Research Article Open Access

Pradeep et al., J Clin Cell Immunol 2015, 6:5http://dx.doi.org/10.4172/2155-9899.1000370

Research Article Open Access

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ImmunologyISSN: 2155-9899

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Volume 6 • Issue 5 • 1000370J Clin Cell ImmunolISSN: 2155-9899 JCCI, an open access journal

*Corresponding author: Dr. A R Pradeep, Proteomics Division, Seribiotech Research Laboratory, CSB-Kodathi Campus, Carmelaram P.O; Bangalore – 560035, Karnataka, India, Tel: 91-80-28440651; Fax: 91-80-28439597; E-mail: arpradeepnair@gmail.com

Received July 24, 2015; Accepted October 28, 2015; Published October 30, 2015

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

Copyright: © 2015 Pradeep AR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AbstractHeterogeneous group of 19 host – response proteins were activated in commercially important silkworm,

Bombyx mori after infestation by dipteran parasitoid, Exorista bombycis. The proteins include components of Toll and melanisation pathways, autophagy and apoptosis regulators, chaperones, cytokines and proteolytic enzymes. We elucidated phylogenetic relation within each host – response proteins belong to different insect orders. Multiple sequence alignment with most similar sequences showed large proportion of amino acid conservation in stress proteins, melanisation components, cactus, chitinase and autophagy 5 – like whereas signal proteins and cytokines showed ~30 % amino acid conservation. Phylogenetic analysis of the proteins revealed divergence which is an adaptive mechanism to provide immunity against parasitic attacks. In order to analyze phylogenetic position of the host – response proteins, amino acid sequences of all the proteins from B. mori and similar sequences from representative insects were aligned and constructed a phylogenetic tree based on maximum likelihood method using MEGA 5.05 program. Bootstrap values for Tree building method were obtained from 1000 replicates. The phylogenetic tree revealed three clusters. On the phylogenetic tree, cluster A showed early divergence of caspase and later divergence of BmToll whereas cluster B showed early divergence of prophenol oxidase activating enzyme (PPAE) through an independent lineage. PPAE expression showed pleiotropic correlation with different genes indicating initiation of diverse immune processes by PPAE at different time points in the evolutionary tree. NF κB transcription factors, dorsal and relish were diverged from common ancestor with high bootstrap value (83%) however showed 58% amino acid similarity. Relish showed long insertions revealing amino acid variations from dorsal. Cluster C illustrated divergence of autophagy 5- like, apoptosis - inducing factor and prophenol oxidase in temporal fashion to protect cells initially or to induce programmed cell death at later stages of parasitism.

Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive EvolutionPradeep AR1*, Anitha J1, Panda A2, Pooja M1, Awasthi AK1, Geetha NM1, Ponnuvel KM1 and Trivedy K1

1Proteomics Division, Seribiotech Research Laboratory, Central Silk Board, CSB-Kodathi Campus, Carmelram P.O, Bangalore – 560035, Karnataka, India2Bioinformatics Center, Bose Institute, Kolkata, West Bengal, India

Keywords: Host-response proteins; Immunity; Functional divergence; Phylogeny; Parasitism; Bombyx mori

IntroductionIn insects, immune functions play pivotal role in controlling

pathogenic and parasitic attack. The immune mechanism comprised of activation of humoral immune system, epithelial responses, cellular immunity, non-specific melanisation reactions, cytokine changes, inflammation, proteolysis and stress responses [1,2]. Though host responses after microbial infection are well analyzed [3,4], those after eukaryotic parasite attack in insects are seldom reported [5,6]. Parasitic infection is known to be recognized by circulating hemocytes causing transient activation of immune genes to synthesize effector molecules as well as to induce encapsulation, phagocytosis, melanisation etc [7,8].

During large scale rearing of commercially important silk- producing lepidopteran insect, Bombyx mori, the worms are often attacked by bacteria, virus and fungus. After the infection, innate immunity components including peptidoglycan recognition proteins (PGRP), C-type lectins, fibrinogen-related proteins (FRP) and scavenger receptors were activated [4,9]. In addition to pathogenic attacks, B. mori larvae are infested by a dipteran fly, Exorista bombycis (Tachnidae). The fly lays eggs on larval cuticle. On hatching, maggot invade the body and live as an endoparasitoid that induced organismal variations like loss of biomass, less production of silk protein, biochemical changes etc., [10]. Our recent studies showed differential production of host-response proteins, upregulation of associated genes and activation of immunocompetence in B. mori induced by the infestation [11]. The host-response proteins comprised of stress proteins, cytokines, Toll-pathway components, proteolytic enzymes,

apoptosis – associated proteins and melanisation pathway components [12,13]. Genome-wide analysis showed that some of the immune proteins are paralogous [4,14] however structural differences indicated that the genes encoding them might have originated from a common ancestor by duplication or as splice variants [4] under different types of selection pressure [15]. Some genes like hemolin evolved as lineage-specific in different lepidopterans, Hyalophora cecropia, Samia cynthia ricini, Antheraea pernyi and B. mori [4,16]. Altogether, immune components of different insects belong to orthologous and paralogus proteins as well as independent lineage proteins indicating its evolutionary conservation [17,18].

Phylogenetic analsis involving various proteins of different families and functions point to its origin and divergence. Analysis of major intrinsic proteins (MIPs), plasma membrane intrinsic proteins (PIPs) and nodulin 26 like intrinsic proteins (NIPs) revealed protein

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Volume 6 • Issue 5 • 1000370J Clin Cell ImmunolISSN: 2155-9899 JCCI, an open access journal

phylogeny in eukaryotes [19,20]. Besides, bootstrap analysis allowed potential comparison of proteins that are conserved across different species and to divide them among different groups based on divergence [21].

In this report we examined conservation of host response proteins among different insect groups as well as divergence among the proteins through phylogenetic analysis using multiple host – response proteins identified from B. mori larvae and its similar sequences from few representative insect species, belong to different orders. The new approach using phylogenetic analysis using multiple host – response proteins revealed temporal divergence of the host – response proteins which is essential in vulnerable organisms for adaptation against parasitic attack.

