Highlighting Potential Physical and Chemical Cues Involved in Conspecific Recognition System in a Predator Nematode, Seinura caverna.

Conspecific recognition is the ability to distinguish and respond to individuals of the same species. In nematodes, this behavior can mediate aggregation, feeding behavior, or mating. Here, we investigated whether and how the predatory nematode Seinura caverna recognizes and avoids conspecifics to prey on. In predation assays, S. caverna did not kill conspecifics, but killed nematodes of three heterospecific species. Interestingly, S. caverna did not kill Ektaphelenchoides spondylis nematodes. Seinura caverna did not eject its stylet when encountering conspecifics or E. spondylis. The characterization of the internal cuticle structure of 13 nematode species suggested that the cuticle may play a role in the preying decision, as E. spondylis and S. caverna exhibited similar, type III, cuticle layers. Chemical extracts from S. caverna further repelled conspecifics. We discuss the potential hierarchical use of physical and chemical cues in S. caverna predation behavior and provide insights into the evolutionary adaptations and behavior of this organism.

The Aphelenchoides genomes reveal substantial horizontal gene transfers in the last common ancestor of free-living and major plant-parasitic nematodes.

Aphelenchoides besseyi is a plant-parasitic nematode (PPN) in the family Aphelenchoididae capable of infecting more than 200 plant species. A. besseyi is also a species complex with strains exhibiting varying pathogenicity to plants. We present the genome and annotations of six Aphelenchoides species, four of which belonged to the A. besseyi species complex. Most Aphelenchoides genomes have a size of 44.7-47.4 Mb and are among the smallest in clade IV, with the exception of A. fujianensis, which has a size of 143.8 Mb and is one of the largest. Phylogenomic analysis successfully delimited the species complex into A. oryzae and A. pseudobesseyi and revealed a reduction of transposon elements in the last common ancestor of Aphelenchoides. Synteny analyses between reference genomes indicated that three chromosomes in A. besseyi were derived from fission and fusion events. A systematic identification of horizontal gene transfer (HGT) genes across 27 representative nematodes allowed us to identify two major episodes of acquisition corresponding to the last common ancestor of clade IV or major PPNs, respectively. These genes were mostly lost and differentially retained between clades or strains. Most HGT events were acquired from bacteria, followed by fungi, and also from plants; plant HGT was especially prevalent in Bursaphelenchus mucronatus. Our results comprehensively improve the understanding of HGT in nematodes.

Coprinopsis cinerea dioxygenase is an oxygenase forming 10(S)-hydroperoxide of linoleic acid, essential for mushroom alcohol, 1-octen-3-ol, synthesis.

1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. The biosynthetic pathway forming 1-octen-3-ol from linoleic acid via the linoleic acid 10(S)-hydroperoxide was characterized 40 years ago in mushrooms, yet the enzymes involved are not identified. The dioxygenase 1 and 2 genes (Ccdox1 and Ccdox2) in the mushroom Coprinopsis cinerea contain an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Herein, we show that recombinant CcDOX1 is responsible for dioxygenation of linoleic acid to form the 10(S)-hydroperoxide, the first step in 1-octen-3-ol synthesis, whereas CcDOX2 conceivably forms linoleic acid 8-hydroperoxide. We demonstrate that KO of the Ccdox1 gene suppressed 1-octen-3-ol synthesis, although added linoleic acid 10(S)-hydroperoxide was still efficiently converted. The P450-related domain of CcDOX1 lacks the characteristic Cys heme ligand and the evidence indicates that a second uncharacterized enzyme converts the 10(S)-hydroperoxide to 1-octen-3-ol. Additionally, we determined the gene KO strain (ΔCcdox1) was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the WT strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on WT mycelia. Thus, 1-octen-3-ol seems to be an attractive compound involved in emitter-receiver ecological communication in mushrooms.

Evaluation of indigenous entomopathogenic nematodes in Southwest China as potential biocontrol agents against Spodoptera litura (Lepidoptera: Noctuidae).

