Genetic determinants of antiviral immunity in dipteran insects - Compiling the experimental evidence.

The genetic basis of antiviral immunity in dipteran insects is extensively studied in Drosophila melanogaster and advanced technologies for genetic manipulation allow a better characterization of immune responses also in non-model insect species. Especially, immunity in vector mosquitoes is recently in the spotlight, due to the medical impact that these insects have by transmitting viruses and other pathogens. Here, we review the current state of experimental evidence that supports antiviral functions for immune genes acting in different cellular pathways. We discuss the well-characterized RNA interference mechanism along with the less well-defined JAK-STAT, Toll, and IMD signaling pathways. Furthermore, we highlight the initial evidence for antiviral activity observed for the autophagy pathway, transcriptional pausing, as well as piRNA production from endogenous viral elements. We focus our review on studies from Drosophila and mosquito species from the lineages Aedes, Culex, and Anopheles, which contain major vector species responsible for virus transmission.

Effects of Quercetin on the Growth and Expression of Immune-Pathway-Related Genes in Silkworm (Lepidoptera: Bombycidae).

Quercetin is a flavonoid produced as a defense by plants. The effects of 1% quercetin on the growth and development of Bombyx mori were studied. The activities of the enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), carboxy-lesterase (CarEs), and glutathione S-transferase (GST) were all measured at 24, 48, 72, and 96 h after quercetin exposure. The results show that quercetin induces the activities of antioxidant and detoxification enzymes. With longer exposure times, enzyme activity first increased and then decreased. The relative expressions of AMP (defensin, CecA), the Toll pathway (cactus, Spatzle, and Rel), the IMD pathway (Imd, Fadd, and Dorsal), the JAK-STAT pathway (STAT, HOP, and Pi3k60), and the Melanization gene (DDC and PAH) were analyzed using quantitative polymerase chain reaction (qPCR). The results indicated that long-term exposure to quercetin could inhibit the expression of immune-related pathway genes in silkworms. This suggests that it can inhibit the activities of antioxidant and detoxifying enzymes, thus inhibiting the immune system and affecting the growth and development, resulting in an increase in the death rate in silkworm. This study provides the novel conclusion that quercetin accumulation inhibits the immune system of silkworm and increases its death rate, a result that may promote the development and utilization of better biopesticides that avoid environmental pollution.

Partner-specific induction of Spodoptera frugiperda immune genes in response to the entomopathogenic nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophila.

The Steinernema carpocapsae-Xenorhabdus nematophila association is a nematobacterial complex used in biological control of insect crop pests. The infection success of this dual pathogen strongly depends on its interactions with the host's immune system. Here, we used the lepidopteran pest Spodoptera frugiperda to analyze the respective impact of each partner in the induction of its immune responses. First, we used previously obtained RNAseq data to construct the immunome of S. frugiperda and analyze its induction. We then selected representative genes to study by RT-qPCR their induction kinetics and specificity after independent injections of each partner. We showed that both X. nematophila and S. carpocapsae participate in the induction of stable immune responses to the complex. While X. nematophila mainly induces genes classically involved in antibacterial responses, S. carpocapsae induces lectins and genes involved in melanization and encapsulation. We discuss putative relationships between these differential inductions and the pathogen immunosuppressive strategies.

Silkworm storage protein Bm30K-19G1 has a certain antifungal effects on Beauveria bassiana.

Storage proteins in the 30 K family are ubiquitous in the hemolymph of insects and play important roles in adult metamorphosis, development, egg formation, carrier transport and even host immunity. Some studies have shown that the 30 K proteins can inhibit apoptosis and have certain antifungal effects. The silkworm protein Bm30K-19G1 is a low molecular weight apolipoprotein that is abundant in hemolymph of fifth instar larvae. Our previous transcriptome sequencing, real-time PCR analysis and proteomic studies showed that the expression level of the 30 K protein gene was significantly up-regulated in the silkworm infected with Beauveria bassiana. In this study, the ORF sequence of Bm30K-19G1 was amplified by PCR. The sequence is 1311 bp in length and encodes a 436 amino acid peptide. Bm30K-19G1 was expressed in all tested tissues of fifth instar larvae, with highest expression in fat body and the lowest expression in the midgut. Bm30K-19G1 protein was successfully expressed in the prokaryotic expression system using pET-28a(+) as vector and E. coli Arctic Express (DE3) as the expression bacterium strain. The expressed recombinant Bm30K-19G1 protein has an inhibitory effect on the conidial germination and hyphal growth of B. bassiana. Bm30K-19G1 also inhibited the growth and reproduction of B. bassiana in vivo; the median lethal time of infected silkworms was postponed by 6.4 h and the time for death of all infected larvae was postponed by 10 h. The results indicated that the silkworm storage protein 30K-19G1 is an antifungal protein against B. bassiana and help to elucidate the molecular mechanism of silkworm resistance against B. bassiana.

