MMaarriinnee EEnnvviirroonnmmeenntt aanndd EEccoollooggyy

Molecular tools for detection of marine pests:

Musculista senhousia, Corbula gibba and Perna canaliculus quantitative PCR assays.

Nathan J. Bott and Danièle Giblot-Ducray

SARDI Publication No. F2010/000991-1 SARDI Research Report Series No. 522

SARDI Aquatic Sciences PO Box 120 Henley Beach SA 5022

February 2011

Report prepared for Biosecurity SA

ii

Molecular tools for detection of marine pests: Musculista senhousia, Corbula gibba and Perna

canaliculus quantitative PCR assays.

Report prepared for Biosecurity SA

Nathan J. Bott and Danièle Giblot-Ducray

SARDI Publication No. F2010/000991-1 SARDI Research Report Series No. 522

February 2011

This publication may be cited as: Bott, N. J. and Giblot-Ducray, D (2011). Molecular tools for detection of marine pests: Musculista senhousia, Corbula gibba and Perna canaliculus quantitative PCR assays. Report prepared for Biosecurity SA. South Australian Research and Development Institute (Aquatic Sciences), Adelaide. SARDI Publication No. F2010/000991-1. SARDI Research Report Series No. 522. 22pp. South Australian Research and Development Institute SARDI Aquatic Sciences 2 Hamra Avenue West Beach SA 5064 Telephone: (08) 8207 5400 Facsimile: (08) 8207 5406 http://www.sardi.gov.au DISCLAIMER The authors warrant that they have taken all reasonable care in producing this report. The report has been through the SARDI Aquatic Sciences internal review process, and has been formally approved for release by the Chief, Aquatic Sciences. Although all reasonable efforts have been made to ensure quality, SARDI Aquatic Sciences does not warrant that the information in this report is free from errors or omissions. SARDI Aquatic Sciences does not accept any liability for the contents of this report or for any consequences arising from its use or any reliance placed upon it. © 2011 SARDI This work is copyright. Apart from any use as permitted under the Copyright Act 1968 (Cth), no part may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owner. Neither may information be stored electronically in any form whatsoever without such permission. Printed in Adelaide: February 2011 SARDI Publication No. F2010/000991-1 SARDI Research Report Series No. 522 Author(s): Nathan J. Bott and Danièle Giblot-Ducray Reviewer(s): Marty Deveney and Kathryn Wiltshire Approved by: Jason Tanner Principal Scientist – Marine Environment & Ecology Signed: Date: 10 February 2011 Distribution: Biosecurity SA, SAASC Library and University of Adelaide Library Circulation: Public Domain

iii

http://www.sardi.gov.au/

Table of Contents Executive Summary.............................................................................................................1 Acknowledgements .............................................................................................................2 Introduction..........................................................................................................................3

Musculista senhousia ...................................................................................................4 Corbula gibba ...............................................................................................................4 Perna canaliculus .........................................................................................................5 Molecular testing methods for marine pests .................................................................6

Methods...............................................................................................................................8 Quantitative PCR (qPCR) assay design .......................................................................8 Samples .......................................................................................................................9 DNA extractions ...........................................................................................................9 Quantitative PCR........................................................................................................10

Results ..............................................................................................................................10 Primers and probes........................................................................................................10

Musculista senhousia .................................................................................................12 Corbula gibba .............................................................................................................14 Perna canaliculus .......................................................................................................16

Discussion .........................................................................................................................17 Conclusions and future approaches ...........................................................................18

References ........................................................................................................................20 Glossary of Terms .............................................................................................................22 Figure 1: Asian date mussel, Musculista senhousia ............................................................4 Figure 2: European clam, Corbula gibba .............................................................................5 Figure 3: New Zealand Greenlip mussel, Perna canaliculus ...............................................6 Figure 4: SARDI Diagnostics laboratory ..............................................................................9 Figure 5: Amplification plot for Musculista senhousia qPCR assay specificity testing. ......13 Figure 6: Amplification plot for Corbula gibba qPCR assay specificity testing. ..................15 Figure 7: Amplification plot for Perna canaliculus qPCR assay specificity testing .............17

Table 1: Primers and TaqMan MGB probes.................................................................................... 11 Table 2: Results of specificity testing for M. senhousia qPCR assay.............................................. 13 Table 3: Results of specificity testing for C. gibba qPCR assay...................................................... 14 Table 4: Results of specificity testing for P. canaliculus qPCR assay............................................. 16

iv

Executive Summary The National System for the Prevention and Management of Marine Pest Incursions requires tools

for the detection and monitoring of marine pests. Specific, robust molecular assays for the

identification and quantification of marine pests (including eggs and larval stages) from

environmental samples facilitate rapid, low-cost surveillance to be undertaken and effective control

strategies to be implemented where marine pest incursions are detected. The use of quantitative

Polymerase Chain Reaction (qPCR) techniques will be suitable for this purpose. The polymerase

chain reaction (PCR) is an enzymatic technique used for the amplification of nucleic acids (e.g.