MethodsRearing of the silkworm, B. mori, infestation by E. bombycis and

collection of integument, haemolymph and hemocyte samples were carried out as described earlier [11-13] and briefed here. Protein samples were extracted from integument using total protein extraction reagent (T-PER; Pierce). For protein from hemocytes, the cells were pelletized from haemolymph by centrifugation at 3000 rpm at 4oC and plasma was separated. The hemocytes were treated with lysis buffer containing Triton-X 100 (Sigma), protease inhibitor (Sigma; 100 x) and phosphatase inhibitor (Sigma; 1 x) for 10 minutes on ice and the supernatant was separated by centrifugation at 10000 RPM for 10

minutes at 4oC and quantified [22]. The protein samples were collected at different time points from control and E. bombycis – infested final instar larvae of B. mori. The protein samples were denatured by 2M urea and reduced by 1M DTT before resolving on 10% SDS-PAGE. For 2D electrophoresis analysis, the protein was extracted in 2D buffer [23,24], followed by focussing on 11 cm strip (pH 3-10) on Protean IEF cell (Biorad), reduced in DTT and alkylated in iodoacetamide as described earlier [11]. The second dimension was resolved on a 12% precasted PAGE gel (Biorad). Exclusive bands/ spots were identified from the infested tissues, cut and digested by trypsin (In-gel digestion) before analysing by mass spectrometry (MALDI-TOF-MS) and denovo sequencing [25,26]. List of the peptides of different host response proteins identified from all the three tissues is presented in Supplementary Table 1 A-D.

Phylogenetic analysis

Amino acid sequences of 19 host-response proteins of B. mori were obtained from NCBI database. The sequences were used for homology search by BLASTp program (NCBI). In order to identify similarities and evolutionary relationships, amino acid sequences of the homologous proteins from representative insect species belonging to different orders, Lepidoptera, Diptera, Coleoptera and Hymenoptera collected from NCBI website were used for multiple sequence alignment with the B. mori sequences using Clustal W2 [27] (www.ebi.ac.uk/tools/msa/clustalw2). Fully conserved amino acids (*), conserved substitutions (:), semi conserved substitutions (.) and amino acid replacements were

Proteins identified by mass spectrometry Biological function

Heat shock protein 70 Chaperone: Protein folding and transport; maintain cell homeostasisChaperoninsubunit 4 delta Chaperone: assists in the folding of actin, tubulin and other cytosolic proteins

Prophenoloxidaseactivating factor 3 Haemolymph prophenoloxidase activating factor, induces lethal melanization response by modulating innate immune response

Prophenoloxidase-2(pro-PO2) (Copper bindingregion)

Induces melanization, implicated in the defense against microbes and woundingPO oxidizes DOPA, and dopamine to melanin to produce quinones which are cytotoxic and facilitate killing of encapsulated pathogens/ parasites

Paralyticpeptide (PP) Cytokine: involved in defense reaction and growth blocking activity

Spatzle-1 Cytokine: Spätzle acts as the ligand of the Toll receptor to induce transcription of antimicrobial peptides

Notch homolog (Notch) Notch signaling controls numerous cell-fate specification events. Dysregulated Notch signaling causes several diseases. Notch mediates anti-apoptotic activity

DOPA decarboxylase DDC is an intermediate enzyme in the melanization pathway which decarboxylate DOPA to dopamine and is one of the essential enzymes for melanization events

Serpin-4 SERPINs inhibit the serine protease activity, thereby regulating the melanization activity

18 wheeler (18w)/ Toll Toll is a type I transmembrane receptor located on the plasma membrane that controls the dorsal-ventral patterning of the Drosophila embryo and involved in host resistance against fungal and Gram-positive bacterial infections

Chitinase (Chi),transcript variant 3

chitinase play a crucial role in postembryonic development and it is activated after the ecdysone surge during larval–pupal transformation. Chitinase (Endochitinase) catalyzes the hydrolysis of β-(1-4)-glycosidic bonds of chitin polymers to produce chitooligomers

Nedd2-like caspase Caspases induces inflammation as well as apoptosisApoptosis-inducing factor (AIF) AIF induces chromatin condensation and DNA fragmentation leading to apoptosis, in the absence of caspase

Autophagy 5-like (Atg5) Atg5 plays a role in autophagosome formation, and is also involved in a pro-apoptotic signalling pathway through cytochrome c release and caspase activation

β –N acetyl glucosaminidase Chitooligomers produced from chitinase are subsequently converted to monomers by exochitinase β-N-acetylglucosaminidases

Trehalase-2(membrane trehalase) The trehalose is converted into glucose by an enzyme, alpha,alpha-trehalase present in insect tissues

Candidate protein components of Toll pathwayCactus Inhibitor of NF-κB transcription factorsDorsal NF-κB transcription factor activated during signal transduction in Toll pathway to induce immune reactionRelish NF-κB transcription factor activated during signal transduction in IMD pathway

Table 1: Biological function of the host-response and immune-response proteins identified by mass spectrometry and three candidate proteins from integument, haemolymph and hemocytes of the final instar larvae of the silkworm, B. mori infested with maggot of E. bombycis.

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Volume 6 • Issue 5 • 1000370J Clin Cell ImmunolISSN: 2155-9899 JCCI, an open access journal

identified from each protein set. Phylograms based on similarity were realized for each of the proteins and the branch strength was evaluated by Bootstrap values of 1000 replicates.

In order to analyze phylogenetic position of the host-response proteins, amino acid sequences of all the 19 proteins from B. mori and its similar sequences from the representative insects from different insect orders were aligned. The aligned sequences were used to construct phylogenetic tree based on maximum likelihood method using molecular evolutionary genetic analysis (MEGA version 5.05) program [28]. Bootstrap values for Tree building method were obtained from 1000 replicates. The branch length information was obtained based on genetic distances derived from Jones-Taylor-Thornton (JTT) model [29]. Branch lengths are proportional to sequence divergence. The bar represents 0.2 or 0.5 substitutions per site.

Bioinformatics analysis

Transmembrane regions of each protein were predicted by DAS (Dense Alignment Surface method [30] (http://www.sbc.su.se/~miklos/DAS/) and Signal peptides were predicted by SignalP 4.0 [31] http://www.cbs.dtu.dk/services/SignalP/). Map position of each gene encoding the respective host- response protein in chromosomes was identified from KAIKOBASE (http://sgp.dna.affrc.go.jp/KAIKObase/). Conserved domains of each protein were identified from “CDD: NCBI’s conserved domain database” [32].

