carbonic Anhydrase proteins (CA) B. Phylogenetic tree of Na+/K+-ATPase α-subunit (NKA) C. Phylogenetic tree of V-type-H+-ATPase 116 kDa subunit a (HAT-A)
Cherax quadricarinatus CAc (AIW68600) Cherax cainii CAc (AJO70180) Cherax destructor CAc (AJO69996)
Crayfish
Halocaridina rubra CAc (KF650062) Callinectes sapidus CAc (EF375490) Portunus trituberculatus CAc (JX524149)
Crabs
Litopenaeus vannamei CAc(HM991703) Penaeus monodon CAc (EF672697)
Shrimps
Drosophila melanogaster CAc (NM 078837) Apis florea CAc (XM 003696244) Bombus terrestris CAc (XM 003395453)
Insecta
Danio rerio CAc (NP 571185) Cyprinus carpio CAc (AAZ83743) Oncorhynchus mykiss CAc (NP 001117693) Salmo salar CAc (NP 001133769)
Fish
Homo sapiens CAc (NP 001729) Pan troglodytes CAc (NP 001182062) Rattus norvegicus CAc (NP 001101130) Mus musculus CAc (NP 033929)
Mammals
CAc
Litopenaeus vannamei CAg (JX975725) Halocaridina rubra CAg (KF650061)
Shrimps
Cherax quadricarinatus CAg (AIW68601) Cherax cainii CAg (AJO70181) Cherax destructor CAg (AJO69997)
Crayfish
Carcinus maenas CAg (EU273944) Callinectes sapidus CAg (EF375491) Portunus trituberculatus CAg (JX524150)
Crabs
CAg
Cherax quadricarinatus CAb (AIW68602) Cherax cainii CAb (AJO70182) Cherax destructor CAb (AJO69999)
Crayfish
Drosophila melanogaster CAb (NM 141592) Bombyx mori CAb (XM 004922942) Apis dorsata CAb (XM 006612942) Megachile rotundata CAb (XM 012283184) Bombus terrestris CAb (XM 003402502)
Insecta
CA-beta
Cherax quadricarinatus (KM538168) Cherax cainii (KP221718) Cherax destructor (KP299966)
Crayfish
Lobster Homarus americanus (AAN17736) Pachygrapsus marmoratus (ABA02166) Callinectes sapidus (AAG47843) Portunus trituberculatus (JX173959)
Crabs
Halocaridina rubra (KF650059) Exopalaemon carinicaudagi (JQ360623) Litopenaeus stylirostris (JN561324) Penaeus monodon (ABD59803) Fenneropenaeus indicus (ADN83843)
Shrimps
Bactrocera dorsalis (XM 011214827) P. humanuscorporis (XP 002427714) Locusta migratoria (KF813097) Leptocoris trivittatus (JQ771499)
Insects
Arachnida I. scapularis (XP 002404061) H. sapiens (NP 001243143) B. taurus (XP 002695120)
Mammals
S. salar (ACN10460) O. mykiss (NP 001117930) F. heteroclitus (AAL18003)
Fish
Wasmannia auropunctata (XM 011701129)
Pogonomyrmex barbatus (XM 011643683)
Camponotus floridanus (XM 011263300)
Cerapachys biroi (XM 011350646)
Megachile rotundata (XM 012284907)
Bombus terrestris (XM 003397650)
Bombus impatiens (XM 003486192)
Nasonia vitripennis (XM 001607768)
Insecta
Cherax quadricarinatus
Cherax destructor (KP299969)
Cherax cainii (KP221721)
Crayfish
Shrimp Litopenaeus vannamei (HM163157)
Arthropoda
Esox lucius (XP 010899534)
Takifugu rubripes (XP 003978025)
Danio rerio (XP 005163855)
Fish (Osteichthyes
Homo sapiens (NP 001123492)
Bos taurus (NP 777179)
Mus musculus (NP 058616)
Rattus norvegicus(NP 113792)
Mammals
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4.4.2.2 Na+/K+-ATPase (NKA)
Full-length nucleotide sequences and predicted aa sequences of the Na+/K+-ATPase alpha
subunit from C. quadricarinatus, C. destructor and C. cainii (CqNKA, CdNKA and CcNKA) were
functionally characterised (Table 4.2). The CqNKA, CdNKA and CcNKA ORFs all produced a
predicted protein of 1038 aa in length. No signal-peptide was present, but eight
transmembrane domains were detected through hydrophobicity analysis of the sequences
(Fig. 4.3).
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Figure 4.3 Multiple alignment of three Cherax Na+/K+-ATPase α-subunit amino acid sequences with other crustaceans Multiple alignment of three Cherax Na+/K+-ATPase α-subunit amino acid sequences with other
crustaceans; Cherax quadricarinatus (), C. destructor (), C. cainii (), Homarus americanus (AAN17736), Callinectes sapidus (AAG47843), Paeneus monodon (ABD59803) and Portunus trituberculatus (AGF90965). Putative transmembrane domains are marked by black-boxed; likely ATP-binding site, by red-boxed ; and phosphorylation site, by dashed-black boxed
Page 100
The NKA proteins from Cherax species were highly conserved (97-99% aa identity) and
showed greatest similarity to NKA from other decapod crustacean species (for example;
96% with Homarus americanus (AY140650); 95% with Eriocheir sinensis (KC691291); 95%
with C. sapidus (AF327439) and 93% with P. monodon (DQ399797)). Genetic similarity
among all pairs of sequences is presented in Appendix 9.
Multiple alignment of NKA with the homologous proteins of other crustaceans revealed that
all active sites are highly conserved (Fig. 4.3). The phylogenetic tree based on the α-subunit
of NKA showed the existence of three distinct groups; arthropods, mammals, and
osteichthyes (Fig 4.2). The arthropods further clustered into three clades; crustaceans,
insects and arachnids. All the NKA α-subunit sequence obtained from C. quadricarinatus, C.
destructor and C. cainii formed a distinct monophyletic group that was sister to a Homarus
americanus sequence (AAN17736). Crayfish sequences were more closely related to those
of the crab species Callinectes sapidus (AAG47843), Portunus trituberculatus (JX173959) and
Pachygrapsus marmoratus (ABA02166) than to shrimp species Penaeus monodon
(ABD59803), Fenneropenaeus indicus (ADN83843), Halocaridina rubra (KF650059),
Litopenaeus stylirostris (JN561324) and Exopalaemon carinicaudagi (JQ360623) (Fig. 4.2).
4.4.2.3 V-type H+ATPase (HAT)
In the Cherax datasets, multiple H+-ATPase (HAT) subunits were identified (Table 4.2),
however, only HAT 116kDa subunit A was used for phylogenetic analyses. Subunit A was
chosen as it is the main catalytic subunit in proton translocation. Translation of ORFS
revealed that the predicted proteins from HAT 116kda subunit A transcripts were similar in
length across the three Cherax species (831 aa, Table 4.2).
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Conserved domains were identified in HAT subunit A including V_ATPase_I domain between
26–818 aa (protein family: pfam01496). Another predicted feature of the HAT-A predicted
protein was seven putative transmembrane regions at positions: 398–420, 441–461, 532–
550, 567–587, 640–660, 733–751 and 765–785aa.
The HAT-A predicted protein was highly conserved in C. quadricarinatus, C. destructor and C.
cainii (97-98% aa identity), as well as across arthropods in general (Fig 4.4). The HAT-A from
Cherax species had the greatest similarity with the HAT-116kDA from other arthropods (for
examples; 63-64% with L. vannamei (AEE25939); 71-72% with B. terrestris (XP_003397698);
70-71% with M. rotundata (XP_012140297); and 70-71% with C. floridanus
(XP_011261602)).
The phylogenetic tree based on the translated amino acid sequences revealed that V-type-
H+-ATPase subunit-A formed three distinct groups; arthropods, fish and mammals.
Arthropods further clustered into two clades; decapod crustaceans and insects. All crayfish
species, C. quadricarinatus, C. destructor and C. cainii formed a monophyletic group that
was sister to a group comprised of insects; and were distantly related to the shrimp L.
vannamei, (AEE25939, Wang et al., 2012)). The arthropod clade was distinct from a second
well resolved clade of vertebrates (Fig 4.2c).
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Figure 4.4 Multiple alignment of three Cherax V-type-H+-ATPase subunit-a amino acid sequences with other species.
Multiple alignment of three Cherax V-type-H+-ATPase 116kDa subunit-a amino acid sequences with other closely related species. Putative transmembrane domains are marked by black-boxed
Page 103
4.4.2.4 Other genes
Comparative sequence analyses showed that all genes including Na+/K+/2Cl− cotransporter
(NKCC), Na+/Cl-/HCO3- cotransporter (NBC), Na+/H+ exchanger 3 (NHE3), Na+/Ca+2 exchanger
1 (NCX1), Arginine kinase (AK), Sarcoplasmic Ca+2-ATase (SERCA) and Calreticulin (CRT) had
the putative proteins (the deduced amino acid composition) similar in length and weight
across the three species (Table 4.2). All predicted proteins shared about 97-99% amino acid
identity with the corresponding genes across the Cherax species (Appendix 9) and all the
functional sites were conserved in the predicted proteins (Table 4.2 and Appendix 9).
4.4.3 Comparative molecular evolutionary analyses
4.4.3.1 Evolutionary analyses of ion transport genes in Cherax sp.
The molecular evolutionary analyses at amino acid/codon level between C. quadricarinatus,
C. destructor and C. cainii showed that all candidate genes had a number of synonymous
substitutions (Table 4.3). In terms of protein changing mutations, CAc, CAg and β-CA
contained 9, 4 and 5 nonsynonymous sites, respectively (Table 4.3). The other candidate
genes examined in detail, NKA, HAT-A, NKCC, NBC, NHE3, NCX1, AK, SERCA, CRT had 3, 12,
37, 24, 91, 15, 10, 23 and 7 nonsynonymous sites respectively (Table 4.3). Relative
evolutionary analysis showed that C. quadricarinatus, C. destructor and C. cainii have had an
equal evolutionary rate for all of the candidate genes (the null hypothesis of unequal
evolutionary rate were rejected at p<0.05, (Table 4.3)).
