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A key aspect of parasitic nematode infection is the nematodes' ability to evade and/or suppress host immunity. This immunomodulatory ability is likely driven by the release of hundreds of excretory/secretory proteins (ESPs) during infection. While ESPs have been shown to display immunosuppressive effects on various hosts, our understanding of the molecular interactions between individual proteins released and host immunity requires further study. We have recently identified a secreted phospholipase A2 (sPLA2) released from the entomopathogenic nematode (EPN) Steinernema carpocapsae we have named Sc-sPLA2. We report that Sc-sPLA2 increased mortality of Drosophila melanogaster infected with Streptococcus pneumoniae and promoted increased bacterial growth. Furthermore, our data showed that Sc-sPLA2 was able to downregulate both Toll and Imd pathway-associated antimicrobial peptides (AMPs) including drosomycin and defensin, in addition to suppressing phagocytosis in the hemolymph. Sc-sPLA2 was also found to be toxic to D. melanogaster with the severity being both dose- and time-dependent. Collectively, our data highlighted that Sc-sPLA2 possessed both toxic and immunosuppressive capabilities.

A key component to understanding host-parasite interactions is the molecular crosstalk between host and parasite. Excreted/secreted products (ESPs) released by parasitic nematodes play an important role in parasitism. They can directly damage host tissue and modulate host defense. Steinernema carpocapsae, a well-studied parasite of insects releases approximately 500 venom proteins as part of the infection process. Though the identity of these proteins is known, few have been studied in detail. One protein family present in the ESPs released by these nematodes is the ShK family. We studied the most abundant ShK-domain-containing protein in S. carpocapsae ESPs, Sc-ShK-1, to investigate its effects in a fruit fly model. We found that Sc-ShK-1 is toxic under high stress conditions and negatively affects the health of fruit flies. We have shown that Sc-ShK-1 contributes to host immunomodulation in bacterial co-infections resulting in increased mortality and microbial growth. This study provides an insight on ShK-domain-containing proteins from nematodes and suggests these proteins may play an important role in host-parasite interactions.

BackgroundNematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria.ResultsA de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified.ConclusionsThe symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.

Nematodes in the genus Phasmarhabditis can infect and kill slugs and snails, which are important agricultural pests. This useful trait has been commercialized by the corporation BASF after they mass produced a product labeled Nemaslug®. The product contains Phasmarhabditis hermaphrodita, which has been cultured with Moraxella osloensis, a bacterial strain that was originally thought to be responsible for causing mortality in slugs and snails. The exact mechanism leading to death in a Phasmarhabditis infected host is unknown but may involve contributions from nematode-associated bacteria. The naturally occurring microbial community of Phasmarhabditis is unexplored; the previous Phasmarhabditis microbial community studies have focused on laboratory grown or commercially reared nematodes, and in order to obtain a deeper understanding of the parasite and its host interactions, it is crucial to characterize the natural microbial communities associated with this organism in the wild. We sampled Phasmarhabditis californica, Phasmarhabditis hermaphrodita, and Phasmarhabditis papillosa directly from their habitats in Central and Southern California nurseries and garden centers and identified their native microbial community via 16S amplicon sequencing. We found that the Phasmarhabditis microbial community was influenced by species, location, and possibly gastropod host from which the nematode was collected. The predominant bacteria of the Phasmarhabditis isolates collected included Shewanella, Clostridium perfringens, Aeromonadaceae, Pseudomonadaceae, and Acinetobacter. Phasmarhabditis papillosa isolates exhibited an enrichment with species belonging to Acinetobacter or Pseudomonadaceae. However, further research must be performed to determine if this is due to the location of isolate collection or a species specific microbial community pattern. More work on the natural microbial community of Phasmarhabditis is needed to determine the role of bacteria in nematode virulence.

Fatty acid-and retinol-binding proteins (FARs) belong to a unique family of excreted/secreted proteins (ESPs) found exclusively in nematodes. Much of our understanding of these proteins, however, is limited to their in vitro binding characteristics toward various fatty acids and retinol and has provided little insight into their in vivo functions or mechanisms. Recent research, however, has shown that FARs elicit an immunomodulatory role in plant and animal model systems, likely by sequestering lipids involved in immune signaling. This alludes to the intricate relationship between parasitic nematode effectors and their hosts.