Transcriptome analysis of differentially expressed genes involved in innate immunity following Bacillus thuringiensis challenge in Bombyx mori larvae.

In this study, we describe RNA-seq expression profiling of larval Bombyx mori response to hemocoel injection of Bacillus thuringiensis (Bt). Two transcriptomes were generated from the hemocytes of the PBS- and Bt-injected B. mori larvae. More than 49 million 100-bp paired-end reads, encompassing over 7.3 Gb of sequence data, were generated for each library. After filtering the raw reads and removing the rRNA mapped reads, more than 89% of the reads in each library could be mapped to the silkworm genome reference sequence. Comparison of gene expression levels revealed that a total of 133 unigenes were upregulated while 84 unigenes were downregulated in PBS vs Bt. To further investigate the biological functions of different expression genes (DEGs), gene ontology (GO) and functional enrichment analysis were performed to map all the DEGs to terms in the GO, euKaryotic Ortholog Groups of proteins (KOG) and Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database. Among these DEGs, many genes involved in immunity against Bt challenge were identified. These included genes participated in pattern recognition, antimicrobial peptides, insecticide resistance or detoxification, immune melanization, cytoskeleton reorganization and many other immune effectors. To confirm the gene expression patterns identified by the RNA-seq data, the transcript levels of 10 immune related DEGs were examined by quantitative real-time PCR (qRT-PCR). The results showed that the DEGs obtained from the deep sequencing data were accurate and gene expression profiles from RNA-Seq data were reliable. Our studies provide insights into the to immune response of B. mori underling the stress of Bt, which is valuable to understand how Bt affects the innate immune system of silkworm and provide new approaches to control insect pests by using Bt as a biological insecticide.

Important roles played by TGF-ß member of Bmdpp and Bmdaw in BmNPV infection.

Transforming growth factor (TGF)-ß superfamily members inhibit Bombyx mori nucleohedrovirus (BmNPV) multiplication in silkworm are not determined. In this study, we first found that BmNPV RNA transcription and protein expression level were regulated by TGF-ß members, Decapentaplegic (Bmdpp) and Dawdle (Bmdaw) in the domesticated silkworm, B. mori and silkworm ovary-derived cells. Furthermore, subcellular localization showed that Bmdpp and Bmdaw were mainly presented in cytomembrane of the cultured BmN cells. Tissues expression pattern analysis found that the highest expression levels of Bmdpp and Bmdaw genes were in the hemocyte of fifth instar larvae. During the immune response, the expression level of Bmdpp gene was elevated and Bmdaw gene was declined in BmNPV infected BmN cells and silkworm. The multiplication of BmNPV was inhibited by overexpression of Bmdpp and Bmdaw genes in BmN cells. RNA interference experiments found that the multiplication of BmNPV was raised with specific siRNAs of Bmdpp and Bmdaw genes in BmN cells. The antiviral immune pathways were not significantly regulated by the TGF-ß superfamily members. Taken together, these findings provided a clue to understand the function of Bmdpp and Bmdaw gene in response to the BmNPV infection in silkworm.

An Evolution-Based Screen for Genetic Differentiation between Anopheles Sister Taxa Enriches for Detection of Functional Immune Factors.

Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with broad specificity a rarity.

Genome-wide characterization and expression profiling of immune genes in the diamondback moth, Plutella xylostella (L.).