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Table 4.3 Molecular evolutionary analyses between three Cherax crayfish (C. quadricarinatus, C. destructor and C. cainii) at amino acid/codon level
CAc CAg CAb NKA HAT-116k NKCC NBC NHE3 NCX1 AK SERCA CRT Total sites/positions in analysis 271 277 257 1038 831 848 1145 921 846 357 1002 402 Identical sites in all three species 262 273 252 1035 819 811 1121 830 831 347 979 395 Nonsynonymous sites 9 4 5 3 12 37 24 91 15 10 23 7 Synonymous substitutions at codon 54 57 18 71 85 506 107 143 91 26 55 39 Divergent sites in all three species 0 0 0 0 0 1 1 9 1 1 1 0 Unique differences in Redclaw 3 0 0 1 6 11 7 29 7 1 6 1 Unique differences in Yabby 3 2 3 2 1 14 7 20 2 3 9 3 Unique differences in Marron 3 2 2 0 5 11 9 33 5 5 7 3 Equal evolutionary rate between Redclaw & Yabby?
Yes (1.00)*
Yes (0.16)
Yes (0.08)
Yes (0.56)
Yes (0.06)
Yes (0.55)
Yes (0.8)
Yes (0.20)
Yes (0.10)
Yes (0.32)
Yes (0.44)
Yes (0.32)
Equal evolutionary rate between Redclaw &Marron?
Yes (1.0)
Yes (0.16)
Yes (0.16)
Yes (0.32)
Yes (0.76)
Yes (1)
Yes (0.8)
Yes (0.61)
Yes (0.56)
Yes (0.10)
Yes (0.78)
Yes (0.32)
Equal evolutionary rate between Yabby & Marron?
Yes (1.0)
Yes (1.00)
Yes (0.65)
Yes (0.16)
Yes (0.10)
Yes (0.55)
Yes (0.6)
Yes (0.07)
Yes (0.26)
Yes (0.48)
Yes (0.62)
Yes (1.0)
Average Evolutionary Divergence 0.022 0.010 0.013 0.002 0.010 0.03 0.05 0.072 0.012 0.02 0.016 0.012
Note: CAc=Cytoplasmic carbonic anhydrase, CAg=GPI-linked carbonic anhydrase, CAb=Beta carbonic anhydrase, NKA=Na+/K+-ATPase alpha subunit, HAT-116k=Vacuolar type H+-ATPase 116 kda, NKCC = Na+/K+/2Cl- cotransporter, NBC=Na+/HCO3
- cotransporter, NHE3 =Na+/H+ exchanger 3, NCX1 =Na+/Ca+2 exchanger 1 , AK=Arginine kinase, SERCA=Sarco/endoplasmic reticulum Ca+2-ATPase, CRT=Calreticulin, * Values in brackets are p-values used to reject the null hypothesis of equal evolutionary rate
Page 105
4.4.3.2 Evolutionary analyses across crustacean species
Comparative analyses across arthropods, which included mostly crustaceans (around 15
species including three Cherax sp.), revealed a large number of non-synonymous mutations
in all the sequences (173 in CAc, 128 in CAg, 143 in CAb, 243 in NKA, 355 in HAT-116k, 586 in
NKCC, 506 in NBC, 504 in NHE3, 529 in NCX1, 78 in AK, 276 in SERCA and 193 in CRT) (see
Table 4.4). Despite a large number of nonsynonymous mutations, HyPHy analysis (codon-by-
codon natural selection estimations for each codon) detected that no candidate genes
showed patterns of nucleotide variation consistent with the action of natural selection
(p<0.05).
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Table 4.4 Molecular evolutionary analyses across crustacean species at Amino acid level
Enzymes Gene No. of species/ Sequences in
analysis
Total sites in analysis
Total segregating
sites
Total Identical
sites
Rate of segregation/site
Nucleotide diversity
In-Del
Cytoplasmic carbonic anhydrase CAc 15 265 173 92 0.65283 0.38225 0
GPI-linked carbonic anhydrase CAg 8 287 128 159 0.445993 0.24029 0
Beta carbonic anhydrase CAb 16 254 143 111 0.562992 0.2691 0
Na+/K+-ATPase alpha subunit NKA 16 1001 243 758 0.242757 0.08577 0
Vacuolar type H+-ATPase 116 kda HAT-116k 15 803 355 448 0.442092 0.19255 0
Na+/K+/2Cl- cotransporter NKCC 16 838 586 252 0.699284 0.39172 0
Na+/HCO3- cotransporter NBC 16 1033 506 527 0.489835 0.23662 0
Na+/H+ exchanger 3 NHE3 15 843 504 339 0.597865 0.25904 0
Na+/Ca+2 exchanger 1 NCX1 15 793 529 264 0.667087 0.28318 0
Arginine kinase AK 15 355 78 277 0.219718 0.07329 0
Sarco/endoplasmic reticulum Ca+2-ATPase SERCA 15 989 276 713 0.27907 0.12366 0
Calreticulin CRT 15 396 193 203 0.487374 0.212 0
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4.4.4 Tissue-specific expression analyses
A total of 80 important genes that are directly or indirectly associated with either acid-base
balance or osmotic/ionic regulation expression was investigated in seven different tissue
types (gills, hepatopancreas, heart, kidney, liver, nerve and testes) in C. quadricarinatus
(Appendix 10). The results showed that 46% of the genes (37) were expressed in all types of
tissues; 11% (9 genes) in 6 tissue types, 5% (4 genes) in 5 tissue types, 8% (6 genes) in 4
tissue types; 6% (5 genes) in 3 tissue types; 14% (11 genes) in 2 tissue types (Fig 4.5 ). About
10% of the genes (8) were expressed in only one tissue type, i.e. they were unique in that
particular type of tissue. The highest number of genes were observed in the nerve (84%, 67
genes) followed by the gills (78%, 62 genes). The percentage of genes expressed in other
tissues are 70% for heart (56 genes), 70% for kidney (56 genes), 69% for liver (55 genes),
67.5% for hepatopancreas (54 genes), and 66% for testes (53 genes) (Fig 4.5).
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Figure 4.5 Tissue-specific expression/occurrences of important genes associated with pH balance or ion-regulation in Redclaw crayfish (Cherax quadricarinatus)
A. Tissue-specific percentage of genes expressed B. Maximum types of organs with % of genes expressed therein
4.5 Discussion
Overall this study has demonstrated that most of the candidate genes involved in systemic
acid-base balance and/or ion transport examined in this study are highly conserved in the
genus Cherax and in crustaceans in general. While these genes showed little variation across
the three target species, we found no evidence of purifying selection shaping nucleotide
variation in any of the genes. The majority of these genes were expressed across multiple
tissue types indicating they play an important and potentially multiple roles in a number of
different tissues.
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4.5.1 Comparative molecular analyses
4.5.1.1 Carbonic anhydrase
Comparative analysis across the three Cherax species showed that both cytoplasmic and
membrane linked CA were highly conserved, which may be explained by the conserved
function of CA. Carbonic anhydrase is a Zn-metalloenzyme that plays a pivotal role in pH
balance, ion transport and gas exchange in all metazoan species (Freire et al., 2008; Henry et
al., 2012; Romano and Zeng, 2012). The molecular structure of CA comprises four primary
components: the Zn binding sites, the substrate association pocket, the threonine-199 loop
and the proton shuttling mechanism (Christianson and Alexander, 1989; Merz, 1990; Krebs
et al., 1993). In our study, we did not find any mutations in these important functional sites
at the amino acid level in both cytoplasmic and membrane-associated isoforms of CA across
the Cherax species. We did, however, find amino acid replacements outside of these
functional sites and these may be important for adaptation to different pH environments in
Cherax species.
Previous research has shown that many Cherax species, including C. quadricarinatus, C.
destructor and C. cainii, naturally occur in locations with water of different pH levels
(Macaranas, 1995; Bryant and Papas, 2007; Baker et al., 2008). Therefore, amino acid
replacements in cytoplasmic and membrane linked CA may be associated with the
differences in water chemistry experienced by different species. Adaptation to acidic water
chemistries has been demonstrated in the freshwater fish, Tribolodon hakonensis, which
contain high concentrations of CA in the chloride cells of its gill (Hirata et al., 2003). In fact,
Hirata et al. (2003) used functional and physiological studies to show that CA protein levels
were increased under low pH conditions in this species and contributed to its ability to
Page 110
survive in this environment. While we lack functional data, the changes in amino acids we
observed in the Cherax species may alter gene expression in the different species and play
an important role in coping with different pH environments. Conversely, these amino acid
replacements may be beneficial to protein function under different water pH environments
or they may also have no effect on protein function and be effectively neutral. Before any of
these hypotheses can be supported more genomic, functional and physiological
experiments will need to be undertaken to validate our findings here.
4.5.1.2 Na+/K+-ATPase
All the expected features of NKA were highly conserved in the three Cherax species,
including eight transmembrane domains (Mitsunaga-Nakatsubo et al., 1996), the putative
ATP-binding site (Horisberger et al., 1991) and a phosphorylation site. In fact, only two
conservative amino acid replacements were observed among the three Cherax species, an
alanine to glycine replacement in C. destructor and an alanine to threonine replacement in
C. quadricarinatus. Given that these Cherax species are naturally distributed in
environments with diverse salinity and pH ranges (Bryant and Papas, 2007), you might
expect to find more adaptive amino acid replacements in this gene. The lack of variation at
the amino acid level may indicate other mechanisms are more important than amino acid
differences for the response to salinity and pH changes in Cherax species. Changes in the
level of expression of NKA associated with salinity or pH changes may be a plausible
hypothesis for this lack of amino acid variation among Cherax species. A number of studies
have demonstrated that NKA expression is strongly induced by changes in salinity and/or pH
in the gills of decapod crustaceans lending support to this hypothesis (Luquet et al., 2005;
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Serrano and Henry, 2008; Wang et al., 2012; Han et al., 2015; Leone et al., 2015; Li et al.,
2015). This hypothesis needs to be further tested, however, before it can be supported.