Results and DiscussionIn invertebrates, immune system recognizes and gets rid of

invading micro- and macroparasites through activation of humoral immunity and cell-mediated events. Invertebrates lack adaptive immunity represented by antibody production and key cellular players like T-cell and B-cell system present in mammals [33]. However humoral immune mechanism in invertebrates and vertebrates showed

significant conservation in immune pathways indicated common evolutionary origin [34]. In the lepidopteran model and commercially important silkworm, B. mori, infestation by the eukaryotic parasitoid, E. bombycis induced enhanced expression of immune proteins and other host-response proteins. Protein extracts of integument, hemocytes and haemolymph of B. mori larvae infested by E. bombycis and control larvae were resolved on 10% SDS-PAGE or by 2D electrophoresis. The gels showed exclusive bands and spots in the extracts of infested larvae and were absent in the tissue protein extracts of non-parasitized controls injected with 0.9% NaCl and in non-parasitized non-injected controls (Supplementary Figure 1). Mass spectrometry of these exclusive bands/spots from all the tissues showed specific peptides of proteins that perform different functions associated with humoral immunity, signalling, stress responses, cell homeostasis, autophagy, apoptosis, and proteolysis as well as melanisation reactions (Table 1). Observed and calculated mass, amino acid sequences of the peptides and intensity are given in Supplementary Tables 1A-1D. This indicated activation of heterogeneous group of host-response proteins which exhibit co-regulation, cross talk and synergetic activation after the infestation. Similar activation of Toll- and IMD pathways was reported from the honey bee Apis mellifera [3] and Drosophila [35,36]. Genes encoding these proteins (except paralytic peptide) have already been mapped on different chromosomes of B. mori (KAIKOBASE; Table 2).

Fourteen peptides of the stress protein, heat shock protein (Hsp70) were identified from tissues of infested larvae of B. mori indicated its abundance (Supplementary Table 1A-D) as the measured mass spectrometry signals showed linear dependence on the amount of materials present in the entire sample analysed [37]. On the other hand, some of the proteins like DDC, paralytic peptide, Autophagy – 5 like (Atg5) and apoptosis-inducing factor (AIF) were represented by few peptides indicating low abundance. However few peptides might not represent all isoforms of a specific protein [37].

Figure 1: Distribution of number of amino acids of host-response proteins that are activated in the final instar larva of B. mori after infestation by the dipteran parasitoid, E. bombycis. Note that signal proteins possess maximum number of amino acids and cytokines possess minimum number.

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Volume 6 • Issue 5 • 1000370J Clin Cell ImmunolISSN: 2155-9899 JCCI, an open access journal

information among the orders as well. Therefore similarity and divergence of the host – response proteins of B. mori was compared with similar proteins of other insects, identified by BLASTp (NCBI). Representative species of insects that showed highest level of similarity were selected from the insect orders, Lepidoptera, Diptera, Hymenoptera and Coleoptera (Supplementary Table 2). Out of them, search for immune genes that were activated after microbial infection, were performed in Drosophila melanogaster, Anopheles gambiae, Aedes aegypti, Apis mellifera and Tribolium castaneum in addition to B. mori [3,4,43-47]. In D. melanogaster, a similar search for immune genes had been made after infection by the eukaryotic parasitoid, Asobara tabida (Hymenoptera: Braconidae) [5]. In the present study, the amino acid similarity was analysed by multiple sequence alignment of each host response protein from B. mori with its similar sequences and the divergence was assessed through unrooted phylogram (Figures 2A-2S) which showed independent groupings with high bootstrap values revealing adaptive evolution in the immune proteins, depending on its functional differences [48].

B. mori Hsp70 is a secretory protein that is grouped with homologues of other lepidopterans with high bootstrap value (91-95%) (Figure 2A). The B. mori Hsp70 showed early divergence whereas that of other three lepidopteran species (Danaus plexippus, Helicoverpa armigera and Spodoptera frugiperda) showed orthology based on high bootstrap value (91–96%). In the coleopteran, T. castaneum, Hsp70 showed an independent genetic differentiation from other species. Similarly, chaperonin of B. mori and D. plexippus grouped independently with

Amino acid length of the host – response proteins varied from 131 (paralytic peptide) to 2463 (Notch) (Table 3). Two cytokines viz., paralytic peptide and spatzle were with shorter sequence length whereas signalling proteins (Toll and Notch) were with longest sequence (Figure 1). Average length of all except the signalling proteins was 501 ± 195 amino acids. Many of the major intrinsic proteins of insects (e.g., aquaporins) are over 600 amino acids length due to extended C-terminal ends [19] which support greater receptor-dependent signalling [38]. Out of the 19 proteins identified from B. mori, 18 had one or more transmembrane (TM) regions. Notch showed eight TM regions, AIF with four, Toll with three and trehalase with two TM regions. The TM domains prevent proteins moving out of the locale [39] and are essential for transmembrane interactions and stability [40] thereby enhances immune reactions at specific infected locations. Divergence and mutations in these regions affect functional variability of the immune proteins [41]. Seven proteins were with signal peptides and presumed to be transmembranous and/ or secretory in nature (Table 4). However, TM region/ signal peptide was not detected in phenol oxidase (PPO 2) sequence corroborate the view that PPO 2 may be released to haemolymph by cell rupture [4,42].

Similarity and DivergenceThe similar/orthologous sequences possess highest amino

acid conservation and low interferences due to long indels and larger divergence [19]. The amino acid substitutions in them could provide differences within a particular insect order and phylogenetic

Genes Chromosome number Scaffold Hit length (Hit

Rate) e-value (score) Hit query position

Hit scaffold position (nucleotide)

Hit chromosome position (nucleotide)

Bombyx mori autophagy 5-like (Atg5) 15 Bm_scaf3 208 (99.52%) 5e-111 (404) 155-362 4437616-4437823 chr15:5516648-5516441Bombyx mori apoptosis-inducing factor (Aif) 14 Bm_scaf155 65 (98.46%) 3e-32 (140) 156-220 337355-337549 chr14:337355-337549

Bombyx mori Nedd2-like caspase 10 Bm_scaf30 100 (83.00%) 5e-71 (164) 162-261 492940-492650 chr10:18710435-18710725

Bombyx mori serpin-4 28 Bm_scaf62 323 (99.69%) 7e-173 (607) 1-323 199963-200931 chr28:10271474-10272442

Bombyx mori prophenoloxidase activating factor 3 (PPAE-3) 25 Bm_scaf32 100 (99.00%) 1e-59 (206) 196-295 4253184-4253483 chr25:8534658-8534359

Bombyx mori prophenoloxidase-2 (pro-PO2) 16 Bm_scaf124 147 (97.28%) 4e-83 (310) 189-335 504911-504471 chr16:10124499-10124059

Bombyx mori dopa decarboxylase (LOC692675) 4 Bm_scaf13 261 (93.49%) 9e-146 (517) 152-408 5822539-5823321 chr4:12516195-12515413