Theba pisana is an invasive snail pest which has established itself in San Diego County and some areas of Los Angeles County, California. The snail has grown to large populations in some areas and mitigation is becoming necessary to stop the spread of the species. In a previous study, three US strains of Phasmarhabditis species (P. californica, P. papillosa, and P. hermaphrodita) effectively killed juvenile (0.25 gram each, 4-6 mm wide) T. pisana in laboratory conditions at 5 times (150 IJs/cm2) the recommended dose. Based on laboratory assays, we demonstrated that the same three US strains of Phasmarhabditis can effectively kill larger adult T. pisana (0.4-1.2 gram, 11.5-15mm wide) in two weeks at the same dose. The strains were more efficient at killing T. pisana than the compared molluscicide Sluggo Plus®. Results further showed that the most virulent P. californica did not effectively kill T. pisana at lower doses of 30 IJs/cm2 and 90 IJs/cm2. Additional research is needed to develop the most efficient means of application of Phasmarhabditis to mitigate T. pisana in the field.

Parasitic nematodes cause significant morbidity and mortality globally. Excretory/secretory products (ESPs) such as fatty acid- and retinol- binding proteins (FARs) are hypothesized to suppress host immunity during nematode infection, yet little is known about their interactions with host tissues. Leveraging the insect parasitic nematode, Steinernema carpocapsae, we describe here the first in vivo study demonstrating that FARs modulate animal immunity, causing an increase in susceptibility to bacterial co-infection. Moreover, we show that FARs dampen key components of the fly immune response including the phenoloxidase cascade and antimicrobial peptide (AMP) production. Our data also reveal that FARs deplete lipid signaling precursors in vivo as well as bind to these fatty acids in vitro, suggesting that FARs elicit their immunomodulatory effects by altering the availability of lipid signaling molecules necessary for an efficient immune response. Collectively, these data support a complex role for FARs in immunosuppression in animals and provide detailed mechanistic insight into parasitism in phylum Nematoda.

Chemosensation plays a role in the behaviors and life cycles of numerous organisms, including nematodes. Many guilds of nematodes exist, ranging from the free-living Caenorhabditis elegans to various parasitic species such as entomopathogenic nematodes (EPNs), which are parasites of insects. Despite ecological differences, previous research has shown that both EPNs and C. elegans respond to prenol (3-methyl-2-buten-1-ol), an odor associated with EPN infections. However, it is unclear how C. elegans responds to prenol. By utilizing natural variation and genetic neuron ablation to investigate the response of C. elegans to prenol, we found that the AWC neurons are involved in the detection of prenol and that several genes (including dcap-1, dcap-2, and clec-39) influence response to this odorant. Furthermore, we identified that the response to prenol is mediated by the canonically proposed pathway required for other AWC-sensed attractants. However, upon testing genetically diverse isolates, we found that the response of some strains to prenol differed from their response to isoamyl alcohol, suggesting that the pathways mediating response to these two odorants may be genetically distinct. Further, evaluations leveraging natural variation and genome wide association revealed specific genes that influence nematode behavior and provide a foundation for future studies to better understand the role of prenol in nematode behavioral ecology.

Entomopathogenic nematodes (EPNs) are lethal parasites of insects that have become valuable in biological control and as a model system for studying host-parasite interactions, behavioral ecology, neurobiology, and genomics, among other fields. Their ability to locate hosts is paramount to successful infection and host seeking has been extensively studied in many species in the lab. Here, we explored the usefulness of pluronic gel as a medium to assess EPN host seeking in the lab by characterizing the response of Steinernema carpocapsae, S. feltiae, S. glaseri, S. riobrave, Heterorhabditis bacteriophora, and H. indica to the odor prenol. We found that the infective juveniles (IJs) of these species were repelled by prenol in pluronic gel. We then evaluated how storing the IJs of S. carpocapsae, S. feltiae, and S. glaseri for different amounts of time affected their behavioral responses to prenol. The response of S. carpocapsae was significantly affected by the storage time, while the responses of S. feltiae and S. glaseri were unaffected. Our data support the notion that pluronic gel is a useful medium for studying EPN behavior and that the response of S. carpocapsae to informative odors is significantly affected by long-term storage.