DNA), and qPCR is a PCR technique monitored in real-time through changes in fluorescence.

Currently, detection of marine pests is based primarily on traditional survey techniques such as

observational walks, snorkel and dive surveys, plankton trawls, traps and netting, with manual

sorting and identification. The use of qPCR offers the ability to conduct testing of very large

numbers of samples to rapidly identify the genetic material of the targeted organisms (referred to

as high-throughput screening). The successful development and implementation of these methods

will allow for the testing of plankton samples to rapidly verify the presence or absence of potential

pest species in marine waters. This study details the development and assessment of qPCR

assays for the Asian Date Mussel, Musculista senhousia, the New Zealand Greenlip Mussel, Perna

canaliculus and the European clam, Corbula gibba.

We have designed a qPCR assay that is, based on available controls, specific to M. senhousia.

We have adapted a specific PCR assay for P. canaliculus to a qPCR assay that is, based on

available controls, specific to P. canaliculus. We have further tested the specificity of the C. gibba

qPCR assay, developed by Ophel-Keller et al. (2007), with closely related corbulid species, and

based on available specimens this assay is specific to C. gibba.

In the future, these qPCR assays, along with other marine pest qPCR assays, are expected to be

used to monitor ports for marine pests, using DNA extracted from plankton samples. Specific

diagnosis of marine pests is central to rapidly establishing the distribution and prevalence of

marine pest species to manage and restrict their spread, and monitoring changes in marine pest

distribution spatially and temporally facilitates targeted eradication and control programmes where

feasible.

1

Acknowledgements Assistance from the following individuals is greatly acknowledged:

• Dr Paul Fisher and Bradley Pease (Queensland Department of Employment, Economic

Development and Innovation) for the provision of mytilid bivalve samples.

• Dr Richard Willan (NT Museum) for identifiying Musculista senhousia samples and Dr

Jawahar Patil (CSIRO Marine Research) for providing M. senhousia DNA.

• Mr Anders Hallan (University of Wollongong) for providing corbulid specimens.

• Dr Alan McKay, Dr Kathy Ophel-Keller, Dr Herdina and Ms Teresa Mammone (SARDI

Diagnostics) for assistance and advice in the laboratory.

• Dr Maylene Loo (SARDI Aquatic Sciences) for assistance in obtaining M. senhousia

samples from Singapore.

• Dr Marty Deveney and Ms Kathryn Wiltshire (SARDI Aquatic Sciences)

• Dr Michael Sierp, Mr John Gilliland and Mr Vic Neverauskas (Biosecurity SA).

2

Introduction The National System for the Prevention and Management of Marine Pest Incursions requires tools

for the detection and monitoring of marine pests. Specific, robust molecular assays for the

identification and quantification of marine pests (including eggs and larval stages) from

environmental samples facilitate rapid, low-cost surveillance, and inform effective control strategies

where marine pest incursions are detected.

Marine pests have the potential to cause significant harm to endemic biodiversity and habitats

(Galil, 2007; Wallentinus and Nyberg, 2007). Marine pests can be translocated and introduced by

numerous vectors including ship ballast, hull fouling, floating debris and man-made structures such

as drilling platforms and canals (Bax et al., 2003). Marine pest introductions continue to occur and

threaten the marine environment and associated industries (Hayes and Sliwa, 2003). With

increasing globalisation comes faster and more frequent shipping and air transport of live seafood.

Propagule pressure is only likely to increase unless effective strategies are employed for early

detection, prevention and control. Central to such strategies is the ability to rapidly identify the

presence of a particular pest species.

The development and implementation of rapid, sensitive and accurate diagnostic techniques for

the identification and surveillance of marine pests from environmental samples (e.g. sea water,

sediments, and ship ballast), is an essential step in early detection and control of marine pests, to

maintain the status of pest-free areas and to limit the economic impacts of management in areas

where pest are established and cannot be eradicated..

