The entomopathogenic nematode, Heterorhabditis bacteriophora, and its mutualistic bacterium, Photorhabdus luminescens, are important biological control agents of insect pests 1 and represent an emerging model for research on mutualistic and parasitic symbiosis 23. The use of H. bacteriophora as a biological control agent is hampered by its susceptibility to environmental extremes including temperature, desiccation, and UV radiation, differences in virulence towards different insect pests, and short shelf life. Elucidation of molecular mechanisms underlying these biological processes may serve as a foundation for improving the biological control potential of the nematode-bacterium complex.

The entomopathogenic nematode H. bacteriophora has a distinct life style. The infective juveniles (IJs) or dauer juveniles (DJs) persist in soil in search of a suitable insect host 4. Following entry into the insect host through natural body openings and cuticle, the IJs regurgitate the symbiotic bacteria into the insect hemocoel 5. The bacteria kill the insect host via septicemia, usually within 24–48 h 5. The nematodes feed on the multiplying bacteria and disintegrated host tissues and produce 1 to 3 generations within the cadaver. When the food source depletes and nematode density reaches a threshold, next-generation IJs are formed which exit the cadaver in search of a new host 3.

In contrast to the closely related genetic model Caenorhabditis elegans, few genomic resources are available for H. bacteriophora. However, some progress has been made over the past few years with the generation and analysis of ~1,000 ESTs from H. bacteriophora GPS11 strain 36, the start of an H. bacteriophora complete genome sequence project (supported by the National Human Genome Research Institute), and the development of a reverse genetics tool using RNA interference 7. Release of the genomes of C. elegans 8, C. briggsae 9, Brugia malayi 10, bacterium Photorhabdus luminescens subsp. laumondii TTO1 (obligate endosymbiont) 11 and over 1 million ESTs from various nematode species deposited in GenBank offers unprecedented opportunities for the genetics of entomopathogenic nematodes. Here, we report on the construction of cDNA libraries from H. bacteriophora TTO1 adult hermaphrodites and the generation and analysis of 31,485 ESTs. The TTO1 strain is different from GPS11 strain in insect toxicity and their symbiotic bacteria (Grewal et al., unpublished). ESTs are valuable for gene discovery and can also be used in the identification of microsatellite markers 12. Therefore, we also identified microsatellite loci in H. bacteriophora ESTs for use in population genetic studies. In addition, ESTs will also aid the prediction of protein-coding genes in the annotation of complete genomes. However, domain identification, secretome prediction, phylogenetic and evolution analysis on the ESTs that are of short-length and low coverage are not very informative, and therefore were not performed.

This work produced a total of 31,485 high quality ESTs representing 10,886 distinct sequences. Sequence similarity searches of H. bacteriophora distinct ESTs showed 71.9% (7,828) matches to proteins from GenBank's nr database. The remaining 28.1% H. bacteriophora distinct ESTs represented novel genes yet to be assigned a function, demonstrating enormous novel gene discovery potential of this EST study. Among H. bacteriophora distinct ESTs having matches to proteins of other organisms in GenBank's nr database, a vast majority (95.9%) matched nematode proteins. About 71% (7,699) H. bacteriophora distinct ESTs match to 4,460 proteins from Wormpep190 that contains 23,771 extensively curated C. elegans proteins. H. bacteriophora homologs in C. elegans represent 18.8% of proteins in C. elegans. This finding suggests that H. bacteriophora and C. elegans have vastly different in proteomes, which may be explained in part by free-living versus parasitic life styles.

Interestingly, 26 distinct ESTs (0.3%) matched to proteins from various prokaryotic organisms (Table 2), all of which had less than 100% local sequence identities to prokaryotic sequences. These transcripts could result from horizontal gene transfer from bacteria encountered by H. bacteriophora during its life cycle. None of these ESTs matched to genes or proteins of P. luminescens subsp. laumondii TTO1, the natural symbiont of H. bacteriophora TTO1 11. Given the fact that poly(A) RNA was used in EST sequencing and the prokaryotic sequences were less than 100% identical to known prokaryotes, the possibility that these sequences are contaminants from other bacteria is low, although the possibility cannot be ruled out completely. The identification of sequences of putative prokaryotic origin in H. bacteriophora ESTs are consistent with our previous observations 6 and those observed in plant parasitic nematodes 14. The putative prokaryotic origin of these sequences could be tested more rigorously once the complete genome sequence becomes available.