Bombyx mori paralytic peptide (Pp) not mapped Bm_scaf419 48 (100.00%) 4e-18 (90.1) 14-61 18649-18792 not mappedBombyx mori heat shock protein 70 15 Bm_scaf3 518 (99.61%) 0.0 (866) 1-518 8362740-8364293 chr15:1591524-1589971Bombyx mori chaperonin subunit 4 delta (LOC692795) 12 Bm_scaf84 108 (89.81%) 1e-27 (125) 299-406 886257-886580 chr12:11693168-11693491

Bombyx mori spatzle-1 (SPZ1) 9 Bm_scaf14 58 (98.28%) 1e-25 (116) 150-207 3501020-3500847 chr9:15751894-15751721Bombyx mori BmToll7-2 (mature peptide region) 23 Bm_scaf31 1225 (100.00%) 0.0 (2258) 1-1225 2043906-2040232 chr23:5689254-5685580

Bombyx mori trehalase-2 17 Bm_scaf33 497 (46.68%) 2e-119 (430) 1-496 2868937-2870424 chr17:1557757-1556270

Bombyx mori Notch homolog (Notch) 15 Bm_scaf3 760 (99.74%) 0.0 (1369) 1065-1824 7592734-7595013 chr15:2361530-2359251

Bombyx mori chitinase (Chi), transcript variant 3 7 Bm_scaf15 99 (98.99%) 6e-53 (209) 245-343 3012593-3012889 chr7:8201344-8201640

Bombyx mori β-N-acetyl glucosaminidase 19 Bm_scaf36 563 (99.47%) 0.0 (1097) 1-563 3292100-3290412 chr19:10348231-10346543Bombyx mori Cactus 12 Bm_scaf125 327 (99.69%) 0.0 (640) 276-602 437272-436946 chr12:20246033-20246362Relish; Bombyx mori relish (Rel),transcript variant 1 9 Bm_scaf14 219 (100.00%) 2e-119 (434) 2372-

2590 1017957-1018175 chr9:13268831-13269049

Dorsal; Bombyx mori embryonic polarity protein dorsal 12 Bm_scaf6 349 (98.85%) 0.0 (660) 1203-

1551 161838-162186 chr12:1320156-1320506

Table 2: Details of chromosome mapping of the genes of immune response/host response proteins induced in fifth instar larva of B. mori after infestation by E. bombycis (source:KAIKOBASE).

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Host-response proteins identified from B. mori

Accession number Total length

(amino acids)

Number of fully conserved substitutions

Number of conserved substitutions

Number of semiconserved substitutions

Total substitutions (%)

Amino acid replaced at position*

Conserved domains at which substitution occurred**

Functions of the conserved domains**

Heat shock protein 70 ACL36370 658 354 123 48 525 (79.78%) ---- ----

Chaperonin subunit 4 delta NP_001040107 537 263 101 28 392

(72.99%)

p.Thr217Ala p.Ile283Met p.Ala436Ser

T Complex protein 1(cd03338)

To sequester non-native proteins inside their central cavity and promote folding

SERPIN-4 ACZ81437 410 27 47 18 92 (22.44%) p.Ile143Leu p.Leu297Met

SERPIN(cd00172)

Serine protease inhibitors which regulate coagulation cascades.

Paralytic peptide precursor NP_001036883 131 21 5 17 43 (32.82%) ----- ----- ------

Prophenoloxidase activating Enzyme AAL31707 386 79 40 21 140

36.27% p.Val229AlaTrypsin – like Serine protease(cd00190)

Serine protease

Prophenoloxidase-2 AAG09303 693 185 142 44 371 (53.53%)

p.Ile98Leu p.Trp214Tyr p.Asp281Ala p.Asp306Glu p.Asp314Glu p.Phe582Tyr

Hemocyanin(Pfam00372)

Hemocyanins and insect larval storage proteins

Aromatic-L-amino-acid decarboxylase(dopa decarboxylase)

NP_001037174 478 230 96 20 346 (72.38%)

p.Lys205Arg p.Arg207Lys p.Ala250Thr p.Lys359Glu p.Arg410Lys

DOPA decarboxylase family(cd06450)

Catalyzes decarboxylation of tyrosine.

Spatzle-1 precursor NP_001108066 277 11 10 05 26 (9.38%) ----- -- --

18 Wheeler precursor(Toll7-2)

NP_001116821 1295 185 78 34 297 (22.93%)

p.Leu240Met p.Leu293lIe p.Ile327Val p.Gln492Leu p.Ala1044Val p.Glu1137Asp

Amino acids up to the position 492 are in Leucine rich repeats (Pfam13855).Alanine at 1044 and Glutamine at 1137 are in the Toll/interleukin-1 receptor (TIR) homology domain (Pfam01582)

TIR is an intracellular signalling domain that mediates protein-protein interactions between the Toll-like receptors and signal-transduction components

Embryonic polarity protein dorsal isoform B

NP_001036896 572 99 50 25 174 (30.42%)p.Ile90Leup.Arg128Lysp.Ile301Val

Isoleucine and arginine are substituted in the N-terminal sub-domain of the Rel homology domain (RHD; cd07887) whereas Isoleucine at 301 is substituted in the IPT domain of Dorsal (cd01177).

RHD is transcription factors, which play roles in mediating innate immunity.Immunoglobulin-like fold, Plexins, Transcription factors (IPT) domain is part of NF kappa B transcription factor activated by stressful conditions

Nuclear factor NF-kappa-B p110 subunit isoform 1 (Relish/BmRel)

NP_001095935 937 75 58 45 178 (18.99%)

p.Asp134Glup.Asn194Aspp.Asn224Serp.Thr230Serp.Val670Ala

Substitution upto 224th position is in N-terminal sub-domain of the Rel homology domain (RHD) (cd07884). Valine substitution is at 670 in the Transient Receptor Potential Ca2+ Channel (TIGR00870) or in the ankyrin repeats (COG0666)

RHD domains are transcription factors that mediate innate immunity.TIGR00870 are members of the family that mediate entry of extracellular Ca2+ into cells in response to depletion of intracellular Ca2+ .Ankyrin repeats associated with signal transduction.

Cactus BAI67121 326 24 31 130 185 (56.75%) p.Ser23Thr Ankyrin repeat protein (PHA02874)

Associated with signal transduction

Notch homolog NP_001157370 2463 173 47 25 245 (9.95%)p.Leu622Val p.Asp742Asn p.Thr847Ser

Calcium binding EGF – like domain (smart00179; cd00054)

Calcium binding site

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Chitinase isoform 3 precursor NP_001166832 544 185 78 34 297 (54.59%)

p.Arg173Lys p.Pro404Ala p.Thr410Ala

Substitution at 173 is in the catalytically inactive chitolectin-chitotriosidase (cd02872).