Theba pisana is a serious snail pest in many parts of the world and affects diverse crops including grain, vegetables, grapevines, and ornamental plants and shrubs. Due to its gregarious nature, ability to reproduce rapidly, and the difficulty of controlling it by conventional methods, it has the potential to become a significant pest where introduced. Mitigating this pest is an important challenge that must be addressed. Phasmarhabditis hermaphrodita, is a gastropod-killing nematode that is commercially available only in Europe (Nemaslug ®) and Sub-Saharan Africa (Slugtech ® SP). The use of effective gastropod-killing nematodes in the genus Phasmarhabditis (P. hermaphrodita, P. californica and P. papillosa) in California may provide one strategy for alleviating the potential damage and further spread of these snails, which are currently limited to San Diego and Los Angeles counties. Laboratory assays demonstrated for the first time that US isolates of P. hermaphrodita, P. californica and P. papillosa at 150 DJs/cm2 caused significant mortality and are equally lethal to T. pisana. Molluscicidal efficacy of these nematodes are comparable with those of iron phosphate, at the recommended high dose of 4.88 kg/m2. Additional trials are needed to determine their effects at lower dose and whether they are dependent on the size or age of the snails.

Helminths stage a powerful infection that allows the parasite to damage host tissue through migration and feeding while simultaneously evading the host immune system. This feat is accomplished in part through the release of a diverse set of molecules that contribute to pathogenicity and immune suppression. Many of these molecules have been characterized in terms of their ability to influence the infectious capabilities of helminths across the tree of life. These include nematodes that infect insects, known as entomopathogenic nematodes (EPN) and plants with applications in agriculture and medicine. In this review we will first discuss the nematode virulence factors, which aid parasite colonization or tissue invasion, and cause many of the negative symptoms associated with infection. These include enzymes involved in detoxification, factors essential for parasite development and growth, and highly immunogenic ES proteins. We also explore how these parasites use several classes of molecules (proteins, carbohydrates, and nucleic acids) to evade the host's immune defenses. For example, helminths release immunomodulatory molecules in extracellular vesicles that may be protective in allergy and inflammatory disease. Collectively, these nematode-derived molecules allow parasites to persist for months or even years in a host, avoiding being killed or expelled by the immune system. Here, we evaluate these molecules, for their individual and combined potential as vaccine candidates, targets for anthelminthic drugs, and therapeutics for allergy and inflammatory disease. Last, we evaluate shared virulence and immunomodulatory mechanisms between mammalian and non-mammalian plant parasitic nematodes and EPNs, and discuss the utility of EPNs as a cost-effective model for studying nematode-derived molecules. Better knowledge of the virulence and immunomodulatory molecules from both entomopathogenic nematodes and soil-based helminths will allow for their use as beneficial agents in fighting disease and pests, divorced from their pathogenic consequences.

Theba pisana is a serious snail pest in many parts of the world and affects diverse crops including grain, vegetables, grapevines, and ornamental plants and shrubs. Due to its gregarious nature, ability to reproduce rapidly, and the difficulty of controlling it by conventional methods, it has the potential to become a significant pest where introduced. Mitigating this pest is an important challenge that must be addressed. Phasmarhabditis hermaphrodita, is a gastropod-killing nematode that is commercially available only in Europe (Nemaslug ®) and Sub-Saharan Africa (Slugtech ® SP). The use of effective gastropod-killing nematodes in the genus Phasmarhabditis (P. hermaphrodita, P. californica and P. papillosa) in California may provide one strategy for alleviating the potential damage and further spread of these snails, which are currently limited to San Diego and Los Angeles counties. Laboratory assays demonstrated for the first time that US isolates of P. hermaphrodita, P. californica and P. papillosa at 150 DJs/cm2 caused significant mortality and are equally lethal to T. pisana. Molluscicidal efficacy of these nematodes are comparable with those of iron phosphate, at the recommended high dose of 4.88 kg/m2. Additional trials are needed to determine their effects at lower dose and whether they are dependent on the size or age of the snails.

Chemosensory cues are crucial for entomopathogenic nematodes (EPNs)-a guild of insect-killing parasitic nematodes that are used as biological control agents against a variety of agricultural pests. Dispersal is an essential element of the EPN life cycle in which newly developed infective juveniles (IJs) emerge and migrate away from a resource-depleted insect cadaver in order to search for new hosts. Emergence and dispersal are complex processes that involve biotic and abiotic factors, however, the elements that result in EPN dispersal behaviors have not been well-studied. Prenol is a simple isoprenoid and a natural alcohol found in association with EPN-infected, resource-depleted insect cadavers, and this odorant has been speculated to play a role in dispersal behavior in EPNs. This hypothesis was tested by evaluating the behavioral responses of five different species of EPNs to prenol both as a distal-chemotactic cue and as a dispersal cue. The results indicate that prenol acted as a repulsive agent for all five species tested, while only two species responded to prenol as a dispersal cue.