4.5.1.3 H+-ATPase
Inter-species evolutionary analyses between the three Cherax crayfish and other arthropod
species showed that V-H+-ATPase subunit A (HAT-A) was largely conserved at the protein
sequence level, but had patterns of nucleotide variation consistent with neutral evolution.
The small amount of amino acid variation found at this enzyme in Cherax species is probably
because of its conserved function, as it is one of key enzymes via which many crustaceans
maintain their internal acid-base balance (Kitagawa et al., 2008; Faleiros et al., 2010; Lee et
al., 2011; Muench et al., 2011; Towle et al., 2011; Wang et al., 2012; Boudour-Boucheker et
al., 2014; Marshansky et al., 2014; Lucena et al., 2015; Rawson et al., 2015). As the
maintenance of acid-base balance is very important in aquatic crustaceans, amino acid
replacements in important functional domains might have a deleterious effect on the
function and activity of this enzyme. Alternatively, amino acid replacements in V-H+-ATPase
subunit A that increase its activity under different pH conditions may be beneficial to the
different Cherax species. A pattern of adaptive amino acid replacement has been observed
in fish gills under low pH conditions in a Japanese lake (Hirata et al., 2003).
4.5.1.4 Other genes
The comparative molecular analyses showed that most other genes including Na+/K+/2Cl-
cotransporter (NKCC), Na+/Cl-/HCO3- cotransporter (NBC), Na+/H+ exchanger 3 (NHE3),
Na+/Ca+2 exchanger 1 (NCX1), Arginine kinase (AK), Sarcoplasmic Ca+2-ATase (SERCA) and
Calreticulin (CRT) were conserved across the three Cherax crayfish (Table 4.3 and 4.4). The
underlying reason may be that the these genes encode a set of enzymes that are actively or
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passively associated with the fundamental and common physiological processes of ion-
regulation in crustaceans (Lucu, 1989; Onken et al., 1991; Onken et al., 2003; Ahearn et al.,
2004; Serrano et al., 2007; Mandal et al., 2009; Lv et al., 2015; Ren et al., 2015; Xu et al.,
2015). The high level of amino acid conservation in these predicted proteins may be
attributed to the fact that they are found in important biochemical pathways that influence
systemic acid-base balance and/or ion transport (Uda et al., 2006; Hwang et al., 2011; Hiroi
and McCormick, 2012; McNamara and Faria, 2012b). These genes have previously been
demonstrated as important enzymes involved in osmoregulatory organs such as gills and
epipodites in a number of crustaceans (Towle and Weihrauch, 2001; Weihrauch et al.,
2004a; Freire et al., 2008; Mandal et al., 2009). In fact, gene expression data for Na+/K+/2Cl-
cotransporter (Luquet et al., 2005; Havird et al., 2013) and Calreticulin (Luana et al., 2007; Lv
et al., 2015; Xu et al., 2015) show that they are induced under different ionic conditions and
arginine kinase has also been shown to be induced under different pH and salinity
conditions (Serrano et al., 2007; Xie et al., 2014; Ali et al., 2015b).
4.5.2 Tissue-specific expression analyses
Tissue-specific expression of the candidate genes involved in systemic acid-base balance
revealed that most of the major genes involved in this process including carbonic anhydrase
(CA), Na+/K+-ATPase (NKA), Vacuolar type H+-ATPase (HAT), Na+/H+ exchanger (NHE),
Na+/K+/2Cl- cotransporter (NKCC), Na+/Cl-/HCO3- cotransporter, Arginine kinase (AK) and
Sarco/endoplasmic reticulum Ca+2-ATPase (SERCA) were expressed in most tissue types. The
two tissue types with highest number of candidate genes expressed were the gills and
nervous system. A large number of genes involved in acid-base balance and osmic-/ionic-
regulation expressed in the gills was expected based on previous studies in decapod
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crustaceans (Freire et al., 2008; Romano and Zeng, 2012). The largest number of transport
related genes were expressed in nervous tissue and studies on the expression of these
genes in crustacean nervous system is still lacking. Data from vertebrate species, however,
has shown that many ion transport related genes are highly expressed in nervous tissue as
they play an important role in generating action potential (Fry and Jabr, 2010; Benarroch,
2011; Deval and Lingueglia, 2015; Hertz et al., 2015; Shrivastava et al., 2015; Wu et al., 2015;
Zhang et al., 2015). It is evident that dynamic voltage changes like action potential (nerve
impulses) are mainly caused by changes in membrane permeability for Na+, K+, Ca+2, and Cl-
ions, which in turn result from changes in functional activity of various ion transporters, ion
channels and ion exchangers (Barnett and Larkman, 2007; Fry and Jabr, 2010). For example,
Na+/K+-ATPase maintains the Na+ and K+ potential inside and outside the cell membrane
that are of fundamental importance for the maintenance of neural excitability and
conduction of action potential and secondary transport mechanisms involved in synaptic
uptake of neurotransmitters and regulation of cell volume, pH and Ca+2 concentration
(Benarroch, 2011). Action potentials in nervous tissues are triggered by specific voltage-
gated ion channels fitted in plasma membrane of a cell. When an action potential travels
towards the axon of a neuron, it results in changes in polarity across the membrane. Na+
and K+ gated ion channels open and close accordingly, in response to a signal from another
neuron. At the beginning of the process, the Na+ channels open and Na+ ions move down
the axon, causing depolarization. Afterwards, K+ channels open causing repolarization and K+
ions move out of the axon. Thus, Na+/K+-ATPase, Na+ and K+ channels plus other transport
enzymes play vital role in nervous system; and the corresponding genes encoding theses
enzymes are highly expressed in mammalian nerve cells. Similar patterns of gene expression
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in the nervous tissue of crustaceans are highly likely, but require further study and
functional validation to support this idea.
4.6 Conclusions
We analysed the molecular differences and similarities at amino acid and nucleotide levels
in most of the major genes involved in acid-base balance and osmotic/ionic regulation in
three freshwater Cherax crayfish. The majority of these genes were expressed across most
tissue types and were highly conserved at the amino-acid level. These findings indicate that
these genes are important and probably have diverse functions related to ion exchange and
pH balance across different tissues in freshwater crayfish.
4.7 Nucleotide sequence accession number
All raw sequence data was deposited in the Sequence Read Archive under the Bio-project
numbers PRJNA275170, PRJNA275038 and PRJNA275165.
4.8 Supplementary data
Supplementary data 1 (Available online: http://dx.doi.org/10.1016/j.margen.2015.03.004)
Supplementary data 2 (Available online: http://dx.doi.org/10.1016/j.margen.2015.03.004)
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General Discussion and Conclusion Chapter 5.
In the study we used Cherax crayfish as a model organism for genetic research considering
their physiological and economic importance. These species make representative species for
other freshwater crayfish as well as other freshwater crustaceans. Crustacean aquaculture
has been growing rapidly because of its high demand, availability of commercially viable
species and the wide range of culture environments they can be produced under (Saoud et
al., 2013). Significantly, over the last three decades, crustacean culture has expanded at a
faster rate than either finfish or mollusc aquaculture (FAO, 2011; FAO, 2012; Saoud et al.,
2012; Saoud et al., 2013). Amongst the presently available farmed freshwater crustacean
species, crayfish are the most highly produced (FAO, 2012). Cherax quadricarinatus
(Redclaw), Cherax destructor (Yabby) and Cherax cainii (Marron) are economically important
freshwater crayfish, and the culture of these species have been developed in many
countries of the world (Macaranas, 1995; FAO, 2010; Saoud et al., 2012; Saoud et al., 2013).
Thus the selection of the Cherax species as a model organism for freshwater crustaceans is
well justified and needed. The findings from our study, will potentially contribute to better
growth and production of crayfish, especially by addressing the stress-related genes and
their expression under different pH and salinity conditions. In addition, it contributes
valuable information in the larger scientific community who can undertake genomic
research in these species as a result of the genetic resources now developed specifically for
the genus Cherax.
In Chapter one, we have critically reviewed all relevant research that have been undertaken
in the field of ion transport physiology in crustaceans; and identified the research gaps
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relevant to our study. It can also be used as ready information to assist in finding out
suitable research objectives for further physiological genomics study in freshwater crayfish.