The domain includes a large number of catalytically inactive chitinase-like lectins

Beta-N-acetylglucosaminidase BAJ20189 633 76 66 25 167 (26.38%)

p.Leu140Ile p.Ala343Thr p.Trp351Phe

The substitutions are in the domain beta-acetyl hexosaminidase and glycosyl hydrolase (cd06562; Pfam14845 & 00728; COG3525)

Beta-N-acetylhexosaminidases catalyze the removal of beta-1,4-linked N-acetyl-D-hexosamine residues.Glycosyl hydrolase (GH) family 20 is a catalytic domain with a triose phosphate isomerase (TIM) barrel fold that catalyse GH reactions

Autophagy 5-like NP_001135959 264 48 68 16 132 (50%) p.Gly211Asn p.Leu219Met

Autophagy protein 5 (Pfam04106)

Apg5 is directly required for the import of aminopeptidase I via the cytoplasm-to-vacuole targeting pathway.

Apoptosis-inducing factor NP_001189462 597 85 101 47 233 (39.03%)

p.Gly93Ala p.Phe408Val p.Pro442Ala p.Phe449Tyr

Phenylalanine substitution at 408th position is in pyridine nucleotide – disulphide oxidoreductase (Pfam07992).Substitution at 442 and 449 are in the domain Apoptosis inducing factor (AIF; Pfam14721).

Class I and class II oxidoreductases and also NADH oxidases and peroxidases.AIF is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis

Caspase Nc NP_001182396 438 21 34 26 81 (18.49) p.Leu297Phe p.Asp359Asn

Caspase domain, interleukin-1 beta converting enzyme (ICE) homologues (cd00032’ smart 00115; pfam006560

Cysteine-dependent aspartate-directed proteases that mediate programmed cell death (apoptosis)

Trehalase-2 BAE45249 642 168 99 47 314 (48.91%)

p.Asn148Ser p.Val161Phe p.Phe199Leu p.Val391Leu

Trehalase (pfam 01204)

It is known to recycle trehalose to glucose and protects of proteins and membranes against a variety of stresses

*p.Thr215Ala represents threonine at 215th position is substituted by alanine; **Accession number and details from NCBI Conserved domains

Table 3: Key to amino acid substitutions and conservations in the host- response proteins of B. mori when aligned with the similar proteins from representative species of other insect orders presented in supplementary Table 2.

Immune/ Host response protein Accession no.Transmembrane segment Position in sequence

Start Stop Length Signal peptide Cleavage site

Heat shock protein 70 ACL36370 235 251 17 1 - 20 20 and 21VCA-DD

Chaperonin subunit 4 delta NP_001040107 109 118 10 Not predictedSerpin-4 ACZ81437 52 64 13 Not predicted

Paralytic peptide precursor NP_001036883 9 22 14 1 - 23 23 and 24VNA-GV

Prophenol oxidase activating factor 3 AAL31707 18 31 14 1 - 24 24 and 25VVG-QK

DOPA decarboxylase NP_001037174 97 113 17 Not predicted

Chitinase isoform 3 precursor NP_001166832 16 31 16 1 - 20 20 and 21VQC-AD

β-N-acetylglucosaminidase BAJ20189 32 45 14 Not predicted

Spatzle-1 precursor NP_001108066 8 20 13 1 - 18 18 and 19 ISA-YK

18 wheeler precursor(BmToll7) NP_001116821

8 23 161 - 17 17 and 18

GWT-LM316 327 12529 539 11

Apoptosis inducing factor NP_001189462

33 49 17

Not predicted87 97 11

261 270 10354 364 11

Autophagy 5 like NP_001135959 46 50 5 Not predictedNedd2-like caspase NP_001182396 195 206 12 Not predicted

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Notch homolog NP_001157370

25 40 16

Not predicted

348 357 10544 553 10

1538 1547 101679 1700 221847 1857 111934 1946 131964 1974 11

Trehalase-2 BAE4524914 30 17

1 - 18 18 and 19VVA-DR591 615 25

Cactus BAI67121138 156 19

Not predicted145 154 10

Dorsal NP_001036896 212 216 5 Not predicted

Relish NP_001095935746 757 12

Not predicted924 935 12

Note: Immune proteins without transmembrane region and Signal peptides were not mentioned; Transmembrane region was predicted by DAS (Dense Alignment Surface method; Cserzo et al. 1997); Signal peptides were predicted by SignalP 4.0 (Petersen et al. 2011).

Table 4: Potential transmembrane region and Signal peptides from immune and host response proteins induced in fifth instar larva of B. mori through infection by E. bombycis.

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Figure 2A-2S: Phylogram realised after performing multiple sequence alignment by CLUSTAL W using total amino acid sequence of host-response proteins that are activated in the final instar larva of B. mori and its homologues from representatives of different insect orders: The proteins analysed were (A) – Hsp70, (B) – Chaperonin subunit 4 Delta, (C) – SERPIN – 4, (D) – paralytic peptide (PP), (E) – prophenol oxidase activating enzyme (PPAE), (F) – DOPA decarboxylase (DDC), (G) – prophenol oxidase 2 (PPO2), (H) – Chitinase, (I) – β-N-Acetylglucosaminidase, (J) – Spatzle, (K) – BmToll (18 wheeler), (L) – Nedd2 like Caspase, (M) – Autophagy 5- like (Atg5), (N) – Apoptosis - inducing factor (AIF), (O) – Trehalase, (P) – Notch, (Q) Dorsal, (R) – Relish and (S) – Cactus. Details of accession numbers of proteins, name of the insects and similarity exhibited are given in the Supplementary Table 2. Numbers in the nodes represent bootstrap values computed from 1000 replications.

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Volume 6 • Issue 5 • 1000370J Clin Cell ImmunolISSN: 2155-9899 JCCI, an open access journal

large bootstrap value (100%) (Figure 2B). However, the group showed delayed divergence from ancestor.

The serine protease inhibitor, Serpin-4 of B. mori showed close proximity with that of D. plexippus and grouped separately with high bootstrap value (98%) (Figure 2C). SERPIN-4 of dipterans formed a separate cluster.

Paralytic peptide (PP) is an insect cytokine belongs to a group of growth inhibitors. Multiple sequence alignment of BmPP showed similarity with that of Samia cynthia pryeri, but with a low bootstrap value (23%). PP of Spodoptera litura and Mythimna separata formed separate grouping with high bootstrap value of 81% (Figure 2D).