Spodoptera litura is a notorious leaf feeding insect pest in the Asia-Pacific region and leads to a significant economic loss in vegetable and field crop production. Entomopathogenic nematodes (EPNs), lethal parasites of insects, are used as biocontrol agents. Yunnan Province in China is a well-known region due to its rich biodiversity. In the present study, a survey of EPNs using the Galleria-baiting technique was conducted in 2017 and 2018 throughout the entire Yunnan province. In total, 789 soil samples were collected from 232 sites, of which 75 samples were positive for EPNs. Phylogenetic analyses of ITS, D2D3 expansion region of the 28S rRNA gene, as well as mitochondrial cytochrome c oxidase subunit I (COI), were performed to identify isolated nematode species and evaluate their genetic diversity. In total, 13, 3, and 58 identified populations belong to Steinernema, Heterorhabditis, and Oscheius, respectively. The phylogenetic relationships of EPN species in the three genera were analyzed with the Neighbor-Joining method. The virulence of the trapped isolates in the genera of Steinernema, Heterorhabditis, and Oscheius against S. litura was evaluated. Ten new indigenous isolates from Steinernema and Heterorhabditis showed prominent virulence to S. litura within 48 hr which is equivalent to that of commercial EPNs populations. The present study provides background information on indigenous EPN resources for S. litura control in Asia-Pacific region.

The suppressive effect of bacterial-feeding nematodes on hemocyte spreading of Galleria mellonella.

Insect parasitic nematodes have developed a mechanism to escape from the cellular immunity of their insect hosts for successful parasitism. However, the detailed mechanism whereby they achieve this remains unclear. In our previous study, we demonstrated that non-parasitic nematodes such as Caenorhabditis elegans potentially have the ability to escape from the cellular immunity of the greater wax moth Galleria mellonella. Here we aimed to clarify the effect of non-parasitic and parasitic nematodes on the spreading of hemocytes-an essential cellular reaction for adhering to a foreign substance -from G. mellonella larvae. The hexane/methanol extract of C. elegans inhibited the spreading of hemocytes. Using 2D-TLC and reversed-phase HPLC, we detected a single peak that inhibited the spreading of hemocytes. In addition, the spreading of hemocytes recovered from C. elegans-injected insects was significantly delayed. Western blotting analysis showed that phosphorylated extracellular signal-regulated protein kinase (ERK) -an essential signaling component for spreading in hemocytes-was decreased by the injection of C. elegans, and that plasma from nematode-injected insects contained the factor that causes the decrease of phosphorylated ERK. We also observed this phenomenon using other non-parasitic and parasitic bacterial-feeding nematodes. These results suggest that the factors inhibiting hemocyte adhesion and delaying the spreading of hemocytes are conserved in bacterial-feeding nematodes and could be a pre-adaptation for parasitism.

Abnormal increases in reactive oxygen species in dying insects infected with nematodes.

Stress enhances the concentration of reactive oxygen species (ROS) in animal plasma. Increased ROS alter various physiological functions, such as development and the immune response, but excessive increases could be harmful. In this study, we tested the hypothesis that abnormally increased plasma ROS levels are associated with animal death. Injection of the nematode Caenorhabditis elegans into insect larvae caused high mortality in Galleria mellonella, and the plasma ROS concentration was four times higher than M9 buffer-injected larvae. There was no difference in plasma antioxidant activity after nematode injection. However, coinjecting nematodes with an antioxidant (ascorbic acid or N-acetylcysteine) suppressed increases in ROS concentrations by the nematodes and increases in the number of nematodes in the larvae, which increased G. mellonella survival. These results suggest that the abnormal elevation of ROS associated with the stress caused by nematode propagation is lethal for G. mellonella.

Bacterial feeding nematodes ingest haemocytes in the haemocoel of the insect Galleria mellonella.