In study one (Chapter 2), we have identified and characterised most of the major pH-
balance and osmoregulatory genes, and shed light on the potential role of select genes in
response to pH changes. The outcomes of these experiments have potential implications in
enhancing crayfish production. Because, in the areas where Cherax species naturally occur
and are commercially cultured, pH and salinity levels fluctuate for many reasons including;
the condition of surrounding soils, duration of daylight, seasonal weather changes, heavy or
low rainfall, flooding, acid precipitation, as well as long term climate change (Haines, 1981;
Boyd, 1990). As crayfish are farmed in a wide range of culture conditions, optimisation of
water quality parameters, particularly pH and salinity, are crucial for their maximum growth
and survival. In fact, fluctuations in pH and salinity have been demonstrated to have a great
impact on the distribution, growth, behaviour and physiology in many crustaceans including
Cherax crayfish (Chen and Chen, 2003; Pavasovic et al., 2004; Pan et al., 2007; Yue et al.,
2009; Haddaway et al., 2013; Kawamura et al., 2015; Kim et al., 2015). For example,
significantly reduced growth performance was observed at sub-lethal pH and salinity ranges
in euryhaline and marine shrimp including Penaeus monodon (Ye et al., 2009), Litopenaeus
vannamei (Pan et al., 2007) and Penaeus latisulcatus (Minh Sang and Fotedar, 2004); and in
the freshwater species Macrobrachium rosenbergii (Chand et al., 2015; Kawamura et al.,
2015) and Austropotamobius pallipes (Haddaway et al., 2013). Reduced growth
performance in these species was suggested to result from higher energy requirements
diverted from growth to allow individuals to maintain pH balance and internal ionic
concentration. Increased energy expenditure on osmoregulation can produce many
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problems in cultured organisms including increased moisture content in muscle tissue,
increased oxygen consumption and higher ammonia-N excretion (Silvia et al., 2004). It is
also evident that environmental pH has a great impact on acid-base balance and electrolyte
concentrations in the haemolymph of freshwater crayfish (Morgan and McMahon, 1982;
Wood and Rogano, 1986; Zanotto and Wheatly, 1993; Wheatly et al., 1996). For example,
water with a low pH level has been shown to cause acid–base imbalance (decreased pH in
haemolymph) and disturbed ion regulation (decreased Na+ and Cl- concentrations and
increased K+) in freshwater crayfish (Morgan and McMahon, 1982; Wood and Rogano, 1986;
Wheatly et al., 1996). Previous research also showed that physiological response of
crustaceans to varied water quality parameters are mainly controled by a conserved set of
physiological processes including osmoregulation and systemic acid-base balance. These
important physiological processes are undertaken by a range of proteins encoded by ion
transport-related genes of which carbonic anhydrase (CA), Na+/K+-ATPase (NKA), V-type-H+-
ATPase (HAT) and Na+/K+/2Cl- cotransporter (NKCC) are among the best studied (Serrano et
al., 2007; Tsai and Lin, 2007; Freire et al., 2008; Pongsomboon et al., 2009; McNamara and
Faria, 2012a; Havird et al., 2013; Tongsaikling, 2013; Havird et al., 2014). In the study, we
have not only sequenced, identified and characterised all of these genes, but also shed light
on the expression and molecular evolution of these and other candidate genes that have
potential roles in pH balance, salinity regulation, or stress-response. To the best of our
knowledge, the molecular characterization and expression studies on these candidate genes
are the first undertaken in freshwater crayfish from the genus Cherax. All the previously
published data are largely limited to either patterns of enzyme activity or population
genetics in wild populations of Cherax species (Miller et al., 2004; Austin et al., 2014; Gan et
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al., 2014). Thus, the data generated here will serve as a foundation for future molecular and
genomic research in freshwater crustaceans, and in particular freshwater crayfish.
In second study (Chapter 3), we have assessed spatial effects of pH changes on expression
patterns of some master genes involved in systemic pH balance and salinity regulation. As
with the first study (Chapter 1) suggested potential role of the genes in pH balance, study at
different time points over a period of time further elucidated their roles in response to pH
challenges. The findings of the study showed that two forms of carbonic anhydrase (CA),
Na+/K+-ATPase (NKA) and V-type-H+-ATPase (HAT) were induced after transfer to different
pH conditions (at pH 6.2 and 8.2 from control pH 7.2) . The cytoplasmic form of carbonic
anhydrase (CqCAc) showed a significant increase in expression levels at low pH (6.2) and
decreased expression levels at high pH condition (8.2). Expression of CAc increased in low
pH after 12 h, and reached their maximum level by 24 h. The GPI-linked membrane-
associated form of carbonic anhydrase (CqCAg) showed changes in expression levels more
quickly than CAc, and the expression remained elevated at low pH (6.2) until the end of the
experiment. The beta form of CA (CA-beta) was very low at all pH levels, so it was excluded
from the analysis. Expression levels of Na+/K+-ATPase (CqNKA) and V-type-H+-ATPase
(CqHAT) showed similar patterns, where expression significantly increased 6 h post transfer
at low pH conditions and remained significantly elevated throughout the experiment (over
24 hrs) (Fig. 3.3 and 3.4). Overall, observed expression patterns of all the genes reported in
our study were similar to those documented for other crustacean species under different pH
or salinity treatments (Pan et al., 2007; Serrano et al., 2007; Tsai and Lin, 2007; Serrano and
Henry, 2008; Pongsomboon et al., 2009; Havird et al., 2013; Han et al., 2015; Leone et al.,
2015; Liu et al., 2015). This indicates that these genes play a very similar role and are
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expressed in a similar way across gills of decapod crustacean species in response to pH
change. The findings of our gene expression study suggest that CAc, CAg, NKA and HAT gene
expression are induced at low pH conditions and are likely to have an important role for
systemic pH balance in freshwater crayfish. These outcomes may have a great practical
application in crayfish farming, in a way that it helps to address stress-related issues due to
environmental pH fluctuations.
Two factors, however, that may have affected our gene expression study are small sample
size and the inter-moult cycle of crustacean species. In this study, we used inter-moult
crayfish in the gene expression study, but could not determine the exact stage of moulting
of the experimental animals. The stage of inter-moult could have affected patterns of gene
expression we observed, as the moulting cycle is known to influence gene expression in
decapod crustaceans. Although we could not control for this, to minimise the potential
influence the inter-moult cycle may have has on gene expression patterns, we applied our
best judgment to pick animals of equal size and age. Given that the results of our study were
consistent with most other studies (Pan et al., 2007; Serrano et al., 2007; Leone et al., 2015;
Liu et al., 2015) on decapod crustaceans, it would seem that the inter-moult cycle has had a
limited impact on our gene expression results. In our study, we also used three individuals at
each sampling point over a period of 24 hours. Small sample size may have obscured some
patterns of gene expression, as we observed some variance in gene expression between
technical replicates. In addition, it seemed from our results that a longer term experiment
may have been beneficial, as gene expression patterns were yet to stabilize at the end of
the of the experiment. Therefore, future experiments evaluating gene expression on the
chosen candidate genes should be undertaken with larger sample size (> eight individuals)
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at each sampling points and expose animals to stress conditions for longer time periods (at
least three days to a few weeks) to determine the duration of expression effects (Pan et al.,
2007; Serrano et al., 2007; Chaudhari et al., 2015; Liu et al., 2015). Similarly, analysing the
expression of these genes across other tissue types may have an effect on how they are
expressed in the gills, while also providing a more comprehensive analysis of the distribution
of expression of these important osmoregulatory and systemic acid-base balance genes in
response to pH change.
In the third study (Chapter 4), we generated gill transcriptomes from two other important
freshwater crayfish, Yabby (C. destructor) and Marron (C. cainii); and identified and
annotated candidate genes associated with pH balance and osmotic-/ionic regulation. In
addition, we made a comprehensive comparative molecular analysis between three Cherax
species, which were sourced from three distinct and geographically isolated areas (see Fig
1.1). The study has generated large scale genomic resources for the three most
commercially important Cherax species. The gill transcriptomes (from Redclaw, Yabby and
Marron) sequenced in this study are much larger than the genomic resources previously
available for C. quadricarinatus, which are largely limited to a few studies, one that was
sequenced using the Ion Torrent platform (Dammannagoda et al., 2015) and another using
Roche 454 (Glazer et al., 2013). Recently, however, a large scale Illumina Miseq dataset (206
million reads with an average read length of 80 bp) has been generated and published for C.
quadricarinatus (Tan et al., 2015). This dataset is comprised of multiple tissue types
including heart, nerve, muscle, testes and brain (Tan et al., 2015), but each individual
dataset is smaller than our current dataset and they did not sequence gill tissue. Our
Illumina sequenced datasets each comprised between 72–100 million paired-end reads and
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assembled into >100 000 contigs for each species. These assembled gill transcriptomes from
the three Cherax species, provided an excellent resource to identify candidate genes related
to systemic pH balance and osmoregulation as they were largely complete based on the
presence of 248 ultra conserved eukaryote genes. Overall, in all three Cherax species, we
identified most of the major genes associated with pH balance or osmoregulation including:
three forms of Carbonic anhydrase (CAc, CAg and CAb), Na+/K+-ATPase (NKA), V-type-H+-
ATPase (HAT), Na+/K+/2Cl- cotransporter (NKCC), (Na+)Cl−/HCO3− (NCB), Na+/H+ exchangers
(NHE), Arginine kinase (AK), Sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA)
and Calreticulin (CRT). These candidate genes were used in follow up studies to examine
their tissue specific expression and molecular evolution in order to better understand their
function in pH balance in freshwater crayfish species.
The structure-function analysis and comparative molecular study showed that all the
candidate genes were highly conserved in Cherax crayfish and other related decapods. The
genes are highly conserved not only in decapod crustaceans but also in other crustaceans
and insects (Hoskins et al., 2007; Serrano et al., 2007; Serrano and Henry, 2008;
Pongsomboon et al., 2009; Xu and Liu, 2011; Havird et al., 2014; Han et al., 2015; Xu et al.,
2015). It is interesting that all three Cherax species, although naturally distributed in three
distinct environmental conditions and geographically disconnected areas (see natural
distribution in Fig 1.1 and environmental tolerances in Table 1.1), showed a high degree of
conservation in terms of sequence variation and functional structures. This high level of
conservation is probably associated with the conserved function of the genes in ion
transport as well as having multiple other basic physiological functions including cell volume
control, maintaining a concentration gradient, signal transduction and integration in
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different tissue types. Many genes that influence to multiple phenotypic traits (pleiotropic
genes) are highly conserved. This is because a non-synonymous mutation in a pleiotropic
gene can often affect multiple traits simultaneously (Mackay et al., 2009). As a result a non-
synonymous mutation that improves the function of one process may be deleterious to
another process and is often selected against. Therefore, in many pleiotropic genes (genes
that affects multiple traits) we often see only a limited number of mutations and those that
we do are often dominated by synonymous mutations. While we observed that the
candidate genes examined in this study were highly conserved, we found no evidence of
purifying selection acting on these genes. This is contrast to other studies which have found
evidence of purifying selection in other animals (Hughes et al., 2003; Ward and Kellis,
2012).