Parasitic helminths release molecular effectors into their hosts and these effectors can directly damage host tissue and modulate host immunity. Excreted/secreted proteins (ESPs) are one category of parasite molecular effectors that are critical to their success within the host. However, most studies of nematode ESPs rely on in vitro stimulation or culture conditions to collect the ESPs, operating under the assumption that in vitro conditions mimic actual in vivo infection. This assumption is rarely if ever validated. Entomopathogenic nematodes (EPNs) are lethal parasites of insects that produce and release toxins into their insect hosts and are a powerful model parasite system. We compared transcriptional profiles of individual Steinernema feltiae nematodes at different time points of activation under in vitro and in vivo conditions and found that some but not all time points during in vitro parasite activation have similar transcriptional profiles with nematodes from in vivo infections. These findings highlight the importance of experimental validation of ESP collection conditions. Additionally, we found that a suite of genes in the neuropeptide pathway were downregulated as nematodes activated and infection progressed in vivo, suggesting that these genes are involved in host-seeking behavior and are less important during active infection. We then characterized the ESPs of activated S. feltiae infective juveniles (IJs) using mass spectrometry and identified 266 proteins that are released by these nematodes. In comparing these ESPs with those previously identified in activated S. carpocapsae IJs, we identified a core set of 52 proteins that are conserved and present in the ESPs of activated IJs of both species. These core venom proteins include both tissue-damaging and immune-modulating proteins, suggesting that the ESPs of these parasites include both a core set of effectors as well as a specialized set, more adapted to the particular hosts they infect.

Previous research demonstrated that Steinernema carpocapsae infective juveniles (IJs) exposed to a host cuticle were more attracted toward certain host-associated volatile odors. We wanted to test the specificity of attraction that results from exposure to host cuticle. Host recognition behavior was analyzed after stimulating IJs by allowing them to physically interact with Galleria mellonella cuticles. The subsequent behavioral response and the proportion of the population participating in chemotaxis to multiple host odors were measured. We found that exposure to host cuticles resulted in a significantly higher percentage of the population participating in host-seeking behavior, with threefold more nematodes participating in chemotaxis. We tested whether exposure to live or dead host cuticle resulted in a different response and found that a higher percentage of IJs exposed to a live host cuticle participated in chemotaxis than IJs exposed to a dead host cuticle, but that IJs exposed to a dead host demonstrated significantly higher participation than was observed for non-stimulated IJs. To test whether the increase in IJ participation in host-seeking behaviors after exposure to a live host cuticle was specific, we exposed stimulated IJs to a known repulsive odor, a neutral odor, and two predicted attractants. We found that stimulation of IJs through physical contact with a host cuticle induces a specific enhancement of host-seeking behavior to host-specific odors rather than a general increased chemotactic response to all volatile stimuli. However, the nematodes displayed an enhanced response to multiple host-specific odors. Future work should focus on the mechanism through which contact with live host cuticle stimulates increased behavioral response.Previous research demonstrated that Steinernema carpocapsae infective juveniles (IJs) exposed to a host cuticle were more attracted toward certain host-associated volatile odors. We wanted to test the specificity of attraction that results from exposure to host cuticle. Host recognition behavior was analyzed after stimulating IJs by allowing them to physically interact with Galleria mellonella cuticles. The subsequent behavioral response and the proportion of the population participating in chemotaxis to multiple host odors were measured. We found that exposure to host cuticles resulted in a significantly higher percentage of the population participating in host-seeking behavior, with threefold more nematodes participating in chemotaxis. We tested whether exposure to live or dead host cuticle resulted in a different response and found that a higher percentage of IJs exposed to a live host cuticle participated in chemotaxis than IJs exposed to a dead host cuticle, but that IJs exposed to a dead host demonstrated significantly higher participation than was observed for non-stimulated IJs. To test whether the increase in IJ participation in host-seeking behaviors after exposure to a live host cuticle was specific, we exposed stimulated IJs to a known repulsive odor, a neutral odor, and two predicted attractants. We found that stimulation of IJs through physical contact with a host cuticle induces a specific enhancement of host-seeking behavior to host-specific odors rather than a general increased chemotactic response to all volatile stimuli. However, the nematodes displayed an enhanced response to multiple host-specific odors. Future work should focus on the mechanism through which contact with live host cuticle stimulates increased behavioral response.