Insect parasitic nematodes have acquired mechanisms to evade their host immune response for successful parasitism. Despite the importance of understanding of the evolution of evasion mechanisms from host immunity, insect immune response against non-parasitic nematodes has not been well studied. In our previous study, we demonstrated that a non-insect parasitic nematode Caenorhabditis elegans was not encapsulated by haemocytes in the larvae of the greater wax moth Galleria mellonella. To understand how nematodes influence insect haemocytes to escape encapsulation, we examined the effect of C. elegans on haemocytes in the haemocoel of G. mellonella larvae. Injection of nematodes resulted in the decrease of haemocyte density while mortality and spreading ability of haemocytes, the haematopoietic organs were not affected. In vitro co-incubation of haemocytes with nematodes resulted in a decrease of haemocyte density and we observed feeding on haemocytes by nematodes. Injection of C. elegans feeding-delay mutants into insects did not cause the decrease of haemocyte density. The decrease of haemocyte density was due to the nematode's ingestion of haemocytes. Furthermore, an entomopathogenic nematode and other bacterial feeding nematodes also showed similar feeding behaviour. The nematode's ability to feed on haemocytes may have played an important role in the evolution of nematode parasitism in bacterial-feeding nematodes.

Cellular immunity in the insect Galleria mellonella against insect non-parasitic nematodes.

Immunity to microbial infections is well understood; however, information regarding the immunity to parasitic multicellular organisms remains lacking. To understand innate host cellular immunity to nematodes, we compared the cellular response of the greater wax moth (Galleria mellonella) larvae against the non-parasitic, bacterial-feeding nematode Caenorhabditis elegans and pathogenic nematode Heterorhabditis bacteriophora. When intact first-instar or dauer larvae of C. elegans were injected into a G. mellonella larva, most of the nematodes were alive and not confined by the surrounding reaction by insect haemocytes (encapsulation), similarly as the pathogenic nematode, whereas most of the heat-killed nematodes of both species were severely encapsulated by 24 h after inoculation. Other non-parasitic nematodes were also not encapsulated. Surprisingly, C. elegans injected into the insect haemocoel grew and propagated in the live insect, resulting in death of the host insect. Our results suggest that C. elegans has some basic mechanisms to evade immunity of G. mellonenlla and grow in the haemocoel.

Transmission electron microscopic observation of body cuticle structures of phoretic and parasitic stages of Parasitaphelenchinae nematodes.

Using transmission electron microscopy, we examined the body cuticle ultrastructures of phoretic and parasitic stages of the parasitaphelenchid nematodes Bursaphelenchus xylophilus, B. conicaudatus, B. luxuriosae, B. rainulfi; an unidentified Bursaphelenchus species, and an unidentified Parasitaphelenchus species. Nematode body cuticles usually consist of three zones, a cortical zone, a median zone, and a basal zone. The phoretic stages of Bursaphelenchus spp., isolated from the tracheal systems of longhorn beetles or the elytra of bark beetles, have a thick and radially striated basal zone. In contrast, the parasitic stage of Parasitaphelenchus sp., isolated from bark beetle hemocoel, has no radial striations in the basal zone. This difference probably reflects the peculiar ecological characteristics of the phoretic stage. A well-developed basal radially striated zone, composed of very closely linked proteins, is the zone closest to the body wall muscle. Therefore, the striation is necessary for the phoretic species to be able to seek, enter, and depart from host/carrier insects, but is not essential for internal parasites in parasitaphelenchid nematodes. Phylogenetic relationships inferred from near-full-length small subunit ribosomal RNA sequences suggest that the cuticle structures of parasitic species have apomorphic characters, e.g., lack of striation in the basal zone, concurrent with the evolution of insect parasitism from a phoretic life history.

Involvement of Vitamin B6 Biosynthesis Pathways in the Insecticidal Activity of Photorhabdus luminescens.