Tissue specific expression of the candidate genes involved in systemic acid-base balance and
ionic/osmotic-regulation revealed that most of the genes involved in these processes
including carbonic anhydrase (CA), Na+/K+-ATPase (NKA), Vacuolar type H+-ATPase (HAT),
Na+/H+ exchanger (NHE), Na+/K+/2Cl- cotransporter (NKCC), Na+/Cl-/HCO3- cotransporter,
Arginine kinase (AK) and Sarco/endoplasmic reticulum Ca+2-ATPase (SERCA) were expressed
in all tissue types. This indicates that the majority of ion transport genes are expressed in
most tissue types in freshwater crayfish and is something that needs to be taken into
account in future gene expression studies of these genes in specific tissues such as gills. As
all the major genes were expressed in tissues other than gills, it implies an important
physiological role for these genes outside of osmoregulation. For example Na+/K+-ATPase,
an important osmoregulatory gene, has another important function as a signal
transducer/integrator that regulates the mitogen activated stress kinase pathway,
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intracellular calcium concentrations and reactive oxygen species (Xie and Askari, 2002; Yuan
et al., 2005; Bhavsar et al., 2014). Multifunctional proteins such as Na+/K+-ATPase are
required to perform functions in multiple cell and tissue types; therefore the expression of
similar ion transport and systemic pH balance genes is expected in multiple tissue types.
In our analysis, the two tissue types with highest number of candidate genes expressed
were the gills and nervous system. A large number of the genes (78%, 62 genes ) expressed
in the gills was expected based on previous research in decapod crustaceans (Freire et al.,
2008; Romano and Zeng, 2012) as this is one of the principal tissues where osmoregulation
and systemic acid-base balance occur. Interestingly, we observed the highest number of
genes expressed in nervous tissue (84%, 67 genes). However, expression studies of these
genes in the nervous system of crayfish are still scant (Kingston and Cronin, 2015), hence
data from crayfish are still lacking to support our finding. Despite this, studies on the
expression profile of vertebrate orthologs of these candidate genes, has shown that many
are highly expressed in nerve tissue (Fry and Jabr, 2010; Benarroch, 2011; Deval and
Lingueglia, 2015; Hertz et al., 2015; Shrivastava et al., 2015; Wu et al., 2015; Zhang et al.,
2015). In fact, a number of ion transport related genes play a large role in generating and
maintaining the concentration gradient and action potential in nerve cells (Barnett and
Larkman, 2007; Fry and Jabr, 2010; Benarroch, 2011; Hertz et al., 2015), which has been
discussed in details at tissue-specific expression section in Chapter 4. Many of these genes
examined in this study are also involved in muscle tissue (e. g. Sarco/endoplasmic reticulum
Ca+2-ATPase) and/or play key roles in other important cellular processes. The findings of this
study opens up a new avenue to multi-tissue expression study for the genes involved in
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maintenance of systemic pH balance and osmoregulation in freshwater crustaceans and
particularly in freshwater caryfish.
Despite a few limitations in our study, we are confident that the findings from our study will
contribute to the wider community of scientific research and in particular crustacean
aquaculture. Overall, we have successfully identified and characterised a suite of key
osmoregulatory and systemic acid-base balance genes, across three freshwater crayfish
species, of commercial and ecological importance, both in Australia and globally. Our results
demonstrate a high degree of conservation in these candidate genes, despite the ecological
and evolutionary divergence between the species studied. Importantly, we provide the first
report on the distribution of expression of these key genes across multiple tissue types in
freshwater crayfish, suggesting they potentially play important and multiple roles across
these tissue types. Ultimately, data generated in our study will produce a valuable resource
of genomic information for future use in studies on freshwater crayfish as well as wider
comparative genomic studies in decapod crustaceans.
To conclude, outcomes of our study will serve as a basic foundation for future molecular
and genomic studies of transport- and stress-related enzymes in freshwater crayfish. It will
also assist to determine the optimal water quality parameters, particularly pH and salinity,
to avoid any stress related to un-favourable pH and salinity conditions, and thus helping in
achieving better productivity of commercially cultured crayfish under variable
environmental conditions.
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Appendices Chapter 7.
7.1 Appendix 1: Data distribution of the assembled contigs for C. quadricarinatus
S Figure 2.1 Data distribution of the assembled contigs after blasting, mapping and annotation.
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7.2 Appendix 2: Top-hit Species distribution of the assembled contigs for Redclaw.
S Figure 2.2 Major top-hit species distribution on the basis of BALST search using the assembled contigs from Redclaw (C. quadricarinatus)
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
Cherax quadricarinatusPenaeus monodon
Hydra magnipapillataDendroctonus ponderosae
Culex quinquefasciatusBombus impatiens
Apis melliferaDanio rerio
Solenopsis invictaApis florea
Nematostella vectensisBombus terrestris
Aedes aegyptiAcromyrmex echinatior
Danaus plexippusAcyrthosiphon pisum
Harpegnathos saltatorCamponotus floridanus
Saccoglossus kowalevskiiMegachile rotundata
Capitella teletaStrongylocentrotus purpuratus
Nasonia vitripennisCrassostrea gigasIxodes scapularis
Branchiostoma floridaePediculus humanus
Tribolium castaneumDaphnia pulex
Others
# Seq
Spec
ies
Page 142
7.3 Appendix 3: Gene Ontology distribution of the gill transcripts from Redclaw
S Figure 2.3 Gene Ontology distribution of the gill transcripts from Cherax quadricarinatus based on Biological processes, Molecular functions and Cellular components.
Page 143
7.4 Appendix 4: Multiple alignment of partial C. quadricarinatus CA
S Figure 2.4 Multiple alignment of translated amino acid sequences of two partial C. quadricarinatus CA
Multiple alignment of translated amino acid sequences of two partial C. quadricarinatus CA (ChqCAp1: KM610228 and ChqCAp2: KM880150) with CA isoforms from some representative crustaceans such as the pacific white shrimp Litopenaeus vannamei (LvCAg: AGC70493), the littoral crab Carcinus maenas (CmCAg: ABX71209), the horse crab Portunus trituberculatus (PtCAg: AFV46145) and the blue crab Callinectes sapidus (CasCAg: ABN51214). The letter g after CA indicates the Glycosyl-phosphatidylinositol-linked carbonic anhydrase.
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7.5 Appendix 5 : Full length nucleotide sequences of C. quadricarinatus CAg
S Figure 2.5: Full length nucleotide sequences of C. quadricarinatus CAg (ChqCAg)
Full length nucleotide sequences along with corresponding deduced amino acid sequences of a CAg (Glycosyl-phosphatidylinositol-linked carbonic anhydrase, ChqCAg: KM538166) cDNA amplified from the gills Redclaw crayfish, C. quadricarinatus. Sequences are numbered at both sides of each line. The start codon and stop codon are in bold. N-linked glycosylation sites (NXS/T) are shaded and in bold. One putative AU-rich mRNA instability motif (polyadenylylation cleavage signal) (ATTTA) is in bold and underlined. 3’ and 5’ untranslated regions (UTR) are in small letters and expressed codons (CDS) are in capital letters.
Page 145
7.6 Appendix 6: Statistics for contigs from C. cainii (Marron) and C. destructor (Yabby)
S Table 4.1 Summary statistics for assembled contigs greater than 200 bp generated from C. cainii (Marron) and C. destructor (Yabby) gill transcriptomes using Trinity de novo assembler and cd-HIT clustering tool.
Summary statistics bp/number
Marron Yabby
Number of total contigs 147 101 136 622
N50 1380 1326
Mean contig length 747 740
Length of the longest contig 22 324 17725
Number of contigs longer than 500 bp 52 459 49678
Number of contigs longer than 1500 bp 17 486 16 178
Number of clusters 18 325 18 113
Number of singletons 128 776 118 509
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7.7 Appendix 7: Gene Ontology classification in C. cainii and C. destructor
S Figure 4.5 The proportion and number of contigs assigned to Gene Ontology groups in C. cainii and C. destructor The proportion and number of contigs assigned to Gene Ontology terms from biological process, cellular
component and molecular function in C. cainii (Marron) and C. destructor (Yabby).