Prophenol oxidase activating enzyme (PPAE), a serine protease of B. mori is clustered with that of Manduca sexta with 98% bootstrap value (Figure 2E). PPAE of M. sexta and B. mori appeared as orthologs however PPAE of D. plexippus seems originated at an early stage from a common ancestor of all the lepidopterans. PPAE of all dipterans were clustered separately with 76% bootstrap value.

DDC phylogram showed three major groups; a cluster of B. mori and other lepidopterans, a group of dipterans and an isolate of hymenopteran representative with high bootstrap value (Figure 2F). Among the lepidopterans, DDC of B. mori and Manduca mucosa were orthologous to others with higher bootstrap value (78%) and showed phylogenetic differentiation at a later stage.

In B. mori, two subunits of prophenol oxidase (PPO) were reported viz., PPO1 and PPO2 [42]. However in our study, only PPO2 was identified by mass spectrometry indicating its larger expression after the infestation. The PPO2 phylogram consisted of two independent groupings, one of lepidopterans with 100% bootstrap values and another with all other insect species. Among the lepidopterans, PPO2 of Helicoverpa armigera and Spodoptera frugiperda were diverged at a later stage from a common ancestor and formed orthologs of B. mori (Figure 2G). PPO2 of coleopterans and dipterans diverged earlier than lepidopterans from the ancestor.

An endochitinase, chitinase of all the lepidopteran representatives, were grouped together with high bootstrap value (100 %) and showed an independent lineage descended from a common ancestor (Figure 2H). Chitinase of Danaus plexippus showed earlier divergence followed by B. mori and M. sexta. Chitinase of Helicoverpa armigera and Spodoptera litura showed an orthologous development. Lepidopteran chitinases showed later divergence than other insect orders. An exochitinase, β-N-acetylglucosaminidase (βGAm) from B. mori, D. plexippus and Spodoptera frugiperda were grouped separately from other lepidopteran, dipteran, coleopteran and hymenopteran species (Figure 2I). βGAm of Danaus plexippus and Spodoptera frugiperda were orthologs of B. mori with high bootstrap value (100%).

Two components of Toll pathway viz., a cytokine, spatzle and BmToll were identified from the parasitized larvae. Spatzle phylogram showed two major groups, one for all lepidopterans together and a second formed of dipteran, hymenopteran and coleopterans insects together (Figure 2J). Within the lepidopteran cluster, B. mori and M. sexta Spatzle were differentiated into orthologs with high bootstrap value (75%). Notably, Drosophila melanogaster spatzle showed its presence as outgroup to all other insect representatives. BmToll showed two major clusters, one for dipterans alone and another for lepidopterans and hymenopterans together (Figure 2K). B. mori and D. plexippus Toll were orthologus to that of S. frugiperda with high bootstrap value (97%).

The apoptosis-inducing protein, B. mori caspase (Nedd2-like caspase) was clustered with D. plexippus and H. armigera (Figure 2L). Caspase of Dipterans showed separate cluster with high bootstrap value whereas hymenopterans formed another subgroup. Atg5 of B. mori and D. plexippus showed separate subgroup from other lepidopterans, dipterans, coleopterans and hymenopterans (Figure 2M). B. mori Atg5 showed orthology to that of other insects with high bootstrap value (100%). AIF of B. mori and D. plexippus formed a group (Figure 2N) and were orthologous to other insects with higher bootstrap value (100%).

Membrane-bound trehalase-2 precursor of dipterans formed a major cluster. All other insect orders formed a second group including a lepidopteran subgroup. BmTrehalase showed close proximity with that of Spodoptera litura (Figure 2O) and is orthologous to other insects.

The signalling protein, Notch from B. mori and Tribolium castaneum formed a group with a bootstrap value of 69% whereas that of dipterans formed a separate group (Figure 2P).

Though Toll and Spatzle were detected by mass spectrometry, no other components of Toll or IMD pathway were reported from any tissues of B. mori larva after infestation by E. bombycis. Hence two NF κB transcription factors (TF), Dorsal (Toll pathway component) and Relish (BmRel; IMD pathway component) and a NF κB inhibitor, Cactus were considered as candidate proteins for analysis. Phylogram showed that Dorsal and Rel of B. mori as orthologous to other species (Figure 2Q-2R). Dorsal of Lepidopterans grouped separately from other orders whereas Rel of B. mori and H. armigera formed separate group from all other representative insect species. Cactus of B. mori and D. plexippus clustered separately whereas that of dipterans and hymenopterans were subgrouped separately (Figure 2S).

Cluster formation in the phylogram of each host-response protein of B. mori is influenced by the presence of amino acid substitutions and insertions. The conserved, semiconserved and substituted amino acids as well as amino acid replacements in each protein are given in Table 3. The conserved substitutions were higher in the stress proteins Hsp70 and chaperonin (72 to 80%) as well as in melanisation pathway components, Cactus, chitinase and Atg5 (>50%). The signalling proteins such as BmToll and Notch as well as SERPIN and PPAE showed low level (~30%) of amino acid conservation. In PPO2 and Toll, six amino acid replacements were observed in B. mori (Table 3). The cytokines, paralytic peptide and spatzle did not show amino acid variations whereas other proteins showed one to five amino acid replacements. Accumulation of conserved amino acids were reported in proteins that are crucial to reduce host – parasite interactions in the Plasmodium - mosquito system [49,50], whereas, absence of amino acid variation indicates a role of purifying selection against rare allelic variability [51,52]. Marked replacements of amino acids in different immune proteins can also occur due to action of negative selection on the encoding genes [50,51,53,54]. Such proteins may be under selective constraint and no change happens in the protein due to either functional constraints of the protein itself or in their targets [15].

However substitution of amino acids within similar proteins can alter response of the protein towards infection [55,56]. Notably, in most of the proteins, the amino acid substitutions were occurred within the active conserved domains (Table 3) indicative of significant implications on interaction of the respective immune proteins.

Amino acid diversity among identical proteins was observed in the dipteran insects, A. gambiae and several species of Drosophila [43-

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Volume 6 • Issue 5 • 1000370J Clin Cell ImmunolISSN: 2155-9899 JCCI, an open access journal

45], in the coleopteran, T. castaneum [47], hymenopteran, A. mellifera [3] and in the lepidopteran, B. mori [4]. Present study showed amino acid substitutions in specific conserved domains of different immune proteins indicating that the divergences are necessary to defend individual species from species - specific parasites/pathogens and for adaptation to its ecological niche under selection pressure [19,44,57-59].

