Analysis of bacteria-challenged wild silkmoth, Antheraea mylitta (lepidoptera) transcriptome reveals potential immune genes
© Gandhe et al; licensee BioMed Central Ltd. 2006
Received: 04 April 2006
Accepted: 21 July 2006
Published: 21 July 2006
In the recent years a strong resemblance has been observed between the insect immune system and the mammalian innate immune mechanisms suggesting their common origin. Among the insects, only the dipterans (Drosophila and various mosquito species) have been widely investigated for their immune responses towards diverse pathogens. In the present study we constructed and analysed the immune transcriptome of the lepidopteran Antheraea mylitta, an economically important Indian tasar silkmoth with a view to unravel the potential immune-related genes and pathways.
An expressed sequence tag (EST) library was constructed from mRNA obtained from fat bodies of A. mylitta larvae that had been challenged by infection with Escherichia coli cells. We identified 719 unique ESTs from a total of 1412 sequences so generated. A third of the transcriptome showed similarity with previously characterized immune-related genes that included both the known and putative immune genes. Of the four putative novel defence proteins (DFPs) annotated by PSI-BLAST three showed similarity to extracellular matrix proteins from vertebrates implicated in innate immunity, while the fourth was similar to, yet distinct from, the anti-microbial protein cecropin. Finally, we analysed the expression profiles of 15 potential immune-related genes, and the majority of them were induced more prominently with E. coli compared to Micrococcus luteus. We also identified several unknown proteins, some of which could have probable immune-related functions based on the results of the ProDom analysis.
The present study has identified many potential immune-related genes in A. mylitta some of which are vertebrate homologues and others are hitherto unreported putative defence proteins. Several genes were present as members of gene families, as has also been observed in other insect species.
Insects are evolutionarily successful organisms and occupy almost all habitats in nature. An efficient immune system is one of the attributes for this evolutionary success. However, unlike mammals, the insects lack an adaptive immune system. The insect immune response is comprised of cellular and humoral components. The former involves the action of haemocytes in phagocytosis of microbes, encapsulation of large pathogens and nodule formation  whereas the latter involves activation of prophenoloxidase cascade leading to melanisation of invading microorganisms  and synthesis of a battery of anti-microbial peptides .
Insect immunity is well studied in dipterans such as fruit flies and mosquito species [4–7]. Only limited information is available on genes induced on pathogen challenge in a few lepidopteran species that include the domesticated silkmoth, Bombyx mori , Cecropia moth,Hyalophora cecropia  and tobacco hornworm,Manduca sexta  and in these too the immune response pathways employed to combat pathogen infections remain to be fully characterised.
Abundant genetic resources are now available for B. mori, with a 9 X shotgun sequence coverage of its genome and more than 100,000 ESTs in dbEST (NCBI) [11–13]. With reference to insect immunity, the ESTs have been obtained from baculovirus-infected B. mori cultured cells and pupae, but no large scale information on bacteria-induced immune genes is as yet available.
In this study, we have constructed and analysed an immune transcriptome following bacterial challenge of the Indian tasar wild silkmoth, Antheraea mylitta, an economically important lepidopteran cultivated for silk production. Prior information on immune response genes in wild silkmoths is lacking except for a few peripheral studies. Two proteins from A. mylitta – a lysozyme protein, 3-D structure of which is elucidated  and a protease inhibitor have been characterized . We chose to examine the fat body transcriptome since it is a major immune organ in insects, analogous to the mammalian liver. We generated a total of 1412 ESTs, of which 31% could be ascribed to putative immune functions. We also validated the upregulation of a selected subset of genes from the immune transcriptome by semi-quantitative RT-PCR.
Results and discussion
Classification of EST functional class categories based on similarity searches with NCBI protein database.
No. of ESTs
Total no. of ESTs
Housekeeping genes and other genes not involved in immunity
Hypothetical/unknown insect proteins
Hypothetical/unknown non-insect proteins
Weak homology in NCBI*
No homology in NCBI
The sequences, which could not be assigned any function based on homology search in NCBI, were searched for conserved domains in ProDom database. Of the 260 clusters that had no matches with known proteins in NCBI BLAST, we could assign protein domain families to 196 clusters based on the ProDom search (see Additional data file 2). The remaining sequences did not show any hits in ProDom and should be further analysed by other specialized computational tools.
Signal peptide and transmembrane domains
We further screened the unannotated proteins (no hits or hits with hypothetical proteins in NCBI database) for the presence of signal peptide and absence of transmembrane domains. This characteristic of many of the immune proteins has been utilized to screen probable immune-related genes in large-scale transcriptome studies , although the reliability of this criterion to identify the immune related genes remains to be experimentally tested. In the present study, 25 out of 260 genes tested fulfilled these criteria and could be considered as potential immune-responsive genes. However, since all the 260 clusters checked are not all full-length sequences, it is possible that we may have missed some others amongst them that represent gene products harbouring signal peptide without transmembrane domain, and hence the actual number may be higher.