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis, an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (2-arachidonoylglycerol [2-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and 2 (CB1R and CB2R). Compared to findings for vehicle-treated mice, inhibition of CB1R but not CB2R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type 2 cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden.

Resistin-like molecule α (RELMα) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMα dampens Th2 inflammatory responses. RELMα is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα-/ - ) in BM or non BM cells and infected them with Nb. Non BM RELMα-/- chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice. In contrast, both RELMα-/- and BM RELMα-/- mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+ /+ macrophages, RELMα-/- macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMα partially reversed this phenotype. Gene expression analysis revealed that RELMα decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.

Entomopathogenic nematodes (EPNs) are potent insect parasites and have been used for pest control in agriculture. Despite the complexity of the EPN infection process, hosts are typically killed within 5 days of initial infection. When free-living infective juveniles (IJs) infect a host, they release their bacterial symbiont, secrete toxic products, and undergo notable morphological changes. Collectively, this process is referred to as "activation" and represents the point in a nematode's life cycle when it becomes actively parasitic. The effect of different host tissues and IJ age on activation, and how activation itself is related to virulence, are not well understood. Here, we employed a recently developed bioassay, which quantifies IJ activation, as a tool to address these matters. Appreciating that activation is a key part of the EPN infection process, we hypothesized that activation would positively correlate to virulence. Using the EPNs Steinernema carpocapsae and S. feltiae we found that EPN activation is host-specific and influenced by infective juvenile age. Additionally, our data suggest that activation has a context-dependent influence on virulence and could be predictive of virulence in some cases such as when IJ activation is especially low.

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds.

BACKGROUND:Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricket-specialist parasite and together the nematode and bacteria infect and kill crickets. Curiously, X. innexi expresses a potent extracellular mosquitocidal toxin activity in culture supernatants. We sequenced a draft genome of X. innexi and compared it to the genomes of related pathogens to elucidate the nature of specialization. RESULTS:Using green fluorescent protein-expressing X. innexi we confirm previous reports using culture-dependent techniques that X. innexi colonizes its nematode host at low levels (~3-8 cells per nematode), relative to other Xenorhabdus-Steinernema associations. We found that compared to the well-characterized entomopathogenic nematode symbiont X. nematophila, X. innexi fails to suppress the insect phenoloxidase immune pathway and is attenuated for virulence and reproduction in the Lepidoptera Galleria mellonella and Manduca sexta, as well as the dipteran Drosophila melanogaster. To assess if, compared to other Xenorhabdus spp., X. innexi has a reduced capacity to synthesize virulence determinants, we obtained and analyzed a draft genome sequence. We found no evidence for several hallmarks of Xenorhabdus spp. toxicity, including Tc and Mcf toxins. Similar to other Xenorhabdus genomes, we found numerous loci predicted to encode non-ribosomal peptide/polyketide synthetases. Anti-SMASH predictions of these loci revealed one, related to the fcl locus that encodes fabclavines and zmn locus that encodes zeamines, as a likely candidate to encode the X. innexi mosquitocidal toxin biosynthetic machinery, which we designated Xlt. In support of this hypothesis, two mutants each with an insertion in an Xlt biosynthesis gene cluster lacked the mosquitocidal compound based on HPLC/MS analysis and neither produced toxin to the levels of the wild type parent. CONCLUSIONS:The X. innexi genome will be a valuable resource in identifying loci encoding new metabolites of interest, but also in future comparative studies of nematode-bacterial symbiosis and niche partitioning among bacterial pathogens.