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7.8 Appendix 8: List of all possible contigs with their sequence description, length and roles in pH and/or salinity balance
(It is a short list. Full list with GO functions, enzyme codes and InterProScan are available online: http://dx.doi.org/10.1016/j.gene.2015.03.074)
Seq. Name Seq. Description Seq. Length #Hits min. eValue mean Similarity #GOs
contig_13232 alkaline phosphatase 1115 20 1.44E-106 61.80% 1
contig_458 alpha-carbonic anhydrase 3318 20 2.48E-152 63.25% 3
contig_2595 alpha-carbonic anhydrase 549 20 2.04E-49 61.85% 5
contig_30 arginine kinase 1197 20 0 97.15% 3
contig_7797 ca-activated cl channel protein 1735 20 5.23E-42 42.30% 4
contig_9096 calcium channel flower-like isoform 1 724 20 2.28E-42 67.25% 4
contig_2587 calcium homeostasis endoplasmic reticulum protein 2893 20 8.01E-43 61.20% 1
contig_33447 calcium uniporter mitochondrial 894 20 6.04E-96 74.70% 8
contig_22653 calcium uptake protein 1 mitochondrial-like 707 20 6.66E-36 72.95% 5
contig_31373 calcium-activated chloride channel regulator 1 754 20 1.95E-18 52.05% 2
contig_10821 calcium-activated chloride channel regulator 1-like 547 20 3.91E-12 55.25% 0
contig_21528 calcium-activated chloride channel regulator 4 986 20 4.05E-45 47.80% 4
contig_109 calcium-dependent chloride channel-1 6381 20 1.01E-143 49.95% 8
contig_2145 calcium-transporting atpase sarcoplasmic endoplasmic reticulum type-like 4631 20 0 91.15% 14
contig_28533 calcium-transporting atpase sarcoplasmic endoplasmic reticulum type-like 508 20 2.81E-92 86.80% 6
contig_7731 calcium-transporting atpase type 2c member 1-like 3166 20 0 73.45% 12
contig_3781 calmodulin-like isoform 1 863 20 5.18E-73 79.50% 2
contig_6375 calmodulin-like protein 4-like 858 20 3.02E-76 83.15% 3
contig_929 calreticulin 1722 20 0 88.55% 5
contig_30278 calreticulin 1052 20 1.18E-156 95.45% 59
contig_19805 carbonic anhydrase 1021 20 6.15E-119 78.40% 4
contig_720 carbonic anhydrase 2-like 1527 20 1.14E-133 73.65% 3
contig_8131 chloride channel accessory 2-like 1149 20 1.20E-76 51.15% 0
contig_16307 chloride channel calcium activated 2-like 1710 20 1.44E-81 49.55% 8
contig_5606 chloride channel clic-like protein 1-like 488 20 8.65E-16 49.55% 2
contig_24700 chloride channel- isoform a 465 20 1.37E-53 71.20% 4
contig_3994 chloride channel protein 2 2408 20 2.07E-80 72.65% 3
contig_5834 chloride channel protein 2 1543 20 6.11E-115 82.30% 4
contig_1950 chloride channel protein 2-like 1089 20 5.43E-70 69.30% 3
contig_27916 chloride channel protein 2-like 393 9 2.33E-04 58.78% 8
contig_32462 chloride channel protein 2-like 382 20 1.31E-63 85.35% 5
contig_35968 chloride channel protein 2-like 426 20 3.21E-75 92.70% 4
contig_36091 chloride channel protein 3 499 20 1.40E-26 78.60% 4
contig_8662 chloride intracellular channel 5 502 20 1.78E-33 51.45% 4
contig_595 chloride intracellular channel exc-4-like 2565 20 5.11E-150 88.00% 5
contig_8488 epithelial chloride channel 3166 20 6.39E-78 43.15% 4
contig_9234 epithelial chloride channel 1440 20 4.39E-54 45.30% 0
contig_16799 epithelial chloride channel 652 20 5.45E-36 61.00% 0
contig_25768 epithelial chloride channel protein 498 20 7.18E-18 58.50% 8
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Seq. Name Seq. Description Seq. Length #Hits min. eValue mean Similarity #GOs
contig_5434 glutamate-gated chloride channel 1657 20 3.87E-65 56.80% 2
contig_33490 glutamate-gated chloride channel 322 20 1.28E-08 56.10% 7
contig_21149 glutamate-gated chloride channel alpha3a subunit 864 20 2.17E-23 82.50% 10
contig_2596 glycosyl-phosphatidylinositol-linked carbonic anhydrase 390 20 2.95E-13 52.40% 5
contig_21880 h+ transporting atp synthase beta subunit 700 20 6.61E-50 82.70% 8
contig_1935 h+ transporting atp synthase subunit d 816 20 5.03E-50 67.90% 5
contig_33631 k+-dependent na+ ca+ exchanger 709 9 1.05E-13 42.56% 8
contig_1327 mitochondrial sodium hydrogen exchanger nha2 3143 20 6.04E-104 60.75% 1
contig_1328 mitochondrial sodium hydrogen exchanger nha2 1562 20 3.77E-105 63.95% 1
contig_1329 mitochondrial sodium hydrogen exchanger nha2 2035 20 1.81E-100 63.55% 2
contig_11596 mitochondrial sodium hydrogen exchanger nha2 1936 20 1.72E-97 60.05% 1
contig_11896 na(+) h(+) exchange regulatory cofactor nhe-rf2-like 771 12 6.96E-21 62.92% 0
contig_34002 na+ + atpase alpha-subunit 1 537 15 2.17E-11 71.07% 14
contig_246 na+ k+-atpase alpha subunit 2391 20 0 95.30% 22
contig_22854 potassium channel 397 20 4.04E-51 86.30% 5
contig_28074 potassium channel subfamily k member 16-like 327 20 9.93E-19 59.30% 1
contig_21007 potassium chloride symporter 417 20 5.73E-45 63.75% 4
contig_28739 potassium chloride symporter 445 20 4.51E-76 88.75% 6
contig_27320 potassium voltage-gated channel subfamily h member 7 366 19 2.80E-09 68.26% 4
contig_19257 potassium voltage-gated subfamily h (eag-related) member 2 797 20 1.36E-60 83.25% 10
contig_22664 probable sodium potassium calcium exchanger cg1090-like 687 20 1.23E-55 54.10% 3
contig_35624 probable sodium potassium calcium exchanger cg1090-like 515 20 9.57E-09 77.95% 2
contig_4984 sodium bicarbonate cotransporter 2023 20 0 70.70% 8
contig_2402 sodium calcium exchanger 1-like 2727 20 0 66.85% 3
contig_20861 sodium channel modifier 1 713 20 1.69E-28 68.90% 1
contig_11941 sodium channel modifier 1-like 408 5 6.62E-05 56.40% 0
contig_25916 sodium chloride dependent transporter 368 17 5.54E-09 63.24% 8
contig_30914 sodium chloride dependent transporter 446 19 2.35E-13 55.42% 8
contig_5168 sodium hydrogen exchanger 3 2826 20 0 68.10% 5
contig_18243 sodium hydrogen exchanger 7-like 914 20 5.89E-67 70.55% 4
contig_16014 sodium hydrogen exchanger 8 2151 20 0 78.45% 5
contig_25242 sodium leak channel non-selective 403 20 3.42E-59 84.45% 7
contig_322 sodium potassium-dependent atpase beta-2 subunit 649 20 3.35E-121 68.45% 5
contig_10309 sodium potassium-transporting atpase subunit beta-1-interacting 791 17 3.80E-11 58.29% 3
contig_1144 sodium potassium-transporting atpase subunit beta-2-like 1690 20 8.01E-153 62.30% 5
contig_11187 sodium-bile acid cotransporter 1799 20 2.82E-99 62.85% 3
contig_5045 sodium-driven chloride bicarbonate exchanger 1471 20 0 77.40% 3
contig_21006 solute carrier family 12 member 6 428 20 2.48E-73 89.70% 4
contig_31783 solute carrier family 12 member 6 441 20 5.48E-75 90.80% 4
contig_6530 solute carrier family 12 member 9-like 2834 20 0 66.90% 3
contig_24613 solute carrier family 13 member 2 764 20 4.96E-64 72.55% 8
contig_33754 solute carrier family 13 member 3 806 20 4.04E-72 62.55% 4
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Seq. Name Seq. Description Seq. Length #Hits min. eValue mean Similarity #GOs
contig_5410 solute carrier family 15 member 4 454 20 1.67E-21 72.40% 4
contig_5637 solute carrier family 15 member 4-like 382 20 1.74E-25 57.30% 2
contig_7707 solute carrier family 2 680 20 1.37E-34 60.90% 3
contig_17227 solute carrier family 22 member 15-like 917 20 1.03E-70 60.25% 6
contig_33919 solute carrier family 22 member 15-like 357 20 3.92E-16 50.00% 1
contig_8702 solute carrier family 22 member 2 1323 20 3.95E-27 56.80% 1
contig_3312 solute carrier family 22 member 5 2723 20 2.17E-08 44.00% 7
contig_30613 solute carrier family 22 member 7 645 20 3.11E-27 50.80% 7
contig_17820 solute carrier family 23 member 1 348 20 1.86E-44 82.95% 16
contig_21235 solute carrier family 23 member 1 771 20 2.00E-82 72.95% 16
contig_442 solute carrier family 25 (mitochondrial carrier phosphate carrier) member 3 1545 20 1.58E-171 82.20% 6
contig_13773 solute carrier family 25 member 35 386 20 5.08E-21 77.05% 2
contig_711 solute carrier family 25 member 38 2716 20 1.82E-115 74.55% 2
contig_19758 solute carrier family 25 member 40 1082 20 2.14E-97 65.50% 1
contig_16434 solute carrier family 25 member 42 1424 20 7.69E-119 74.65% 11
contig_14925 solute carrier family 25 member 46 1890 20 3.63E-107 67.00% 1
contig_30576 solute carrier family 26 (sulfate transporter) member isoform cra_a 441 20 1.33E-18 63.70% 4
contig_18874 solute carrier family 35 member b1 1287 20 2.66E-133 72.50% 1
contig_29226 solute carrier family 35 member c2 482 20 2.67E-51 73.70% 1
contig_16598 solute carrier family 35 member d3-like 372 20 1.70E-30 60.10% 2