Putative immune proteins
Putative anti-microbial proteins
The abundant genes in A. mylitta transcriptome.
No of ESTs
Defense protein 1
Ribosomal protein S2, Attacin
Elongation factor 1α
The data generated in the present study conformed to the trend of anti-microbial genes being present as multiple gene families as observed in many other insect species and highlighted the essentiality of these genes in the organism . Another interesting finding with respect to immune response was the presence of a protein identical to seroin. This protein is reported to express in the silk glands of B. mori and is known to protect the cocoon from microbes . A homologue of anti-fungal protein, gallerimycin, previously characterized from Galleria mellonella and shown to be induced by LPS injection , was also found.
Putative pattern recognition receptors (PRRs)
PRRs in the insects bind to and detect pathogen associated molecular patterns (PAMPs) like lipopolysaccharide, peptidoglycan, β 1–3 glucan, lipotechoic acid etc [9, 25]. The putative PRRs identified in the present immune transcriptome analysis were hemolins, peptidoglycan recognition proteins (PGRPs), gram-negative binding proteins (GNBPs), lectins and mucins. A. mylitta fat body transcriptome revealed two different proteins resembling hemolin, an immune inducible protein implicated in insect immunity  and it would be worthwhile to study the function and specificities of the different proteins of hemolin family. Three types of PGRP-like proteins were found in the A. mylitta immune repertoire in the present study as compared to D. melanogaster where 13 PGRP genes are known to be involved in the activation of the various effector pathways in immunity .
Putative proteases and protease inhibitors
Various proteases and protease inhibitors regulate the diverse immune mechanisms like melanization, phagocytosis and induction of anti-microbial peptides . A homologue of a prophenoloxidase activating protease characterized in B. mori and M. sexta  was also found in the A. mylitta immune transcriptome. Several other classes of putative proteases like cysteine proteases, serine proteases and metallo-proteases were also identified. As many as thirteen distinct serpin-like (serine protease inhibitors) and twelve potential protease inhibitors were detected in the immune transcriptome. Five different serpins have earlier been identified in M. sexta and shown to differ in the induction pattern upon immune challenge . In the light of these studies, the information on serpins and protease inhibitors from the A. mylitta immune transcriptome will prove to be invaluable in further understanding of various immune pathways in insects.
Putative defence proteins with unknown function
Several potential new members of known protein families were identified in the present study. Among the new members, one was a putative lysozyme-like protein described in the previous section. An array of putative proteinase inhibitors and proteases were also found. Many of them are new, and their study would enhance our understanding of the mechanisms of proteolytic cascades in insect innate immunity. A few immunoglobulin (Ig) like molecules were identified by ProDom search (see Additional data file 2). Ig-like molecules- hemolin  and more recently Dscam have been implicated in insect immunity [28, 29] and it would be interesting to evaluate the role of these putative Ig-like molecules in insects. Among the potential immune proteins, we describe below in more detail four putative defence proteins (DFPs), for two of which (DFP-1 and DFP-4) we have confirmed the induction upon E. coli infection by semi-quantitative RT-PCR.
DFP-1, DFP-2 and DFP-3
Proteins similar to putative defence proteins (DFPs) revealed by PSI-BLAST analysis.
Novel proteins from A. mylitta
Description and function of proteins similar to DFPs.
DFP-1, DFP-2, DFP-3
1. Stromal cell derived factor receptor 2 homologue
Homo sapiens [GenBank:NM_001013660.1]
(catecholamine catabolism) reeler domain
Bos taurus [GenBank:Q9GLX9]
H. sapiens [GenBank:Q9HCB6]
Extracellular matrix, cell adhesion protein that promotes the attachment of spinal cord and sensory neuron cells
3. Reelin precursor
H. sapiens [GenBank:P78509]
Extracellular matrix serine protease. Enzymatic activity is important for the modulation of cell adhesion
Cecropin D precursor
L. obliqua [GenBank:AAV91462]
The immune response in insects is dynamic and different effector genes are likely expressed at different time points during infection, contributing to the ability of the insects to ward off infections in spite of the absence of adaptive immunity. The current transcriptome represents genes likely expressed upon E. coli infection in the A. mylitta fat body at 24 hrs post infection. Unexpectedly, the Imd pathway components that are implicated in the activation of various effector pathways upon gram-negative bacterial infection in Drosophila were not present in our transcriptome. The 24-hour post infection period may have been non-optimal for expression of some genes, and since the EST library was not normalized the less abundant transcripts may have gone undetected. Alternatively, it is possible that other pathways are involved in the immune induction in the moths.
The present study has increased the repertoire of lepidopteran-specific putative immune response genes by several hundred-fold. This will be a valuable resource for lepidopteran-specific immune studies in particular and insect immune studies in general.