The diamondback moth, Plutella xylostella (L.), is a destructive pest that attacks cruciferous crops worldwide. Immune responses are important for interactions between insects and pathogens and information on these underpins the development of strategies for biocontrol-based pest management. Little, however, is known about immune genes and their regulation patterns in P. xylostella. A total of 149 immune-related genes in 20 gene families were identified through comparison of P. xylostella genome with the genomes of other insects. Complete and conserved Toll, IMD and JAK-STAT signaling pathways were found in P. xylostella. Genes involved in pathogen recognition were expanded and more diversified than genes associated with intracellular signal transduction. Gene expression profiles showed that the IMD pathway may regulate expression of antimicrobial peptide (AMP) genes in the midgut, and be related to an observed down-regulation of AMPs in experimental lines of insecticide-resistant P. xylostella. A bacterial feeding study demonstrated that P. xylostella could activate different AMPs in response to bacterial infection. This study has established a framework of comprehensive expression profiles that highlight cues for immune regulation in a major pest. Our work provides a foundation for further studies on the functions of P. xylostella immune genes and mechanisms of innate immunity.

Immune competence in insect eggs depends on the extraembryonic serosa.

Innate immunity is common to all metazoans and serves as a first line of defense against pathogens. Although the immune response of adult and larval insects has been well characterized, it remains unknown whether the insect egg is able to mount an immune response. Contrary to Drosophila, Tribolium eggs develop an extraembryonic epithelium, the serosa. Epithelia are well known for their ability to fight infection, so the serosa has the potential to protect the embryo against pathogens. To test this hypothesis we created serosa-less eggs by Tc-zen1 parental RNAi. We found that the Tribolium egg upregulates several immune genes to comparable levels as adults in response to infection. Drosophila eggs and serosa-less Tribolium eggs, however, show little to no upregulation of any of the tested immune genes. We conclude that the extraembryonic serosa is crucial for the early immune competence of the Tribolium egg.

Next generation sequencing based transcriptome analysis of septic-injury responsive genes in the beetle Tribolium castaneum.

Beetles (Coleoptera) are the most diverse animal group on earth and interact with numerous symbiotic or pathogenic microbes in their environments. The red flour beetle Tribolium castaneum is a genetically tractable model beetle species and its whole genome sequence has recently been determined. To advance our understanding of the molecular basis of beetle immunity here we analyzed the whole transcriptome of T. castaneum by high-throughput next generation sequencing technology. Here, we demonstrate that the Illumina/Solexa sequencing approach of cDNA samples from T. castaneum including over 9.7 million reads with 72 base pairs (bp) length (approximately 700 million bp sequence information with about 30× transcriptome coverage) confirms the expression of most predicted genes and enabled subsequent qualitative and quantitative transcriptome analysis. This approach recapitulates our recent quantitative real-time PCR studies of immune-challenged and naïve T. castaneum beetles, validating our approach. Furthermore, this sequencing analysis resulted in the identification of 73 differentially expressed genes upon immune-challenge with statistical significance by comparing expression data to calculated values derived by fitting to generalized linear models. We identified up regulation of diverse immune-related genes (e.g. Toll receptor, serine proteinases, DOPA decarboxylase and thaumatin) and of numerous genes encoding proteins with yet unknown functions. Of note, septic-injury resulted also in the elevated expression of genes encoding heat-shock proteins or cytochrome P450s supporting the view that there is crosstalk between immune and stress responses in T. castaneum. The present study provides a first comprehensive overview of septic-injury responsive genes in T. castaneum beetles. Identified genes advance our understanding of T. castaneum specific gene expression alteration upon immune-challenge in particular and may help to understand beetle immunity in general.

Resistance to BmNPV via overexpression of an exogenous gene controlled by an inducible promoter and enhancer in transgenic silkworm, Bombyx mori.

The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 10(5) occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity.

Caspar-like gene depletion reduces Leishmania infection in sand fly host Lutzomyia longipalpis.

Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.

A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella.