Current marine pest diagnostics research at SARDI includes the development and refinement of

specific, sensitive, quantitative Polymerase Chain Reaction (qPCR) assays for the detection of

several marine pest species. PCR is an enzymatic technique used for the amplification of nucleic

acids (e.g. DNA) and qPCR is a PCR technique monitored in real-time through changes in

fluorescence. Through consultation between SARDI Aquatic Sciences and Biosecurity SA it was

decided to undertake a study to implement and/or develop qPCR assays for three marine pest

species of significance to Australia, two of which: European clam, Corbula gibba ; and Asian Bag

Mussel, Musculista senhousia, are part of the Consultative Committee on Introduced Marine Pest Emergencies (CCIMPE) Trigger List, which is endorsed by the National Introduced Marine Pest

Coordinating Group (NIMPCG). The third is the New Zealand Greenlip Mussel, Perna canaliculus,

which has high potential as an invasive species and has been previously reported but eradicated

from Outer Harbor and a hopper barge docked in Port Adelaide (Wiltshire et al., 2010).

3

Musculista senhousia Musculista senhousia (Figure 1) is commonly known as the Asian date mussel and is native to the Pacific Ocean, inhabiting coastal areas from Siberia and the Kuril Islands south to Singapore

(Slack-Smith and Brearley, 1987). Aggregations can attain very high densities in intertidal and

subtidal soft sediments, typically 5 000-10 000/m2, and up to 15 000/m2 (Crooks and Soule, 1999;

Dexter and Crooks, 2000; Reush and Williams, 1998). When in high densities, M. senhousia can

cause significant habitat alteration, which can have profound effects on native infaunal

communities, and native seagrasses (Crooks, 1998). Based on seawater temperature, M.

senhousia has significant potential to become established across Australian waters

(http://marinepests.gov.au/__data/assets/pdf_file/0005/952529/Musculista-ncp-08.pdf).

Figure 1: Asian date mussel, Musculista senhousia

Corbula gibba The European clam, C. gibba (see Figure 2), is commonly found in subtidal environments, in

coastal and estuarine silts and muddy gravels, in its native Europe (Holmes and Miller, 2006).

Very high densities of C. gibba have been recorded in Port Phillip Bay (up to 2600/m2), and have

been linked to changes in benthic community structure (Currie and Parry, 1999). In Europe, high

abundances of C. gibba tend to be associated with habitat degradation, although this has not been

adequately studied in Australia.

4

Figure 2: European clam, Corbula gibba

Perna canaliculus Perna canaliculus (Figure 3) is commonly known as the New Zealand greenlip mussel and is

endemic to New Zealand waters, it has been previously reported from Outer Harbor and from a

vessel in Port Adelaide, but it was successfully eradicated (Wiltshire et al., 2010). Perna

canaliculus is commercially harvested as seafood in New Zealand. Potential impacts of P.

canaliculus on native populations are unknown but it would likely include competition with native

bivalve species, fouling structures and potential introduction of diseases to the Australian mollusc

aquaculture industry.

5

Figure 3: New Zealand Greenlip mussel, Perna canaliculus

Molecular testing methods for marine pests Development of rapid testing methods for marine pests has recently focussed on molecular

techniques. A broad range of these techniques have been developed for marine pests (see Bott et

al. 2010 and references therein). Polymerase Chain Reaction (PCR) has revolutionised many

areas of biological research including species and strain delineation. PCR can amplify minute

amounts of template DNA, and its high specificity makes it highly effective for species and strain

identification for a wide range of organisms. The relatively low cost of equipment and reagents

makes PCR accessible to a wide range of laboratories. Quantitative PCR (qPCR) allows the

amplification of a target DNA to be monitored in real-time as amplification occurs. qPCR offers a

relatively rapid analysis (< 2 hours), the potential for high-throughput, allows linear quantification

over a wide dynamic range (>6 orders of magnitude), and has the benefit of not requiring post-

PCR handling (“closed-tube” format), decreasing the likelihood of sample contamination. It is now

routinely used in numerous clinical applications for the detection of a wide range of bacterial,

fungal, parasitic and viral diseases of humans (Espy et al., 2006). Recent advances have seen a

6

number of studies utilising qPCR-based techniques for the identification of marine pests (see

Galluzi et al., 2004; Pan et al., 2008).