UNLABELLED: Photorhabdus luminescens is a Gram-negative entomopathogenic bacterium which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans To understand the virulence mechanisms of P. luminescens, we obtained virulence-deficient and -attenuated mutants against C. elegans through a transposon-mutagenized library. From the genetic screening, we identified the pdxB gene, encoding erythronate-4-phosphate dehydrogenase, as required for de novo vitamin B6 biosynthesis. Mutation in pdxB caused growth deficiency of P. luminescens in nutrient-poor medium, which was restored under nutrient-rich conditions or by supplementation with pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 Supplementation with three other B6 vitamers (pyridoxal, pyridoxine, and pyridoxamine) also restored the growth of the pdxB mutant, suggesting the existence of a salvage pathway for vitamin B6 biosynthesis in P. luminescens Moreover, supplementation with PLP restored the virulence-deficient phenotype against C. elegans Combining these results with the fact that pdxB mutation also caused attenuation of insecticidal activity, we concluded that the production of appropriate amounts of vitamin B6 is critical for P. luminescens pathogenicity. IMPORTANCE: The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans We have obtained several virulence-deficient and -attenuated P. luminescens mutants against C. elegans through genetic screening. From the genetic analysis, we present the vitamin B6 biosynthetic pathways in P. luminescens that are important for its insecticidal activity. Mutation in pdxB, encoding erythronate-4-phosphate dehydrogenase and required for the de novo vitamin B6 biosynthesis pathway, caused virulence deficiency against C. elegans and growth deficiency of P. luminescens in nutrient-poor medium. Because such phenotypes were restored under nutrient-rich conditions or by supplementation with B6 vitamers, we showed the presence of the two vitamin B6 synthetic pathways (de novo and salvage) in P. luminescens and also showed that the ability to produce an appropriate amount of vitamin B6 is critical for P. luminescens pathogenicity.

Activated and inactivated immune responses in Caenorhabditis elegans against Photorhabdus luminescens TT01.

The Gram-negative bacterium Photorhabdus luminescens which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora, has a broad insecticidal and nematicidal activity. The virulence of P. luminescens toward the non-mutualistic nematode Caenorhabditis elegans has not been described. We showed that when fed on P. luminescens, the intestinal cells of C. elegans worms become delicate and some crystal-like structure was developed within the intestinal lumen. Next, we examined the requirement of the p38 mitogen-activated protein kinase (MAPK) and insulin/IGF-1 signaling pathway against P. luminescens. Depletion of pmk-1 by RNAi enhances susceptibility to P. luminescens, and numerous downstream targets regulated by the p38 MAPK pathway were induced when fed on P. luminescens. On the other hand, knockdown of daf-16 has no effects on C. elegans lifespan, but knockdown of daf-2 dramatically increased resistance to P. luminescens in a daf-16-dependent manner. We also revealed one of the daf-2 ligands ins-7 was induced and ins-7 deletion mutant survived longer when fed on P. luminescens. These results suggest the p38 MAPK pathway is activated and required for the host defense against P. luminescens. Insulin/IGF-1 signaling pathway is inactivated by P. luminescens through the overexpression of insulin-like gene.

Host orientation using volatiles in the phoretic nematode Caenorhabditis japonica.

Host orientation is the most important step in host-searching nematodes; however, information on direct cues from hosts to evoke this behaviour is limited. Caenorhabditis japonica establishes a species-specific phoresy with Parastrachia japonensis. Dauer larvae (DL), the non-feeding and phoretic stage of C. japonica, are predominantly found on female phoretic hosts, but the mechanisms underlying the establishment of this phoresy remain unknown. To determine whether C. japonica DL are able to recognize and orient themselves to a host using a volatile cue from the host, we developed a Y-tube olfactory assay system in which C. japonica DL were significantly attracted to the air from P. japonensis but not to the air from three other insects or to CO2. These results demonstrated that C. japonica DL utilize volatiles for host recognition and orientation and that the presence of a specific volatile kairomone released by the host attracts C. japonica DL.

First report of the nematode Leidynema appendiculata from Periplaneta fuliginosa.

The smokybrown cockroach Periplaneta fuliginosa has spread all over the world, and is now one of the most undesired invasive alien pests in Japan. Because cockroaches are generally infected by thelastomatid nematodes, they are being distributed around the world with their parasitic nematodes. Nothing is known about parasitic nematode species in P. fuliginosa differences, or similarity of the parasite's population structures between the different countries of the host cockroaches. Here we investigated the P. fuliginosa invasive to Japan and found that 100% of individuals were infected with one nematode species. According to the morphology and the sequence of the D2/D3 expansion segment of the 28S ribosomal RNA gene, we identified the parasite as Leidynema appendiculata. This nematode reproduced by haplodiploidy and its developmental timing under various conditions is quite divergent. Their population in the hindgut of P. fuliginosa was controlled with a few adult females and a male. This is the first report of the thelastomatid nematode isolated from the smokybrown cockroach, and is the basis for our future research examining the origin, distribution route and immigration history of the cockroach and the impact of L. appendiculata on native Japanese cockroach species.