BACKGROUND: The larvae of the greater wax moth Galleria mellonella are increasingly used (i) as mini-hosts to study pathogenesis and virulence factors of prominent bacterial and fungal human pathogens, (ii) as a whole-animal high throughput infection system for testing pathogen mutant libraries, and (iii) as a reliable host model to evaluate the efficacy of antibiotics against human pathogens. In order to compensate for the lack of genomic information in Galleria, we subjected the transcriptome of different developmental stages and immune-challenged larvae to next generation sequencing. RESULTS: We performed a Galleria transcriptome characterization on the Roche 454-FLX platform combined with traditional Sanger sequencing to obtain a comprehensive transcriptome. To maximize sequence diversity, we pooled RNA extracted from different developmental stages, larval tissues including hemocytes, and from immune-challenged larvae and normalized the cDNA pool. We generated a total of 789,105 pyrosequencing and 12,032 high-quality Sanger EST sequences which clustered into 18,690 contigs with an average length of 1,132 bases. Approximately 40% of the ESTs were significantly similar (E ≤ e-03) to proteins of other insects, of which 45% have a reported function. We identified a large number of genes encoding proteins with established functions in immunity related sensing of microbial signatures and signaling, as well as effector molecules such as antimicrobial peptides and inhibitors of microbial proteinases. In addition, we found genes known as mediators of melanization or contributing to stress responses. Using the transcriptomic data, we identified hemolymph peptides and proteins induced upon immune challenge by 2D-gelelectrophoresis combined with mass spectrometric analysis. CONCLUSION: Here, we have developed extensive transcriptomic resources for Galleria. The data obtained is rich in gene transcripts related to immunity, expanding remarkably our knowledge about immune and stress-inducible genes in Galleria and providing the complete sequences of genes whose primary structure have only partially been characterized using proteomic methods. The generated data provide for the first time access to the genetic architecture of immunity in this model host, allowing us to elucidate the molecular mechanisms underlying pathogen and parasite response and detailed analyses of both its immune responses against human pathogens, and its coevolution with entomopathogens.

Comparison of the humoral and cellular immune responses between body and head lice following bacterial challenge.

The differences in the immune response between body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, were investigated initially by measuring the proliferation rates of two model bacteria, a Gram-positive Staphylococcus aureus and a Gram-negative Escherichia coli, following challenge by injection. Body lice showed a significantly reduced immune response compared to head lice particularly to E. coli at the early stage of the immune challenge. Annotation of the body louse genome identified substantially fewer immune-related genes compared with other insects. Nevertheless, all required genetic components of the major immune pathways, except for the immune deficiency (Imd) pathway, are still retained in the body louse genome. Transcriptional profiling of representative genes involved in the humoral immune response, following bacterial challenge, revealed that both body and head lice, regardless of their developmental stages, exhibited an increased immune response to S. aureus but little to E. coli. Head lice, however, exhibited a significantly higher phagocytotic activity against E. coli than body lice, whereas the phagocytosis against S. aureus differed only slightly between body and head lice. These findings suggest that the greater immune response in head lice against E. coli is largely due to enhanced phagocytosis and not due to differences in the humoral immune response. The reduced phagocytotic activity in body lice could be responsible, in part, for their increased vector competence.

A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori.

A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mori. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mori has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species.

Identification and functional analysis of antifungal immune response genes in Drosophila.

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.

Parasitization by the wasp Eretmocerus mundus induces transcription of genes related to immune response and symbiotic bacteria proliferation in the whitefly Bemisia tabaci.

BACKGROUND: The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and the viruses it transmits, are a major constraint to growing vegetable crops worldwide. Although the whitefly is often controlled using chemical pesticides, biological control agents constitute an important component in integrated pest management programs, especially in protected agriculture. One of these agents is the wasp Eretmocerus mundus (Mercet) (Hymenoptera: Aphelinidae). E. mundus lays its egg on the leaf underneath the second-third instar nymph of B. tabaci. First instars of the wasp hatch and penetrate the whitefly nymphs. Initiation of parasitization induces the host to form a capsule composed of epidermal cells around the parasitoid. The physiological and molecular processes underlying B. tabaci-E. mundus interactions have never been investigated. RESULTS: We used a cDNA microarray containing 6,000 expressed sequence tags (ESTs) from the whitefly genome to study the parasitoid-whitefly interaction. We compared RNA samples collected at two time points of the parasitization process: when the parasitoid first instar starts the penetration process and once it has fully penetrated the host. The results clearly indicated that genes known to be part of the defense pathways described in other insects are also involved in the response of B. tabaci to parasitization by E. mundus. Some of these responses included repression of a serine protease inhibitor (serpin) and induction of a melanization cascade. A second set of genes that responded strongly to parasitization were bacterial, encoded by whitefly symbionts. Quantitative real-time PCR and FISH analyses showed that proliferation of Rickettsia, a facultative secondary symbiont, is strongly induced upon initiation of the parasitization process, a result that supported previous reports suggesting that endosymbionts might be involved in the insect host's resistance to various environmental stresses. CONCLUSION: This is the first study to examine the transcriptional response of a hemipteran insect to attack by a biological control agent (hymenopterous parasitoid), using a new genomic approach developed for this insect pest. The defense response in B. tabaci involves genes related to the immune response as described in model organisms such as Drosophila melanogaster. Moreover, endosymbionts of B. tabaci appear to play a role in the response to parasitization, as supported by previously published results from aphids.