The development of these tests requires that the target organism is taxonomically unambiguous.

Testing of a number of species closely related to the target organism is required as well as testing

of environmental samples containing unknown taxa. Most test development achieves the first

criterion but for implementation, it is important to validate tests on samples exhibiting higher

complexity such as natural water and sediment samples.

Many PCR-based tests are developed based on nuclear ribosomal and mitochondrial gene

sequences (including tests from this study). Genes evolve at different rates and a suitable DNA

region should vary in sequence sufficiently to allow the identification of an individual to the

taxonomic level required. For specific identification, the DNA marker should exhibit little or no

genetic variation within a species but differ sufficiently between species so as to allow unequivocal

delineation.

In nuclear ribosomal genes and spacers, there is typically little variation amongst individuals of a

species within and between populations (Larsen et al., 2005; Livi et al., 2006). The ribosomal

DNA (rDNA) genes, Internal Transcribed Spacers (ITS) and Intergenic Spacer (IGS)/ Non-

transcribed spacer (NTS) regions have been shown to be particularly useful in defining species

specific markers for marine pest assay development. The mitochondrial genome is also utilised for

diagnostic purposes; mitochondria are generally inherited maternally making them particularly

useful as a species-specific marker for the delineation of closely related species (e.g. Blair et al.,

2006, Kamikawa et al., 2008).

In this report we detail the development of qPCR assays for M. senhousia and P. canaliculus, and

further specificity testing of the C. gibba qPCR assay developed by Ophel-Keller et al. (2007).

7

Methods Quantitative PCR (qPCR) assay design Assays were developed in the SARDI Diagnostics laboratory (see Figure 4) as qPCR, using

TaqMan® minor groove binder (TaqMan MGB) chemistry. DNA sequences of the desired genetic marker of target and related organisms were imported into the sequence manipulation software

Bioedit (available from http://www.mbio.ncsu.edu/RNaseP/info/programs/BIOEDIT/bioedit.html.),

and aligned using Clustal W. The genetic marker of choice is defined by the ability for that marker

to delineate the target from heterologous species and also by the availability of sequences from

publicly available databases. A range of DNA sequences was obtained from the public domain

database GenBank (http://www.ncbi.nlm.nih.gov/genbank/). The National Centre for Biotechnology

Information (NCBI), as a division of the National Library of Medicine (NLM) at the National

Institutes of Health (NIH), has developed databases to deal with molecular data, and facilitates the

use of molecular databases by the research and medical community, Genbank is one of these

databases and is an annotated collection of all publicly available nucleotide and amino acid

sequences.

Sequences of target and related taxa were aligned to infer sequence regions that appeared to be

useful diagnostic regions. DNA sequences were identified which were common to the target taxa

but where there were enough differences to distinguish target from related taxa. Specific PCR

primers and TaqMan MGB probes were developed for target taxa using the assay design software

Primer Express v2.0 (Applied Biosystems), an application that designs primers and TaqMan MGB

probes that display suitable thermodynamic properties and nucleotide content for efficient

amplification.

8

Figure 4: SARDI Diagnostics laboratory

Samples

Musculista senhousia samples (preserved in ethanol) were obtained from Singapore (thanks to Dr

Maylene Loo) and M. senhousia DNA was kindly donated by Dr Jawahar Patil (CSIRO). Perna

canaliculus samples were obtained frozen and purchased from an Adelaide fish monger.Other

mytilid material was obtained through Dr Richard Willan, Northern Territory Museum and Dr Paul

Fisher, Primary Industries and Fisheries, Queensland. Specimens of Corbula gibba were

collected from Port Philip Bay for Ophel-Keller et al. (2007). Specimens of Lentidium dalyfluvialis,

L. mediterraneum and Potamocorbula amurensis (Corbulidae) preserved in RNAlater (Ambion),

were obtained from Mr Anders Hallan, University of Wollongong. Other samples utilised for

specificity testing were collected whole (as part of other SARDI marine pest qPCR projects), and

immediately stored frozen, or preserved in ethanol, for genomic DNA (gDNA) extraction.

DNA extractions

gDNA was extracted from target and non-target samples by one of two methods. The first method

was the Root Disease Testing Service (RDTS) commercial DNA extraction method, a service

provided by SARDI Diagnostics, while the second method was the QIAGEN DNeasy Blood and

9

10

Tissue kit following the manufacturer’s instructions. DNA concentration was estimated by

fluorometry (Wallac 1420 multilabel counter) using Quant-iT™ PicoGreen® (Invitrogen). gDNA for

qPCR specificity experiments was typically diluted to 200 pg/μl.