Specialist versus generalist life histories and nucleotide diversity in Caenorhabditis nematodes.

Species with broad ecological amplitudes with respect to a key focal resource, niche generalists, should maintain larger and more connected populations than niche specialists, leading to the prediction that nucleotide diversity will be lower and more subdivided in specialists relative to their generalist relatives. This logic describes the specialist-generalist variation hypothesis (SGVH). Some outbreeding species of Caenorhabditis nematodes use a variety of invertebrate dispersal vectors and have high molecular diversity. By contrast, Caenorhabditis japonica lives in a strict association and synchronized life cycle with its dispersal host, the shield bug Parastrachia japonensis, itself a diet specialist. Here, we characterize sequence variation for 20 nuclear loci to investigate how C. japonica's life history shapes nucleotide diversity. We find that C. japonica has more than threefold lower polymorphism than other outbreeding Caenorhabditis species, but that local populations are not genetically disconnected. Coupled with its restricted range, we propose that its specialist host association contributes to a smaller effective population size and lower genetic variation than host generalist Caenorhabditis species with outbreeding reproductive modes. A literature survey of diverse organisms provides broader support for the SGVH. These findings encourage further testing of ecological and evolutionary hypotheses with comparative population genetics in Caenorhabditis and other taxa.

Propagation of Caenorhabditis japonica in the nest of its carrier insect, Parastrachia japonensis.

We demonstrated the disembarkation of the bacterial-feeding nematode Caenorhabditis japonica dauer larvae (DL) from adult Parastrachia japonensis female insects and observed the propagation of nematodes in artificial insect nests. Our results clarify the process of propagation in this nematode species and provide insights into the nematode-insect relationship. Quiescent C. japonica DL resumed their mobility only at > 99.9% relative humidity (RH) at 25°C in the presence or absence of the carrier insect. In artificial nests with > 99.9% RH, DL resumed their mobility and the number of DL on female insects decreased gradually after oviposition, although numerous DL remained on the insects. Very few DL were detected on mother insects after hatching. Nematode propagation was observed on the egg mass after hatching and on nymphal carcasses; the total number of nematodes in the nest increased dramatically after this point. These results indicate that humidity is an important factor for disembarkation of C. japonica DL and that C. japonica propagates in the nest of P. japonensis where it feeds on the remains of eggs and nymph carcasses, indicating that C. japonica and P. japonensis have a unique phoretic and necromenic association.

Species-specific and female host-biased ectophoresy in the roundworm Caenorhabditis japonica.

Caenorhabditis japonica is a bacteriophagous nematode species that was discovered on the semi-social burrower bug, Parastrachia japonensis, which demonstrates egg-guarding and provisioning behaviors. To understand the life history of C. japonica in relation to P. japonensis, we demonstrated the specificity of this association and fluctuations in nematode number on the insect throughout the year. C. japonica dauer larvae (DL), larvae in a nonfeeding diapause stage, were predominantly found as clumps on the adult female insects but rarely found on the male insects in all populations examined. This female-biased association was consistent throughout the year, but after the nymphs hatched, nematodes were not detected on the mother insects showing provisioning behavior. DL appeared on the nymphs, and the number of DL on the newly emerged female insects gradually increased thereafter. C. japonica has never been detected on other invertebrates collected from the P. japonensis habitat thus far. Our data suggest that the life cycles of C. japonica and P. japonensis are synchronized.

Negative gravitactic behavior of Caenorhabditis japonica dauer larvae.

Gravity on Earth is a constant stimulus and many organisms are able to perceive and respond to it. However, there is no clear evidence that nematodes respond to gravity. In this study, we demonstrated negative gravitaxis in a nematode using dauer larvae (DL) of Caenorhabditis japonica, which form an association with their carrier insect Parastrachia japonensis. Caenorhabditis japonica DL demonstrating nictation, a typical host-finding behavior, had a negative gravitactic behavior, whereas non-nictating C. japonica and C. elegans DL did not. The negative gravitactic index of nictating DL collected from younger nematode cultures was higher than that from older cultures. After a 24 h incubation in M9 buffer, nictating DL did not alter their negative gravitactic behavior, but a longer incubation resulted in less pronounced negative gravitaxis. These results are indicative of negative gravitaxis in nictating C. japonica DL, which is maintained once initiated, seems to be affected by the age of DL and does not appear to be a simple passive mechanism.