Insects, bacterial inoculation and tissue collection
A. mylitta, 5th instar, day 3 larvae were procured from Regional Research Station, Warangal, Andhra Pradesh. Log phase E. coli cells (DH5α), washed and resuspended in saline (0.3 M NaCl, 0.005 M KCl), were injected into the haemocoel of the larvae as described earlier . At 24 hours post infection (hpi), larvae were dissected to isolate fat body, and the tissue was flash frozen in liquid N2 and then stored at -70°C till further use.
cDNA library construction and generation of ESTs
Total RNA was extracted from the fat body using Trizol reagent (Invitrogen). The complementary DNA synthesis was carried out using Stratagene ZAP-cDNA® synthesis kit following manufacturer's instructions. Directional cDNA library was constructed by cloning of cDNA fragments into pBluescript II SK (+) vector and electroporation into E. coli strain DH10B. Insert-containing plasmid clones were sequenced with RV-M primer (5'GAG CGG ATA ACA ATT TCA CAC AGG 3') with the aid of MegaBACE3000 sequencer.
Raw sequences obtained from sequence chromatograms were processed using several programs. A cut-off Phred Quality Value of ≥15 was assigned to extract quality sequences from chromatograms. The quality sequences were screened for the presence of vector sequences using 'Cross Match' program . Then masked vector sequences were automatically removed by in-house developed trimming tool. Sequences shorter than 50 bases were removed. The resulting high-quality sequences were assembled into sequence contigs with the TGICL program , which initially makes clusters using MegaBLAST and thereafter makes an assembly using CAP3 for each cluster generated in the first step. A cluster is defined as a unique sequence obtained either by multiple alignment of many sequences that are > 95% similar or derived from a single sequence. A cluster containing ≥2 ESTs is termed a contig and that containing only one sequence, a singleton.
The unique putative gene sequences obtained by clustering and assembly were annotated by running BLAST  against non-redundant (nr) protein database of NCBI. Further, BLAST output was parsed to classify the putative gene transcripts into different functional classes.
Analysis of unknown proteins
Proteins that did not show any significant hits in NCBI nr database or showed similarity to unknown or hypothetical proteins were characterized by additional computational tools.
a. Domain search
For the sequences showing high similarity to hypothetical and/or unknown proteins, and those showing weak similarity to nr protein database, domain search was performed using ProDom . Putative function was assigned based on the type of domains found.
b. Signal peptide and transmembrane domain analysis
Presence of transmembrane domains and signal peptide analysis was done on the transcripts not showing any significant hits in NCBI database. The signal peptide analysis was done by SignalP software  and trans-membrane domain analysis was done with TMHMM program .
c. Functional annotation with PSI-BLAST
The functional annotation of four novel immune upregulated transcripts (DFPs) was done using PSI-BLAST .
A. mylitta larvae were differentially challenged- a) Unchallenged, b) Saline-injected, c) M. luteus and d) E. coli. Log phase E. coli and M. luteus bacteria, washed and resuspended in insect saline (0.3 M NaCl, 0.005 M KCl) were injected (30 μl) into a set of A. mylitta larvae. One set each of saline-injected and uninjected larvae were kept as a control. Four tissues, namely fat body, epidermis, mid gut, and silk gland were dissected out and flash frozen in liquid nitrogen. Total RNA was isolated using Trizol reagent. To remove genomic DNA contamination, total RNA was treated with RNAse free DNAse (NEB) as prescribed by the manufacturers. cDNA was synthesized using MMLV reverse transcriptase (Invitrogen) and oligo dT primers from 1 μg of total RNA. The primers were designed for the selected ESTs by Primer3 software .
Semi-quantitative RT-PCR was carried out for all the four differentially challenged tissues using an Eppendorf master cycler under the following conditions- 94°C, 2 min- initial denaturation, 27 cycles (94°C – 30 s, 58°C- 30s, 72°C-2 mins) and a final elongation at 72°C for 10 mins. Actin cDNA was amplified as an endogenous control. PCR reaction components included: 1X buffer, 100μM dNTPs, 1.5 mM MgCl2, 0.5 units Taq polymerase (MBI), 0.5 μM primers. Primer sequences are enlisted in additional data (see Additional data file 5).
Obtaining full-length cDNA by 5' RACE
We had obtained full-length coding sequences of the DFP-1, 2 and 3 through EST sequencing. To acquire full-length DFP-4 cDNA, we carried out 5' RACE PCR using the 5' RACE kit (Clontech). The 5' ends were amplified by using an adaptor primer and a reverse gene specific primer. PCR was performed for 25 cycles in an Eppendorf master cycler. A 300 bp band was isolated, sequenced and confirmed to be the 5' DFP-4 sequences.
We acknowledge Ms Riti Mohapatra for her assistance in the bioinformatics analysis. We are thankful to Mr. Riccardo M. Bennett-Lovsey and Mr. Alex D. Herbert, graduate students, Imperial College, London, for their suggestions in annotating the unknown proteins. We also acknowledge Mr. Venkateswara Rao, Department of Sericulture, Andhra Pradesh, India for supplying A. mylitta larvae. JN acknowledges financial support from Department of Biotechnology, India. AG and AKP are recipients of the fellowship from University Grants Commission (UGC) and Council of Scientific and Industrial Research (CSIR), India respectively.
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