Quantitative PCR qPCR reactions were carried out in 384 well plates for analysis on an ABI HT 7900 sequence

detection system (Applied Biosystems, Foster City, CA) using QuantiTect™ qPCR mastermix

(QIAGEN). Each qPCR assay was run with plate controls (no DNA control and positive control for

each assay) and was analysed with ABI SDS 2.3 software (Applied Biosystems). The PCR cycling

conditions were: 15 minutes at 95°C (activation) plus 40 cycles of 15 secs at 95°C (denaturation)

and 60°C at 1 minute (annealing). qPCR results are given as cycle threshold (Ct) values. The Ct

value represents the PCR cycle number at which the fluorescence signal passes a fixed threshold,

displayed as a horizontal green line in plots showing number of qPCR cycles vs magnitude of the

fluorescence signal intensity (∆Rn) (see Figures 5-7).

Results Primers and probes We designed a range of potential qPCR assays for the detection and enumeration of M. senhousia

and P. canaliculus. Primers and TaqMan MGB probes that have exhibited the highest level of

specificity to date are shown in Table 1. Other primer and probe combinations that did not offer

appropriate specificity or amplification efficiency were not considered further (data available on

request). Table 1 lists the genetic marker that the qPCR assay targets, the nucleotide content of

the primers and probes, and the melting temperature (Tm) of the primers and probes, which is

important for determining the reaction conditions of qPCR experiments. The primers and probe for

the C. gibba qPCR assay (developed by Ophel-Keller et al., 2007) are also shown in Table 1.

Tabl

e 1:

Prim

ers

and

TaqM

an M

GB

pro

bes

As

say

Gen

etic

M

arke

r Fo

rwar

d Pr

imer

(5’-3

’) Tm

(°

C)

Rev

erse

Prim

er (5

’-3’)

Tm

(°C

) Ta

qman

MG

B p

robe

Tm

(°

C)

Mus

culis

ta s

enho

usia

28

S

CG

GC

GG

TCA

GA

AG

CC

TGT

61

C

CA

GC

TATA

AA

CTC

CC

GA

CG

60

6FA

M- C

CG

GA

AG

GTG

AC

CC

G -M

GB

68

.1

Cor

bula

gib

ba

28S

G

GG

CA

GC

GTC

GTC

TTG

TG

61

CTA

TCG

GA

CTC

GTG

CC

TGTA

TTTA

G

58

6FA

M- A

TTC

CC

AA

AC

AA

CC

CG

- M

GB

68

Per

na c

anal

icul

us

IGS

C

GTA

ATC

CTC

AG

TAC

TGG

CTA

60

C

TCTA

AC

ATA

AG

GG

CTC

TAA

C

61

6FA

M- A

TAG

AG

TAG

AG

CTA

TTTA

GG

G -

MG

B

69.8

Key:

28S

-28S

ribo

som

al D

NA

. IG

S-In

terg

enic

spa

cer o

f mito

chon

dria

l DN

A. T

m-M

eltin

g te

mpe

ratu

re o

f prim

er/p

robe

. 6FA

M- 6

Car

boxy

fluor

esce

in (f

luor

opho

re),

MG

B-M

inor

Gro

ove

Bin

der n

on

fluor

esce

nt q

uenc

her.

11

Musculista senhousia A TaqMan MGB PCR assay was designed for M. senhousia by Ophel-Keller et al.,

(2007). Subsequent analysis of environmental samples collected from Queensland

showed that the assay lacked specificity, so it has been re-designed and re-

evaluated as part of this project. New primer and TaqMan MGB probe combinations

were designed in silico, based on alignments of 28S rDNA sequences, with the

intention of developing an assay specific to M. senhousia, based on available

controls (see Table 1). The assay was screened against a wide range of

heterologous controls, including mytilids, other bivalves and a range of other

invertebrate species (see Table 2 and Figure 5). We note that with one gDNA

sample of P. viridis, late amplification (i.e. ~ Ct of 40) was exhibited (not shown).

However this result is not consistent across all our P. viridis gDNA samples, and may

reflect a low level contamination of this sample with M. senhousia gDNA.