Though the role of selection is undisputable, how infection interacts with evolutionary forces is not clear. However it is known that linkage and recombination influences gene interaction [60]. Interaction between multiple loci or genes could contribute to phenotypic variability such as occurrence of disease [61,62] which may, in turn, be influenced by linkage or recombination. Genes encoding the host – response proteins of B. mori (present study) were mapped on 13 different chromosomes (Table 2). Three genes each were located in Chromosome numbers 12 and 15 whereas Chromosome 9 had two and other chromosomes had one each. Genes found at larger distance as well as those located in different chromosomes may be evolutionarily independent however those located closer may undergo recombination together. However, “rates of recombination are not a uniform function of physical distance along the DNA” [63,64]. In B. mori, genes encoding the Toll pathway components, NF κB transcription factor Dorsal and its inhibitor Cactus are located on chromosome 12. It is interesting to investigate whether these genes undergo recombination together or not. In B. mori, upregulation of Dorsal is accompanied by down regulation of Cactus at 48 h after the infestation by E. bombycis whereas the reverse trend is noticed at 72 h after infestation indicating a negative correlation in expression pattern of these genes [11,12]. Under normal conditions, Cactus binds with Dorsal to prevent the transcription of antimicrobial proteins (AMP). Following infection, Cactus undergoes degradation and releases Dorsal to induce AMP gene transcription. Moreover, down regulation of Cactus gene was observed after infection [12,65,66] demonstrating that Cactus regulates the functioning of Dorsal through a gene – mediated feedback mechanism also [67]. Presence of Cactus and Dorsal genes together on the chromosome 12 seemed to promote an effective regulation of the Toll pathway which is developed through adaptation and balanced selection [68] to meet antiparasitic responses [69,70]. It has been shown that another NF κB transcription factor, Rel gained function by recombination with a locus encoding cytoplasmic retention domains [71]. However the probable recombinant hotspots [64] of Cactus – Dorsal pair in chromosome 12 of B. mori need to be confirmed.

Amino acid insertions and divergence

Sequence alignment revealed few short insertion sequences in host-

response proteins of B. mori (Table 2). In chaperonin delta subunit, proline, isoleucine and valine were inserted at 190-192nd position in B. mori and at corresponding region in D. plexippus. Insertions of new amino acids in the stress protein at a later evolutionary stage indicated its requirement to adapt under changed cellular milieu induced by infestation [48]. Such adaptive variations happen at a larger speed in immune proteins than that occurred in non-immune proteins [44,58,72]. Similarly, the amino acid sequence, APNLDDFSMVIQV at position from 461 – 474 in AIF of B. mori, was absent in other insect species compared here (Figure 3). Notably, this C-terminal dimerization domain (Pfam 14721) is a part of the conserved domain of mitochondrial AIF which is critical for energy metabolism and induction of caspase-independent - apoptosis by interacting with DNA [73]. Similar ‘indels’ (insertions and deletions) have been reported in active sites of Relish in fruit flies [44,74] which indicates that both AIF of B. mori and Relish of Drosophila are under selective pressure [48,58] induced by respective parasitic interactions.

Phylogenetic analysis

In order to understand functional diversity that evolved among various immune and host –response proteins to resist parasitic attack, a phylogenetic network among the proteins of representative species belonging to different insect orders is obligatory. A phylogram was realized through MEGA analysis with 1000 bootstrap replications using the aligned amino acid sequences of the 19 immune – and host - response proteins of B. mori as well as its similar sequences and orthologues from other insect species (Figure 4). The whole set of host-response proteins were grouped into three clades (Clusters A – C) of heterogeneous proteins which were supported by high bootstrap value (60-95%). Though the proteins were components of melanization pathway, Toll pathway, proteolytic enzymes, apoptosis-associated proteins, cytokines and chaperones from different insect orders, it showed uniform divergence pattern in the phylogeny tree indicating similar tendency of adaptation to develop immunity in different insect orders.

In B. mori, genes encoding the host-response proteins showed differential expression after the parasitic invasion [11-13]. Few immune genes showed regulated expression in control larvae which was deregulated due to the parasitic maggot infestation (Table 5). This is possibly to initiate new physiological roles for the gene products indicating functional divergence under the influence of infestation [75,76]. Thus a co-ordinated net-working of proteins of different functions is essential to elicit host defence mechanism against specific parasite attacks [4,77], which necessitate synchronized evolution of immune-associated proteins.

Figure 3: Multiple sequence alignment of portion of the amino acid sequence of apoptosis – inducing factor (AIF) of B. mori with homologues from other insect species showed an insertion of APNLDDFSMVIQV in B. mori which was absent in all other insects analysed. Symbol details are given in text.

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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All proteins of Cluster A were with TM regions indicating functional significance of TM region in the immune proteins by enhancing the transmembrane interactions [40,56,77]. An exception in this cluster was apoptotic enzyme Nedd2-like caspase, which possess TM region however without signal peptide. Phylogram showed large genetic divergence in this cluster. Caspase showed earlier divergence, whereas, BmToll showed slow divergence revealing that functionally different proteins respond differentially to selection [48]. Multiple sequence alignment of caspase showed merely 18.48% conserved sequences, leading to rapid diversity and evolution [78] possibly to protect the cells through apoptosis induced by infection [78,79].

BmToll is a member of the Toll receptor family that signals attack of different pathogens/parasites in vertebrates and invertebrates. The phylogram showed that in B. mori, Toll had evolved at a later stage of evolution presumably due to slow but accumulated demand (77% varied amino acids and six replacements; Table 3) by pathogen invasion to activate immune responses which is a recent adaptation in holometabolous insects [56,80,81]. Thus the BmToll is under strong evolutionary pressure by infections in different higher organisms including human beings [56, 82,83].

Presence of the cytokines, Spatzle and paralytic peptide in the cluster A and its divergence before BmToll was also key to adaptive

evolution of immune system in B. mori. Functionally, spätzle binds with Toll to activate the pathway [84], whereas, paralytic peptide activates the DOPA decarboxylase (DDC) of the melanisation pathway [85,86] indicating that both specific (Toll) and non-specific (melanisation) immune reactions have diverged synchronously in the B. mori immune system.