Identification of immune-related genes from an apterygote insect, the firebrat Thermobia domestica.

In this study, we report the analysis of the immune-related transcriptome from an apterygote insect, the firebrat Thermobia domestica (Zygentoma, Lepismatidae), which currently emerges as a suitable model insect for evolutionary and developmental studies. The suppression subtractive hybridization method was used for targeted screening of genes that are up-regulated in response to injected bacterial lipopolysaccharide (LPS). A subtracted cDNA library enriched in immune-inducible genes was constructed and analysis of 288 cDNAs resulted in identification of 26 novel genes in T. domestica. Among these immune-related transcripts we found homologues of genes from other insects which are involved in the regulation of signaling cascades and six novel putative antimicrobial peptides. The identified genes implicate the presence of sophisticated regulatory mechanisms in insect immune signaling and give insight into evolutionarily conserved features of insect innate immunity.

Beepath: an ordered quantitative-PCR array for exploring honey bee immunity and disease.

Honey bees and other insects face many important parasites and pathogens against which they have evolved behavioral, morphological, physiological, and immune-based defenses. To help validate honey bee immune-gene candidates and determine their responsiveness to pathogens, a quantitative-PCR array was developed to measure transcript levels for 48 honey bee and pathogen genes in parallel. It is shown that this array can accurately measure host and pathogen transcript abundance, providing a new tool for assessing the environmental and genetic components behind honey bee immunity. By using common platforms and chemistries (e.g., SYBR-Green intercalating dye), quantitative-PCR arrays such as the one described should have diverse uses in invertebrate pathology.

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua.

An entomopathogenic bacterium, Xenorhabdus nematophila, is known to depress hemocyte nodule formation of target insects by inhibiting eicosanoid biosynthesis. This study analyzed the inhibitory effect of X. nematophila on the humoral immunity of the target insects and tested its association with the host eicosanoid pathway. Plasma collected from the fifth instar larvae of Spodoptera exigua, when they were injected with X. nematophila, did not show antibacterial activity against Escherichia coli by a growth inhibition zone assay. In comparison, heat-killed X. nematophila induced significant antibacterial activity in the plasma. The antibacterial humoral activity was further demonstrated by examining a specific potent antibacterial peptide, cecropin. Two cecropin genes ('A' and 'B') were partially cloned from the fifth instar larvae of S. exigua by conserved degenerate primers using nested reverse transcriptase-polymerase chain reaction (RT-PCR). They showed high homologies with known cecropins from other lepidopteran species. Northern analysis using the cecropin probe showed that the injection of the heat-killed X. nematophila induced significant expression of a cecropin mRNA transcript (approximately 1.1 kb), but the larvae injected with the live bacteria did not show the corresponding transcript. Injection of arachidonic acid did not rescue the inhibition of X. nematophila based on either antibacterial activity or cecropin gene expression. The addition of dexamethasone, a specific phospholipase A2 inhibitor, did not inhibit antibacterial activity or cecropin gene expression when the larvae were injected with heat-killed X. nematophila. These results suggest that X. nematophila inhibits the antibacterial humoral immune reaction as well as the cellular immune reaction in S. exigua and that the inhibition of X. nematophila on the expression of the antibacterial peptide is not associated with inhibition of the eicosanoid pathway.