contig_19192 solute carrier family 35 member e1 homolog 1363 20 2.41E-121 76.25% 2
contig_24210 solute carrier family 40 (iron-regulated transporter) member 1 498 20 1.11E-26 60.15% 10
contig_13469 solute carrier family 40 member 1 893 20 5.46E-33 54.95% 6
contig_10680 solute carrier family 41 member 1-like 1977 20 8.78E-158 76.75% 2
contig_23324 solute carrier family 43 member 3-like 483 20 7.48E-09 56.85% 2
contig_25283 solute carrier family 43 member 3-like 668 4 3.06E-05 62.75% 2
contig_11457 solute carrier family 45 member 3 1135 20 3.58E-46 59.60% 2
contig_16093 t family of potassium channels protein 18 580 20 1.07E-35 74.30% 3
contig_25373 t family of potassium channels protein 18 386 20 6.59E-18 77.50% 7
contig_18958 two pore calcium channel protein 1 isoform 1 1438 20 4.33E-123 67.90% 5
contig_4067 two pore calcium channel protein 1-like 1093 20 4.54E-49 66.35% 4
contig_20456 two pore channel 3 475 20 5.29E-50 78.30% 4
contig_395 vacuolar h 2008 20 0 95.90% 12
contig_791 vacuolar h alpha subunit 2978 20 0 75.15% 7
contig_1152 vacuolar h 1127 20 0 91.10% 6
contig_2008 vacuolar h 2070 20 0 80.30% 7
contig_2517 vacuolar h Subunit C 1348 20 0 83.25% 5
contig_24856 vacuolar h 511 20 2.02E-87 94.20% 9
contig_1931 vacuolar h(+)-atpase proteolipid subunit homolog 500 20 5.14E-25 86.20% 5
contig_17208 voltage-dependent calcium channel subunit alpha-2 delta-1 477 20 5.11E-40 57.35% 3
contig_17836 voltage-dependent calcium channel subunit alpha-2 delta-1 1064 20 1.72E-13 46.10% 1
contig_31332 voltage-dependent calcium channel subunit alpha-2 delta-2-like 607 20 1.17E-18 49.95% 3
contig_19298 voltage-gated ion channel 801 20 2.43E-117 80.30% 10
contig_24738 voltage-gated ion channel 1015 20 5.15E-118 76.45% 6
contig_26733 voltage-gated ion channel 576 20 5.82E-87 81.85% 14
contig_15404 v-type proton atpase 116 kda subunit a isoform 1-like isoform 1 1898 20 0 78.40% 3
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Seq. Name Seq. Description Seq. Length #Hits min. eValue mean Similarity #GOs
contig_1525 v-type proton atpase 21 kda proteolipid subunit-like 796 20 1.74E-64 81.25% 5
contig_598 v-type proton atpase catalytic subunit a-like 2354 20 0 91.10% 6
contig_9658 v-type proton atpase subunit d 1-like 412 20 1.94E-26 71.75% 2
contig_397 v-type proton atpase subunit s1-like 1472 20 7.83E-42 45.00% 2
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7.9 Appendix 9: Identity matrix across species based on amino acid compositions (7.9.1-7.9.11)
7.9.1 Amino acid identity across crustacean species based on cytoplasmic CA (CAc)
C. quadricarinatus C. cainii C. destructor L. vannamei P. monodon C. sapidus P. trituberculatus
Cherax quadricarinatus CAc (KM538165)
97.786 97.786 76.015 76.015 73.801 73.063 Cherax cainii CAc (KP221715) 97.786
97.786 76.015 76.015 73.801 73.063
Cherax destructor (KP299962) 97.786 97.786
75.646 75.277 73.801 72.694 Litopenaeus vannamei (HM991703) 76.015 76.015 75.646
97.778 73.432 73.063
Penaeus monodon (EF672697) 76.015 76.015 75.277 97.778
73.432 72.694 Callinectes sapidus (EF375490) 73.801 73.801 73.801 73.432 73.432
94.465
Portunus trituberculatus (JX524149) 73.063 73.063 72.694 73.063 72.694 94.465
7.9.2 Amino acid identity across crustacean species based on membrane-associated CA (CAg)
C. quadricarinatus C. cainii
C. destructor L. vannamei H. rubra C. sapidus P. trituberculatus C. maenas
Cherax quadricarinatus (KM538166) 99.355 92.929 71.104 72.903 73.701 73.377 70.13 Cherax cainii (KP221716) 99.355 92.256 71.429 73.226 73.701 73.377 70.455 Cherax destructor (KP299963) 92.929 92.256 66.78 68.35 68.475 68.136 67.119 Litopenaeus vannamei (JX975725) 71.104 71.429 66.78 75.806 70.779 69.481 69.481 Halocaridina rubra (KF650061) 72.903 73.226 68.35 75.806 71.935 71.613 70.645 Callinectes sapidus (EF375491) 73.701 73.701 68.475 70.779 71.935 95.13 88.636 Portunus trituberculatus (JX524150) 73.377 73.377 68.136 69.481 71.613 95.13 87.338 Carcinus maenas (EU273944) 70.13 70.455 67.119 69.481 70.645 88.636 87.338
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7.9.3 Amino acid identity across crustacean species based on beta CA (CA-beta)
C. quadricarinatus C. cainii
C. destructor
B. terrestris
Apis dorsata
N. vitripennis
Athalia rosae
Cherax quadricarinatus (KM538167) 99.222 98.833 62.745 60.392 63.529 63.137 Cherax cainii (KP221717) 99.222 98.054 63.137 60.784 63.922 63.529 Cherax destructor (KP299965) 98.833 98.054 63.137 61.176 64.314 63.922 Bombus terrestris (XM_003402502) 62.745 63.137 63.137 82.745 84.706 86.275 Apis dorsata (XM_006612942) 60.392 60.784 61.176 82.745 81.569 83.529 Nasonia vitripennis (XM_001606922) 63.529 63.922 64.314 84.706 81.569 94.902 Athalia rosae (XM 012404250) 63.137 63.529 63.922 86.275 83.529 94.902
7.9.4 Amino acid identity across crustacean species based on Na+/K+-ATPase alpha subunit (NKA)
C. quadricarnatus NKA
Cherax cainii (KP221718)
C. destructor
P. monodon
F. indicus
L. stylirostris
C. sapidus
P. trituberculatus
P. marmoratus
E. sinensis
Cherax quadricarnatus NKA 99.904 99.711 93.545 94.027 91.908 95 95 95.568 95.665 Cherax cainii (KP221718) 99.904 99.807 93.545 94.027 92.004 95.096 95.096 95.665 95.761 Cherax destructor (KP299966) 99.711 99.807 93.449 93.931 91.908 95 95 95.568 95.665 Penaeus monodon (|DQ399797) 93.545 93.545 93.449 98.844 95.472 92.212 92.404 92.1 92.004 Fenneropenaeus indicus (HM012803) 94.027 94.027 93.931 98.844 96.243 92.596 92.788 92.775 92.293 Litopenaeus stylirostris (JN561324) 91.908 92.004 91.908 95.472 96.243 90.577 90.865 90.655 90.366 Callinectes sapidus (AF327439) 95 95.096 95 92.212 92.596 90.577 99.326 97.69 97.209 Portunus trituberculatus (JX173959) 95 95.096 95 92.404 92.788 90.865 99.326 97.594 97.113 Pachygrapsus marmoratus (DQ173924) 95.568 95.665 95.568 92.1 92.775 90.655 97.69 97.594 98.457 Eriocheir sinensis (KC691291) 95.665 95.761 95.665 92.004 92.293 90.366 97.209 97.113 98.457
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7.9.5 Amino acid identity across crustacean species based on V- type H+-ATPase (HAT-A)
C. quadricarinatus C. destructor C. cainii
W. auropunctata
P. barbatus C. floridanus B. terrestris
Cherax quadricarinatus
99.158 98.676 71.059 70.824 71.025 71.226 Cherax.destructor (KP299969) 99.158
99.278 70.824 70.588 70.907 70.991
Cherax.cainii (KP221721) 98.676 99.278
70.824 70.588 70.907 70.991 Wasmannia auropunctata (XM 011701129) 71.059 70.824 70.824
96.908 95.838 88.889
Pogonomyrmex barbatus (XM 011643683) 70.824 70.588 70.588 96.908
95.244 88.416 Camponotus floridanus (XM 011263300) 71.025 70.907 70.907 95.838 95.244
89.125
Bombus terrestris (XM 003397650) 71.226 70.991 70.991 88.889 88.416 89.125
7.9.6 Amino acid identity across crustacean species based on Na+/K+/2Cl- cotransporter (NKCC)
C. quadricarinatus C. destructor C. cainii
C. sapidus H. rubra
M. domestica
B. cucurbitae
D. melanogaster
B. terrestris
Cherax quadricarinatus 96.934 97.288 49.25 49.024 49.664 49.943 50 47.119 Cherax destructor (KP299986) 96.934 97.03 50.431 49.893 51.207 51.386 51.86 48.837 Cherax cainii (KP221733) 97.288 97.03 48.448 47.711 48.802 49.101 49.282 47.25 Callinectes sapidus (AF190129) 49.25 50.431 48.448 76.245 49.359 49.118 49.722 47.903 Halocaridinarubra (KF650065) 49.024 49.893 47.711 76.245 48.168 48.37 48.597 47.23 Musca domestica (XM_011298324) 49.664 51.207 48.802 49.359 48.168 84.898 81.795 57.713 Bactrocera cucurbitae (XM 011181369) 49.943 51.386 49.101 49.118 48.37 84.898 81.803 58.079 Drosophila melanogaster (NM_140315) 50 51.86 49.282 49.722 48.597 81.795 81.803 58.318 Bombus terrestris (XM 003399442) 47.119 48.837 47.25 47.903 47.23 57.713 58.079 58.318
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7.9.7 Amino acid identity across crustacean species based on Na+/HCO3- cotransporter (NBC)
C. destructor C. destructor C. cainii C. cainii C. quadricarinatus W. auropunctata P. barbatus
Cherax destructor (KP299976) 99.914 98.497 98.361 97.412 60.371 59.354 Cherax destructor (KP299977) 99.914 98.361 98.447 97.498 60.371 59.354 Cherax cainii (KP221727) 98.497 98.361 99.914 97.323 60.455 59.437 Cherax cainii (KP221728) 98.361 98.447 99.914 97.409 60.455 59.437 Cherax quadricarinatus (KP221728) 97.412 97.498 97.323 97.409 60.796 59.933 Wasmannia auropunctata (XM 011705063) 60.371 60.371 60.455 60.455 60.796 94.187 Pogonomyrmex barbatus (XM 011642543) 59.354 59.354 59.437 59.437 59.933 94.187