12

Table 2: Results of specificity testing for M. senhousia qPCR assay

Phylum Class Genus Species DNA (pg/ul) Ct values Mollusca Bivalvia Musculista senhousia 200 19.90

Corbula gibba 200 UD Musculus cummingianus 200 UD Musculus miranda 200 UD Perna viridis 200 UD Limnoperna securis 200 UD Modiolus micropterus 200 UD Trichomya hirsutus 200 UD Perna canaliculus 200 UD

Echinodermata Asterias amurensis 200 UD Chordata Ciona intestinalis 200 UD

Ascidiella sp. 200 UD Arthropoda Crustacea Carcinus maenas 200 UD Annelida Polychaeta Sabella spallanzanii 200 UD

Undaria pinnatifida 200 UD NTC UD

\ Figure 5: Amplification plot for Musculista senhousia qPCR assay specificity testing.

13

Corbula gibba A TaqMan MGB qPCR assay was designed for C. gibba by Ophel-Keller et al.

(2007). While there have been no reported issues with the specificity of the C. gibba

qPCR assay, it has not previously been adequately tested on DNA from other

corbulid species

The C. gibba assay was further tested with the corbulid species: Lentidium

dalyfluvialis, L. mediterraneum and Potamocorbula amurensis and found not to

cross-react with any of these species (Table 3 and Figure 6). Lack of specificity was

not encountered with the C. gibba qPCR assay during this study.

Table 3: Results of specificity testing for C. gibba qPCR assay Phylum Class Genus Species DNA (pg/ul) Ct values Mollusca Bivalvia Corbula gibba 200 20.73

Lentidium dalyfluvialis 200 UD L. mediterraneum 200 UD Potamocorbula amurensis 200 UD Musculista senhousia 200 UD Musculus cummingianus 200 UD Musculus miranda 200 UD Perna viridis 200 UD Limnoperna securis 200 UD Modiolus micropterus 200 UD Trichomya hirsutus 200 UD Perna canaliculus 200 UD

Echinodermata Asterias amurensis 200 UD Chordata Ciona intestinalis 200 UD

Ascidiella sp. 200 UD Arthropoda Crustacea Carcinus maenas 200 UD

Sabella spallanzanii 200 UD Undaria pinnatifida 200 UD NTC UD

14

Figure 6: Amplification plot for Corbula gibba qPCR assay specificity testing.

15

Perna canaliculus Blair et al. (2006) designed an end-point PCR assay for the specific detection of

Perna canaliculus based on mitochondrial gene sequences. The assay was modified

from Blair et al. (2006), utilising their specific reverse primer and designing a new

forward primer and Taqman MGB probe suitable for use as a qPCR assay. We have

tested the P. canaliculus qPCR assay with a wide range of heterologous taxa,

including Perna viridis. We attempted to obtain sample of P. perna from South Africa

but were unsuccessful. The P. canaliculus qPCR assay did not cross react with the

range of invertebrate taxa shown in Table 4 and Figure 7.

Table 4: Results of specificity testing for P. canaliculus qPCR assay

Phylum Class Genus Species DNA (pg/ul) Ct values Mollusca Bivalvia Perna canaliculus 200 22.37

Corbula gibba 200 UD Musculus cummingianus 200 UD Musculus miranda 200 UD Perna viridis 200 UD Limnoperna securis 200 UD Modiolus micropterus 200 UD Trichomya hirsutus 200 UD Musculista senhousia 200 UD

Echinodermata Asterias amurensis 200 UD Chordata Ciona intestinalis 200 UD

Ascidiella sp. 200 UD Arthropoda Crustacea Carcinus maenas 200 UD

Sabella spallanzanii 200 UD Undaria pinnatifida 200 UD NTC UD

16

Figure 7: Amplification plot for Perna canaliculus qPCR assay specificity testing

Discussion Quantitative PCR (qPCR) assays have been designed for M. senhousia, C. gibba

and P. canaliculus. These assays have been designed in TaqMan MGB format and

have been tested against a range of target and heterologous taxa.

Ophel-Keller et al. (2007) developed a TaqMan MGB qPCR assay for M. senhousia,

but subsequent testing of plankton samples from Queensland indicated that there

were specificity issues with it. This study set out to design a new M. senhousia

qPCR assay; once we had designed a putative assay the first step was sourcing new

target material from Singapore and gDNA from CSIRO Marine and Atmospheric

Research. Closely related native mytilids were also sourced for specificity testing.

Based on available specimens it appears that the qPCR assay described here is

specific to M. senhousia gDNA.