Xenorhabdus ishibashii sp. nov., isolated from the entomopathogenic nematode Steinernema aciari.

Gram-negative bacteria of the genus Xenorhabdus exhibit a mutualistic association with steinernematid entomopathogenic nematodes and a pathogenic relationship with insects. Here we describe two isolates of the entomopathogenic nematode Steinernema aciari collected from China and Japan. 16S rRNA gene sequence similarity and phylogenetic analysis indicated that the isolates obtained from S. aciari belonged to the genus Xenorhabdus. Multilocus sequence analysis based on five universal protein-coding gene sequences revealed that the isolates were closely related to Xenorhabdus ehlersii DSM 16337(T) and Xenorhabdus griffiniae ID10(T) but that they exhibited <97 % sequence similarity with these reference strains, which indicated that the isolates were distinct from previously described species. Based on these genetic differences and several differential phenotypic traits, we propose that the isolates represent a novel species of the genus Xenorhabdus, for which we propose the name Xenorhabdus ishibashii sp. nov. The type strain is GDh7(T) ( = DSM 22670(T)  = CGMCC 1.9166(T)).

Species-specific recognition of the carrier insect by dauer larvae of the nematode Caenorhabditis japonica.

Host recognition is crucial during the phoretic stage of nematodes because it facilitates their association with hosts. However, limited information is available on the direct cues used for host recognition and host specificity in nematodes. Caenorhabditis japonica forms an intimate association with the burrower bug Parastrachia japonensis. Caenorhabditis japonica dauer larvae (DL), the phoretic stage of the nematode, are mainly found on adult P. japonensis females but no other species. To understand the mechanisms of species-specific and female carrier-biased ectophoresy in C. japonica, we investigated whether C. japonica DL could recognize their hosts using nematode loading and chemoattraction experiments. During the loading experiments, up to 300 C. japonica DL embarked on male and female P. japonensis, whereas none or very few utilized the other shield bugs Erthesina fullo and Macroscytus japonensis or the terrestrial isopod Armadillidium vulgare. In the chemoattraction experiments, hexane extracts containing the body surface components of nymphs and both adult P. japonensis sexes attracted C. japonica DL, whereas those of other shield bugs did not. Parastrachia japonensis extracts also arrested the dispersal of C. japonica DL released at a site where hexane extracts were spotted on an agar plate; i.e. >50% of DL remained at the site even 60 min after nematode inoculation whereas M. japonensis extracts or hexane alone did not have the same effect. These results suggest that C. japonica DL recognize their host species using direct chemical attractants from their specific host to maintain their association.

Identification and characterization of host factors interacting with Bombyx mori nucleopolyhedrovirus ORF8.

The orf8 gene (Bm8) in Bombyx mori nucleopolyhedrovirus (BmNPV) is one of 17 genes unique to group I NPVs and is expressed as an early gene. We have reported that Bm8 may play an important role during viral infection and that Bm8 protein co-localized with IE1 to specific nuclear foci throughout infection. It was also demonstrated that both IE1 and BmNPV hr facilitate this localization of Bm8. To investigate further, host proteins interacting with Bm8 were screened using a yeast two-hybrid system. We identified 6 host clones as Bm8-interacting partners from three cDNA libraries derived from BmN cells or B. mori larvae. Further assays showed that the N-terminal region of Bm8 is important for the interaction with most host clones and that two of the clones can associate with IE1. Cloning and sequencing of full-length cDNAs revealed that most of the clones potentially encode either membrane-bound proteins or secreted proteins. Quantitative RT-PCR analysis revealed that some of these host genes were slightly induced during the early stage of infection in BmN cells, and that the expression of all genes was markedly reduced during the late stage of infection. Generation of mutant BmNPVs over-expressing these host genes also identified a gene that potentially functions as a negative factor during BmNPV infection. These features of Bm8-interacting host proteins strongly support that Bm8 is a multifunctional protein involved in multiple signaling pathways in host cells.