Sequence similarity searches of H. bacteriophora distinct ESTs against ESTs of other nematodes and proteins revealed the presence of 554 parasitic nematode-specific ESTs (Additional file 3). Eighty-six percent of these ESTs matched ESTs from parasitic nematodes in clade V. Taken into consideration the fact that most FLNs included in this study were also from clade V, we are confident that these ESTs reflect the differences between parasitic and free-living nematodes and are not the result of phylogenetic constraint. These 554 H. bacteriophora ESTs had sequence similarities with ESTs from other parasitic nematodes, suggesting that these genes may participate in parasitism-related activities. Although the 2,523 ESTs without matches to any nematode ESTs could be H. bacteriophora specific, we hesitate to consider them as parasitic nematode specific at this time because they lack sequence similarity to ESTs of parasitic nematodes. Among these, the 2,371 ESTs without matches to any proteins in the current GenBank nr database represent potentially novel H. bacteriophora genes, and 81 of these were identified in the EST dataset of H. bacteriophora GPS11 strain 36. These findings suggest the enormous potential of discovering new genes and gene functions, genetic networks, and metabolic pathways specific to H. bacteriophora and other entomopathogenic nematodes. The identification of H. bacteriophora ESTs shared with other parasitic nematodes through our EST comparison opens the door for conducting in-depth research on gene functions that will ultimately elucidate the parasitic nematode-specific biological processes.

Among the 554 parasitic nematode-specific ESTs are those encoding F-box-like/WD-repeat protein, theromacin, Bax inhibitor-1-like protein, and PAZ domain containing protein. EST FF678238 encodes a homolog of the F-box-like/ED-repeat protein in Brugia malayi 10. The WD-repeat is commonly associated with F-box domain that mediates protein-protein interactions in a variety of contexts such as polyubiquitination 15. EST FF678397 encodes a protein similar to the PAZ domain containing protein in Brugia malayi 10. The PAZ (Piwi, Argonaut and Zwille) domain has nucleic acid-binding capability and is potentially involved in post-transcriptional gene silencing 16. Further investigation is needed to elucidate the functions of these two ESTs with common domains and whether they are related to parasitic nematode-specific processes.

Two other parasitic nematode-specific ESTs are Contig2528 and Contig1066. Contig2528 encodes a homolog of theromacin in the segmented worm Theromyzon tessulatum 17. Theromacin is a novel antimicrobial peptide acting against Gram-positive bacteria but without any similarities to other known antimicrobial peptides 17. H. bacteriophora TTO1 is obligately symbiotic with Photorhabdus luminescens subsp. laumondii TTO1 in natural environments. The production of an antimicrobial peptide could help establish the symbiotic relationship by selectively eliminating competing microbes. It is also possible that the antimicrobial peptide is a defense mechanism against potentially harmful microbes in the environment. Contig1066 encodes a protein similar to the uncharacterized protein family UPF0005 containing protein in Brugia malayi (GenBank accession number XP_001896958 http://www.ncbi.nlm.nih.gov/protein/170584338) and BAX inhibitor-1-like protein in wasp Nasonia vitripennis (GenBank accession number XP_001605379 http://www.ncbi.nlm.nih.gov/protein/156542785). BAX inhibitor-1 is a member of Bcl-2 family that suppresses programmed cell death 18. Transmembrane BAX inhibitor motif protein (TMBI) homologs have been identified in C. elegans, C. briggsae, C. japonica, C. remanei, and Pristioncus pacifiucs (Wormbase). However, these genes have very low sequence similarities to H. bacteriophora Contig1066. BAX inhibitor-1 is involved in preventing endoplasmic reticulum stress-related programmed cell death in Arabidopsis 19 and humans 20.

GO assignments based on sequence similarity searches aid identification of H. bacteriophora distinct ESTs involved in different biological processes. Here we discuss the genes involved in some biological processes of interest in detail. A number of ESTs related to defense responses and stress responses were identified in these H. bacteriophora distinct ESTs (Additional File 2) based on GO assignments. Among H. bacteriophora distinct ESTs involved in defense response are 3 ESTs encoding a homolog of C. elegans SMEK (Dictyostelium suppressor of MEK null) homolog that is essential for DAF-16-mediated defense response to pathogenic bacteria and increased resistance to oxidative and UV-induced damage 21. EST ES410098 encodes a heat shock protein HSP16-1 that is induced solely in response to heat shock or other environmental stresses 22. Another 13 ESTs encoding 5 different proteins whose C. elegans homologs exhibit a "pathogen susceptibility increased" phenotype when silenced by RNAi 23. However, the molecular functions of these proteins have yet to be elucidated. The defense response transcripts may be involved in the protection of entomopathogenic nematode IJs from bacterial or fungal pathogens and the insect innate immune system.

Five H. bacteriophora distinct ESTs involved in stress response encode a homolog to C. elegans catalase CTL-2 that likely is involved in protecting cells from reactive oxygen species as an antioxidant enzyme 24. Another EST (ES742296) encodes a protein whose C. elegans homolog showed an "organism stress response abnormal" phenotype when silenced with RNAi 25. Functions of other ESTs involved in stress response are yet to be clearly characterized. These transcripts related to stress responses provide workable targets for the improvement of ultraviolet, desiccation, and heat stress tolerance, traits desperately sought for improving the biological pest control potential of H. bacteriophora. Once the functions of these genes are determined, they can be potentially used for genetic manipulations of entomopathogenic nematodes. ESTs involved in dauer larval development, dauer entry, and dauer exit were also identified (Table 4) according to GO assignments. The infective juvenile stage of entomopathogenic nematodes is developmentally similar to the dauer stage in many bacteria feeding nematodes, including C. elegans and C. briggsae. The dauer is a developmentally arrested stage triggered by food deprivation, high population density, and other harsh environmental conditions 26. Elucidation of this process is of specific interest in the case of entomopathogenic nematodes because the dauer juvenile is the only life stage capable of infecting insects 4.