17

We adapted an end-point PCR assay for P. canaliculus (see Blair et al., 2006) to a

qPCR assay suitable for routine use in the SARDI Diagnostics laboratory. The P.

canaliculus qPCR assay is specific, based on available heterologous specimens,

including a range of mytilids. Testing the P. canaliculus qPCR assay with Perna

perna would be advantageous and efforts to source material will continue. It should

be noted that the end-point PCR developed by Blair et al. (2006) was specific to P.

canaliculus and we have utilised the specific element (reverse primer) of that assay

nd developed the forward primer and TaqMan MGB probe to be specific to P.

m Anders

allan, University of Wollongong and the C. gibba qPCR assay did not cross react

ith any of these specimens, or a range of other heterologous specimens.

ip with Biosecurity SA at SARDI’s Diagnostic laboratories; qPCR assays

eveloped for marine pests (including for this study) will be utilised for routine use at

. The

. senhousia, P. canaliculus and C. gibba qPCR assays will be utilised for the

a

canaliculus.

The C. gibba qPCR assay was developed by Ophel-Keller et al. (2007). While this

assay did not exhibit any specificity issues when first developed, there was a need to

test the assay with other corbulid species. Specimens were obtained fro

H

w

Conclusions and future approaches With continued development of these qPCR assays, in conjunction with the

development of assays for other significant marine pest species, it is feasible that

comprehensive surveillance for marine pests in South Australia can be achieved

using DNA based assays. Molecular-based testing of environmental samples (water

and sediments) offers the potential for more rapid and cost effective testing than

more resource and time intensive traditional sampling methods. The Australian

Testing Centre for Marine Pests (ATCMP) is proposed for establishment in

partnersh

d

ATCMP.

In a parallel project (also funded by Biosecurity SA), SARDI has been developing a

plankton collection method, in which samples are filtered and preserved for later

molecular analyses. It is anticipated that this method can be used, in conjunction

with qPCR, to detect a broad range of pest species for surveillance purposes

M

detection of DNA of target larval stages using this plankton collection strategy.

18

Specific diagnosis of marine pests is central to: (a) rapidly establishing the

prevalence and distribution of marine pest species in the environment in conjunction

with traditional sampling and taxonomic techniques; (b) monitoring changes in marine

pest distribution spatially and temporally; and (c) conducting targeted eradication and

control programmes if economics and logistics permit.

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References Bax N., Williamson A., Aguero M., Gonzalez E. and Geeves W. (2003) Marine invasive alien species: a threat to global biodiversity. Marine Polution 27: 313-23. Blair D., Waycott M., Byrne L., Dunshea G., Smith-Keune C., Neil KM. (2006) Molecular discrimination of Perna (Mollusca: Bivalvia) species using the polymerase chain reaction and species-specific mitochondrial primers. Marine Biotechnology. 8: 380-385. Bott N.J., Ophel-Keller K.M,. Sierp M.T., Herdina, Rowling K.P., McKay A.C., Loo M.G.K., Tanner J.E. and Deveney M.R. (2010). Toward routine, DNA-based detection methods for marine pests. Biotechnology Advances 28: 706-714. Crooks, J.A. (1998) Habitat alteration and community-level effects of an exotic mussel, Musculista senhousia. Marine Ecology Progress Series 162, 137-152. Crooks, J.A., and Soule, M.E. (1999) Lag times in population explosions of invasive species: Causes and implications. In Invasive species and biodiversity management (Sandlund, O.T.,et al., eds), Kluwer Academic Publishers, pp. 103-125. Currie, D.R., and Parry, G.D. (1999) Changes to benthic communities over 20 years in Port Phillip Bay, Victoria, Australia. Marine Pollution Bulletin 38, 36-43. Dexter, D.M., and Crooks, J.A. (2000) Subtidal benthic communities and the invasion of an exotic mussel in an urbanized southern California bay: A long-term history. Bulletin Southern California Academy of Science 99, 128-146. Espy M.J., Uhl J.R., Sloan L.M., Buckwater S.P., Jones M.F. et al. (2006) Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clinical Microbiology Reviews 19: 165-256. Galil B.S (2007). Loss or gain? Invasive aliens and biodiversity in the Mediterranean Sea. Marine Pollution Bulletin 55: 314–322. Galluzzi L, Penna A, Bertozzini E, Vila M, Garcés E and Magnani M. (2004) Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a dinoflagellate). Applied and Environmental Microbiology 70: 1199-206. Hayes K. R. and Sliwa C. (2003) Identifying potential marine pests- a deductive approach applied to Australia. Marine Pollution Bulletin 46: 91-8. Holmes, S.P., and Miller, N. (2006) Aspects of the ecology and population genetics of the bivalve Corbula gibba. Marine Ecology Progress Series 315, 129-140. Kamikawa R., Hosoi-Tanabe S., Yoshimatsu S., Oyama K., Masuda I. and Sako Y. (2008) Development of a novel molecular marker on the mitochondrial genome of a toxic dinoflagellate, Alexandrium spp., and its application in single-cell PCR. Journal of Applied Phycology 20: 153-159. Larsen J.B., Frischer M.E., Rasmussen L.J. and Hansen B. W. (2005) Single-step nested multiplex PCR to differentiate between various bivalve larvae. Marine Biology 146: 1119-29.