7.9.8 Amino acid identity across crustacean species based on Na+/H+ exchanger (NHE)
C. quadricarinatus
C. destructor C. cainii
A. echinatior
A. echinatior
V. emeryi
P. barbatus
B. impatiens
Cherax quadricarinatus (KM880153) 92.628 91.161 40.833 40.535 40.457 40.99 40.04 Cherax destructor (KP299982) 92.628 92.259 41.071 40.774 40.597 41.089 40.681 Cherax cainii (KP221730) 91.161 92.259 39.603 39.319 39.242 39.754 38.725 Acromyrmex echinatior (XM_011069477) 40.833 41.071 39.603 96.753 91.516 92.769 78.524 Acromyrmex echinatior (XM_011069496) 40.535 40.774 39.319 96.753 88.37 89.534 76.42 Vollenhovia emeryi (XM 012027471) 40.457 40.597 39.242 91.516 88.37 90.095 78.178 Pogonomyrmex barbatus (XM_011649419) 40.99 41.089 39.754 92.769 89.534 90.095 79.037 Bombus impatiens (XM 003491441) 40.04 40.681 38.725 78.524 76.42 78.178 79.037
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7.9.9 Amino acid identity across crustacean species based on Na+/Ca+2 exchanger (NCX)
C. destructor C. cainii
C. quadricarinatus
P. barbatus
A. cephalotes
W. auropunctata C. floridanus A. dorsata
Cherax destructor (KP299983) 98.94 98.469 38.317 37.721 38 37.751 37.458 Cherax cainii (KP221731) 98.94 98.231 38.206 37.611 37.889 37.639 37.347 Cherax quadricarinatus 98.469 98.231 38.206 37.611 37.889 37.639 37.458 Pogonomyrmex barbatus (XM 011636160) 38.317 38.206 38.206 95.293 93.8 91.734 87.011 Atta cephalotes (XM 012203310) 37.721 37.611 37.611 95.293 95.293 93.456 87.371 Wasmannia auropunctata (XM 011701097) 38 37.889 37.889 93.8 95.293 93.318 87.112 Camponotus floridanus (XM_011255402) 37.751 37.639 37.639 91.734 93.456 93.318 87.112 Apis dorsata (XM 006616508) 37.458 37.347 37.458 87.011 87.371 87.112 87.112
7.9.10 Amino acid identity across crustacean species based on Arginine kinase
C. quadricarinatus
C. destructor
C. cainii P. clarkii H. vulgaris
C. maenas
S. serrata C. sapidus
L. vannamei
P. monodon
Cherax.quadricarinatus 98.599 98.039 96.639 94.944 92.717 92.135 92.997 90.169 90.73 Cherax destructor (KP299970) 98.599 97.479 96.359 94.663 92.437 92.135 92.717 89.607 89.607 Cherax cainii (KP221722) 98.039 97.479 96.078 94.101 92.157 91.573 91.877 89.045 89.607 Procambarus clarkii (JN828651) 96.639 96.359 96.078 95.225 93.838 94.382 94.678 91.292 91.011 Homarus vulgaris (X68703) 94.944 94.663 94.101 95.225 92.697 92.697 93.82 91.011 91.011 Carcinus maenas (AF167313) 92.717 92.437 92.157 93.838 92.697 97.191 97.199 91.292 92.135 Scylla serrata (GQ851626) 92.135 92.135 91.573 94.382 92.697 97.191 98.034 93.258 94.101 Callinectes sapidus (AF233355) 92.997 92.717 91.877 94.678 93.82 97.199 98.034 92.978 92.978 Litopenaeus vannamei (DQ975203) 90.169 89.607 89.045 91.292 91.011 91.292 93.258 92.978 96.067 Penaeus monodon (KF177337) 90.73 89.607 89.607 91.011 91.011 92.135 94.101 92.978 96.067
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7.9.11 Amino acid identity across crustacean species based on Calreticulin
C. quadricarinatus
C. destructor C. cainii
F. chinensis
P. monodon L. vannamei
E. carinicauda
P. leniusculus
S. paramamosain
Cherax quadricarinatus (KM538170) 98.759 99.005 86.946 87.192 86.453 85.714 91.832 86.207 Cherax destructor (KP299971) 98.759 98.263 86.7 86.946 86.207 85.961 91.111 85.714 Cherax cainii (KP221723) 99.005 98.263 87.192 87.192 86.453 85.961 91.337 86.207 Fenneropenaeus chinensis (DQ323054) 86.946 86.7 87.192 98.768 96.798 88.424 86.7 85.222 Penaeus monodon (GU140040) 87.192 86.946 87.192 98.768 98.03 88.67 86.946 83.99 Litopenaeus vannamei (JQ682618.) 86.453 86.207 86.453 96.798 98.03 88.177 86.207 83.99 Exopalaemon carinicauda (JX508647) 85.714 85.961 85.961 88.424 88.67 88.177 85.222 83.99 Pacifastacus leniusculus (HQ596362) 91.832 91.111 91.337 86.7 86.946 86.207 85.222 84.975 Scylla paramamosain (HQ260918) 86.207 85.714 86.207 85.222 83.99 83.99 83.99 84.975
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7.10 Appendix 10: Tissue-specific expression of major pH- and osmoregulatory genes in Redclaw crayfish Cherax quadricarinatus
# Gene Name Gill Hepatopancreas Heart Kidney Liver Nerve Testes
1 cytoplasmic carbonic anhydrase (CAc) p p p p p p p
2 GPI-linked carbonic anhydrase (CAg) p p p p p p p
3 Beta carbonic anhydrase (CAb) p a p p p p a
4 carbonic anhydrase-related protein 10 p a a a a p a
5 na+ k+-atpase alpha subunit p p p p p p p
6 sodium potassium-transporting atpase subunit beta p p p p p p p
7 v-type proton atpase catalytic subunit a p p p p p p p
8 v-type proton atpase 116 kda subunit a p p p a p p p
9 v-type proton atpase subunit B p p p a a a a
10 v-type proton atpase subunit C p p p a p a a
11 v-type proton atpase subunit D p p p p p p p
12 v-type proton atpase subunit E p p p p p p p
13 v-type proton atpase subunit H p p a a a a a
14 v-type proton atpase subunit S p p p p p p a
15 v-type proton atpase 21 kda proteolipid subunit p p p p p p p
16 arginine kinase p p p p p p p
17 alkaline phosphatase p p p p p p p
18 calreticulin p p p p p p p
19 selenophosphate synthetase p p p p p p p
20 calcium-transporting atpase sarcoplasmic endoplasmic reticulum type p p p p p p p
21 calcium-transporting atpase type 2C p p p p p p p
22 plasma membrane calcium-transporting atpase 3 p p p p p p p
23 sodium hydrogen exchanger 2 a a a a a a p
24 sodium hydrogen exchanger 3 p a a p a p a
25 sodium hydrogen exchanger 7 p p p p p p p
26 sodium hydrogen exchanger 8 p p p p p p p
27 sodium hydrogen exchanger 9 p a a a a p a
28 sodium calcium exchanger 1 p a p p p p p
29 sodium calcium exchanger 3 a a p p p p a
30 sodium bicarbonate cotransporter (Na+/HCO3) p a p p p p p
31 sodium-driven chloride bicarbonate exchanger (Cl (Na+)/HCO3) p p p a p a a
32 sodium chloride cotransporter (Na/Cl) p p p p p p p
33 potassium chloride symporter (K/Cl) p p p p a p a
34 sodium channel protein (Na) a a p a a p a
35 sodium channel protein 60e a a a a a p p
36 potassium channels protein 7 (t family ) a a a a p p a
37 potassium channels protein 18 (t family ) p a a a p p p
38 potassium channel 1 (k family) a p a a p p a
39 potassium channel 3 (k family) a a a a a p a
40 potassium channel 9 (k family) a a a a a p p
41 potassium channel 10 (k family) a a a a a p a
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# Gene Name Gill Hepatopancreas Heart Kidney Liver Nerve Testes
42 potassium channel 16 (k family) p a p a p a a
43 potassium channel 18 (k family) a a a a a a p
44 calcium-activated potassium channel a a p p a p p
45 two pore potassium channel protein sup-9 a a a P p p p
46 chloride channel p p p p a p p
47 chloride channel protein 2 p p p p p p p
48 chloride channel protein 3 p a a p p p p
49 chloride channel protein 7 a a a p a a a
50 epithelial chloride channel p p p p p p p
51 glutamate-gated chloride channel p p a a a p a
52 histamine-gated chloride channel a a p a p a a
53 ph-sensitive chloride channel a a a p p a a
54 ca-activated cl channel protein p p p p p p p
55 two pore calcium channel protein 1 p p p p a p p
56 calcium release-activated calcium channel protein p p p p p p p
57 Calmodulin p p p p p p p
58 h+ transporting atp synthase beta subunit p p a p a p p
59 h+ transporting atp synthase subunit o p p p p p p p
60 h+ transporting atp synthase subunit d p p p p p p p
61 Integrin alpha p p p p p p p
62 Integrin beta p p p p p p p
63 ATP-binding cassette transporters A p p p p p p p
64 ATP-binding cassette transporters B p p p p p p p
65 ATP-binding cassette transporters C p p p p p p a
66 ATP-binding cassette transporters D p p p p p p p
67 ATP-binding cassette transporters E p p p p p p p
68 ATP-binding cassette transporters F p p p p p p p
69 ATP-binding cassette transporters G p p p p a p p
70 Calmodulin p p p p p p p
71 p38 mitogen-activated protein kinase p p p p p p p
72 Aquaporin transporter p p p p p p p
73 Aquaporin-1 a a p a a a a
74 Aquaporin-3 a P a p a p a
75 Aquaporin 9 p a a a a p a
76 Aquaporin 10 p a a a a a a
77 Aquaporin 12a a P a a a a a
78 magnesium transporter protein 1 p p p p p p p
79 magnesium transporter nipa p p a p p p p
80 membrane magnesium transporter p p p p p p p