RNA interference represents a powerful technique for analysis of gene function. An RNAi system relying on soaking in double-stranded RNA solution has been established in H. bacteriophora 7. Interestingly, we were able to identify only a small number of known RNAi related genes in H. bacteriophora (Table 3) compared to C. elegans and B. malayi 10.

We have identified genes encoding RNAi induced silencing complex (RISC) components. One EST (EX014403) encodes a homolog of C. elegans TSN-1 (71% similarity at the amino acid level) and another EST (Contig211) encodes a homolog of C. elegans VIG-1 (45% similarity at the amino acid level). TSN-1 (Tudor staphylococcal nuclease) containing 5 staphylococcal/micrococcal nuclease domains and a tudor domain is a RISC component in C. elegans, Drosophila and mammals 27. The purified TSN-1 from C. elegans was shown to have nuclease activity and therefore thought to contribute to RNA degradation in RNAi 27. The product of the vig-1 gene was also shown to be a component of RISC 28. We did not identify a member of Argonaute family in this EST set based on sequence similarity. However, we identified an EST (FF678397) encoding a protein similar to a PAZ domain containing protein from Brugia malayi (57% similarity at the amino acid level). Another EST (FF679415) encodes a putative homolog to Drosophila and human Drosha 29 rather than Dicer in C. elegans. These findings suggest that H. bacteriophora may have structurally different RNAi pathway components than its relative, C. elegans.

Other RNAi related genes we were able to identify are those encoding homologs of SMG-2, SMG-5, RDE-4, GFL-1, and ZFP-1. SMG-2 and SMG-5 are involved in nonsense-medicated mRNA decay (NMD) where eukaryotic mRNAs with premature stop codons are selectively and rapidly degraded 3031. The other three genes, rde-4 32, gfl-1 and zfp-1 33 were shown to be involved in RNAi via RNAi evidence. We currently are not able to identify a gene encoding a SID-1 homolog in H. bacteriophora TTO1 that was shown to be necessary for systemic RNAi 34. However, a sid-1 gene has been found in H. bacteriophora GPS11 3. It is possible that more known genes may be identified when the complete genome of H. bacteriophora TTO1 is sequenced.

This EST project also enabled the development of genetic markers. We have identified 168 microsatellite loci from H. bacteriophora distinct ESTs, of which we were able to design primers for 141 based on the flanking sequences. These microsatellite markers may be useful for genetic mapping, linkage analysis, and population genetic studies. In a separate effort, microsatellite loci with 2- or 3-bp repeat units were selected for microsatellite marker development, along with the microsatellite loci enriched from genomic DNA of H. bacteriophora 35. Eight polymorphic microsatellite loci were demonstrated within a Northeast Ohio population.

We have generated 31,485 high quality H. bacteriophora ESTs representing 10,886 distinct sequences. Among these, 7,828 (71.9%) ESTs matched to proteins in GenBank's nr database. The vast majority (95.9%) of the best matches was to nematode proteins, a small portion (0.3%) to prokaryotic proteins and the remaining 3.8% to other eukaryotic proteins. GO terms were assigned to 6,685 H. bacteriophora distinct ESTs. "Embryonic development ending in birth or egg hatching" and "protein binding" were the most dominant terms in the categories of Biological Process and Molecular Function, respectively. This EST collection offers unprecedented opportunities for research on this unique nematode-bacterium symbiotic complex. The comparison of ESTs of H. bacteriophora TTO1 with those of AHPNs, FLNs, and PPNs resulted in the identification of 554 parasitic nematode-specific ESTs. These ESTs should be valuable for future research related to insect parasitism by these nematodes. We were able to identify a small number of ESTs involved in RNAi, among which is an EST encoding a Drosha homolog, suggesting structurally different RNAi pathway components from those in C. elegans. In addition, we have identified 157 microsatellite loci which may prove valuable once their polymorphisms are tested and validated. Overall, novel, parasitic nematode-specific, and C. elegans homologous genes have been identified in this EST study, greatly facilitating genome annotation, gene functional analysis, population genetic studies, and microarray development.

XB conducted vector sequence removal, assembly and analysis of EST sequences and prepared the manuscript. TAC provided the poly(A) RNA for cDNA library construction. RKW, SC, and JS led groups at the Genome Center at Washington University School of Medicine in St. Louis, MO that conducted cDNA library construction, sequencing, and data deposition. PSG, BJA, TAC, SC, RG, SAH, JS, and PWS initiated the study and obtained funding. PSG supervised the study and assisted in manuscript preparation. All authors read, edited, and approved the final manuscript.