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Livi S., Cordisco C., Damiani C., Romanelli M. and Crosetti D. (2006) Identification of bivalve species at an early developmental stage through PCR-SSCP and sequence analysis of partial 18S rDNA. Marine Biology 149: 1149-1161. Ophel-Keller K, Loo M, Bott NJ, McKay A. (2007) Development of gene probes for introduced marine pest species (Musculista senhousia, Corbula gibba, Sabella spallanzanii). Report prepared for Department of Environment, Heritage and Water.

Pan M., McBeath A.J.A, Hay S.J., Pierce G.J. and Cunnigham C.O.. (2008) Real-time PCR assay for detection and relative quantification of Liocarcinus depurator larvae from plankton samples. Marine Biology 153: 859-870. Reusch, T.B.H., and Williams, S.L. (1998) Variable responses of native eelgrass Zostera marina to a non-indigenous bivalve Musculista senhousia. Oecologia 113, 428-441

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Wallentinus, I. and Nyberg, C.D. (2007) Introduced marine organisms as habitat modifiers. Marine Pollution Bulletin 55: 323–332. Wiltshire, K. Rowling, K. and Deveney, M. (2010) Introduced marine species in South Australia: a review of records and distribution mapping. South Australian Research and Development Institute (Aquatic Sciences), Adelaide. SARDI Publication No. F2010/000305-1. SARDI Research Report Series No. 468. 232p.

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Glossary of Terms ATCMP- Australian Testing Centre for Marine Pests. CCIMPE- Consultative Committee on Introduced Marine Pest Emergencies. Ct-Cycle threshold: qPCR cycle where fluorescence is observed above a threshold level indicating a positive result. DNA- Deoxyribonucleic Acid: genetic information responsible for the development and function of all organisms, with the exception of some viruses. gDNA-genomic Deoxyribonucleic Acid: the total DNA of an organism, or the genome of an organism. IGS- Intergenic Spacer of mitochondrial DNA. ITS-2: second Internal Transcribed Spacer: a region of ribosomal DNA that does not code for any genes. mtDNA- Mitochondrial DNA: the genome of the intracellular organelles called mitochondria. Considered an informative diagnostic region. NIMPCG- National Introduced Marine Pest Coordinating Group NTC- No Template Control: a PCR reaction with no DNA template added, is used to ensure that PCR is not previously contaminated i.e. NTC should not be a positive result. Nucleotide: Molecules, that when joined together make up the functional units of DNA. PCR- Polymerase Chain Reaction: enzymatic technique used for the amplification of nucleic acids (e.g. DNA). qPCR- Quantitative Polymerase Chain Reaction: PCR reaction whereby amplification is monitored in real time through the use of fluorescent dyes or probe based chemistry. TaqMan MGB probe-TaqMan Minor Groove Binder probe: hybridises to specific fragment of DNA and emits fluorescence; used to quantify target DNA in a sample. rDNA- ribosomal Deoxyribonucleic Acid: codes for vital cellular components in Eukaryotes; an informative diagnostic marker. RDTS- Root Disease Testing Service: a commercial diagnostic service at SARDI.

Executive SummaryAcknowledgementsIntroductionMusculista senhousiaCorbula gibbaPerna canaliculusMolecular testing methods for marine pests

MethodsQuantitative PCR (qPCR) assay designSamplesDNA extractionsQuantitative PCR

ResultsPrimers and probesMusculista senhousiaCorbula gibbaPerna canaliculus

DiscussionConclusions and future approaches

ReferencesGlossary of Terms

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