Cluster B showed common ancestry of NF κB transcription factors, Relish and Dorsal. Relish and Dorsal showed divergence by forking from a single ancestor with higher bootstrap value (83%) supporting that it is originated from a generalized ancient signalling system [87] that possesses significant DNA binding preferences [88]. Sequence alignment of Dorsal and Relish showed 58% conservation in the amino acid sequence positioned from 1-344. However, from 345 – 470th amino acid position, most of the amino acids present in Relish were absent in Dorsal showing 11 long deletions that seems to be the reason for functional diversity among the sister genes. Presence of ‘indel’ in Relish had been reported earlier in insects [15]. Similar deletions induced lack of enhancer activity in the desatF gene of D. melanogaster [89].

In cluster B, melanization initiation factor and prophenol oxidase activating enzyme (PPAE; a serine protease) showed early divergence through an independent lineage whereas serine protease inhibitor (SERPIN 4) was evolved at a later stage. Even though PPAE activates

Figure 4: Phylogenetic analysis showing evolutionary relationship among host – response proteins that are activated in the final instar larva of B. mori after infestation by the dipteran parasitoid, E. bombycis: Unrooted phylogram revealed three clusters (A, B, C) which showed protein divergence at different timings of evolution. Oval circle represents central node. Note the early divergence of caspase and late divergence of BmToll in Cluster A, fork formation of Dorsal and BmRel from a common ancestor in Cluster B and co-differentiation of Atg5, AIF and PPO2 in Cluster C. Bold arrow heads indicate the bootstrap values greater than 50% computed from 1000 replications. Accession numbers in red fonts in each group represent protein from B. mori. Details of the proteins are as given in the legend of Figure 2 and Supplementary Table 2.

Gene name Physiological function Co-regulated genes in B. moriDorsal NF κB transcription factor BmToll, SpatzleBmRel NF κB transcription factor PPAE, Hsp70, CaspaseSpatzle Cytokine that binds with Toll receptor to initiate the Toll signalling Dorsal, CaspasePPAE Enzyme that activates prophenol oxidase activity to initiate melanisation DDC, Chaperonin, Hsp70, BmRel, Caspase

Caspase Apoptosis induction Notch, Spatzle, PPAE, BmRel

*Anitha et al. 2013

Table 5: Genes that showed pleiotropic regulation* of expression of different genes in the normal larvae of B. mori and deregulated after infestation with E. bombycis.

Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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prophenol oxidase (PPO) to initiate melanisation [90], PPAE gene expression showed pleiotropic interaction with expression of more than one immune gene (Table 5). Association of PPAE with early and late evolved proteins of different immune functions displayed initiation of diverse immune processes by PPAE in B. mori larvae at different time points in the evolutionary tree.

An earlier activation of melanisation events is a prior requirement against specific or non-specific infection whereas continued melanisation could lead to quinone toxicity which could be prevented by the serine protease inhibitor (SERPIN). Thirty four SERPIN genes were identified from B. mori which might have originated by successive gene duplications at multiple genomic locations and formed gene clusters [91]. SERPIN 4 inhibits PPAE in the PPO activation pathway [92] and regulates quinone production. Moreover a compromised PO activity under the influence of selection was reported from the beetle T. castaneum [93]. This revealed that negative selection acts against immune system component after optimising an adapted trait (melanisation). Further a combination of negative (purifying) and positive selection could not only lead to low genetic variability [57] but interact to induce rapid functional and genetic diversity in immune proteins/genes [51,52,58].

Cluster C proteins did not possess signal peptide indicating that absence of signal peptides affect phylogenetic grouping of proteins. In this cluster, Atg5 and PPO2 formed a subgroup with 66% bootstrap value. Functionally, Atg5 is associated with autophagy [94,95]. Autophagy was induced in the integumental epithelium at 24 h after the E. bombycis maggot infestation in B. mori which was followed by lysis of the integument. The lysis showed a curvilinear relation with phenol oxidase (PO) activity indicating higher PO activity in mid – infestation which was preceded by activation of PPO2 gene [11,12]. Melanisation of the integument and autophagy in integumental epithelium are involved in protecting host larval integument from the early parasitic influence. This supports functional divergence and synchronized evolution of Atg5 and PPO2 from a common ancestor in the phylogram.

ConclusionIn the commercially important lepidopteran silkworm, Bombyx

mori, heterogeneous group of exclusive host-response proteins showed its larger presence after infestation by the dipteran parasitoid, E. bombycis. Multiple sequence alignment of the host-response proteins with its similar sequences showed orthology and accumulation of conserved amino acids which are crucial in reducing the host-parasite interaction to suppress parasitic influence. Few amino acids were replaced from most of the proteins, presumably due to action of negative selection on the encoding genes.

However under the phylogenetic analysis, host-response proteins from B. mori and its homologues from other insect orders showed uniform diversification pattern. The phylogram revealed three clusters of host-response proteins with functional diversity and temporal evolution. Caspase showed earlier divergence and rapid evolution whereas BmToll evolved at a later stage indicating that functionally different proteins respond differentially to selection. Similarly, serine protease, PPAE showed early independent divergence whereas serine protease inhibitor (SERPIN) diverged later and had diverse functions. The NF κB transcription factors, Dorsal and Relish originated early from a common ancestor but diverged in a forked fashion to perform transcription, probably through two (Toll and Immuno deficiency) pathways. Prophenol oxidase, Atg5 and apoptosis inducing factor

were originated from a common ancestor. Expression pattern of the genes encoding these proteins showed significant co-regulation which indicated that regulation of gene expression originated along with functional divergence. The phylogram showed temporal divergence of the cell death-associated proteins indicating its requirement in a temporal fashion to protect the cells from parasitism in the initial stages or to induce cellular death at later stages of infection. Thus the phylogenetic analysis of multiple host-response proteins of B. mori induced after the parasitic attack showed functional divergence and temporal evolution according to the functional requirement induced by parasitism in insects.

Acknowledgement

The authors are thankful to anonymous reviewers for critical reading and suggestions to improve the manuscript and to Central Silk Board for facilities. The authors acknowledge financial support received from Department of Biotechnology (DBT), Government of India, New Delhi, in the form of research projects to ARP (BT/PR11722/PBD/19/197/2008 dated 11/6/2009 and BT/PR6355/PBD/19/236/2012 dated 08/01/2013). AJ and PM are supported by research fellowship from the projects.

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Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370

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Volume 6 • Issue 5 • 1000370J Clin Cell ImmunolISSN: 2155-9899 JCCI, an open access journal

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Citation: Pradeep AR, Anitha J, Panda A, Pooja M, Awasthi AK, et al. (2015) Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution. J Clin Cell Immunol 6: 370. doi:10.4172/2155-9899.1000370