We report the isolation and identification of seven bacterial strains and one fungal strain from dead and diseased Scapteriscus borellii mole crickets collected from a golf course in southern California. Using 16S and 18S rRNA gene sequence analysis we identified the microbes as Serratia marcescens (red), S. marcescens (white), S. marcescens (purple), Achromobacter xylosoxidans, Chryseobacterium sp., Ochrobactrum anthropi, Tsukamurella tryosinosolvens, and Beauveria bassiana. We performed a dose response curve for each of these cricket-associated microbial strains (except T. tryosinosolvens) and two other strains of S. marcescens (DB1140 and ATCC 13880). We found that all of these microbes except O. anthropi were highly pathogenic to D. melanogaster compared to the other strains of S. marcescens. Injecting the mole cricket associated strains of Serratia into flies killed all infected flies in ≤24h. For all other strains, the median time to death of injected flies varied in a dose-dependent manner. In vivo growth assessments of these microbes suggested that the host immune system was quickly overcome. We used disease tolerance curves to better understand the host-microbe interactions. Further studies are necessary to understand in mechanistic detail the virulence mechanisms of these mole cricket associated microbes and how this association may have influenced the evolution of mole cricket immunity.

Entomopathogenic nematodes (EPNs) are insect parasites used as biological control agents. Free-living infective juveniles (IJs) of EPNs employ host-seeking behaviors to locate suitable hosts for infection. We found that EPNs can differentiate between naïve and infected hosts, and that host attractiveness changes over time in a species-specific manner. We used solid-phase microextraction and gas chromatography/mass spectrometry to identify volatile chemical cues that may relay information about a potential host's infection status and resource availability. Among the chemicals identified from the headspace of infected hosts, 3-Methyl-2-buten-1-ol (prenol) and 3-Hydroxy-2-butanone (AMC) were selected for further behavioral assays due to their temporal correlation with the behavioral changes of IJs towards the infected hosts. Both compounds were repulsive to IJs of Steinernema glaseri and S. riobrave in a dose-dependent manner when applied on an agar substrate. Furthermore, the repulsive effects of prenol were maintained when co-presented with the uninfected host odors, overriding attraction to uninfected hosts. Prenol was attractive to dauers of some free-living nematodes and insect larvae. These data suggest that host-associated chemical cues may have several implications in EPN biology, not only as signals for avoidance and dispersal of conspecifics, but also as attractants for new potential hosts.

Entomopathogenic nematodes (EPNs) are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs) of Steinernema carpocapsae (a well-studied EPN species) release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products.

Entomopathogenic nematodes are a subgroup of insect-parasitic nematodes that are used in biological control as alternatives or supplements to chemical pesticides. Steinernema scapterisci is an unusual member of the entomopathogenic nematode guild for many reasons including that it is promiscuous in its association with bacteria, it can reproduce in the absence of its described bacterial symbiont, and it is known to have a narrow host range. It is a powerful comparative model within the species and could be used to elucidate parasite specialization. Here we describe a new method of efficiently producing large numbers of S. scapterisci infective juveniles (IJs) in house crickets and for quantifying parasitic activation of the IJs upon exposure to host tissue using morphological features. We found that parasite activation is a temporal process with more IJs activating over time. Furthermore, we found that activated IJs secrete a complex mixture of proteins and that S. scapterisci IJs preferentially activate upon exposure to cricket tissue, reaffirming the description of S. scapterisci as a cricket specialist.

BackgroundEntomopathogenic nematodes (EPNs) are lethal parasites of insects that are of interest as biocontrol agents for insect pests and disease vectors. Although EPNs have been successfully commercialized for pest control, their efficacy in the field is often inconsistent for reasons that remain elusive. EPN infective juveniles (IJs) actively search for hosts to infect using a diverse array of host-emitted odorants. Here we investigate whether their host-seeking behavior is subject to context-dependent modulation.ResultsWe find that EPN IJs exhibit extreme plasticity of olfactory behavior as a function of cultivation temperature. Many odorants that are attractive for IJs grown at lower temperatures are repulsive for IJs grown at higher temperatures and vice versa. Temperature-induced changes in olfactory preferences occur gradually over the course of days to weeks and are reversible. Similar changes in olfactory behavior occur in some EPNs as a function of IJ age. EPNs also show temperature-dependent changes in their host-seeking strategy: IJs cultured at lower temperatures appear to more actively cruise for hosts than IJs cultured at higher temperatures. Furthermore, we find that the skin-penetrating rat parasite Strongyloides ratti also shows temperature-dependent changes in olfactory behavior, demonstrating that such changes occur in mammalian-parasitic nematodes.ConclusionsIJs are developmentally arrested and long-lived, often surviving in the environment through multiple seasonal temperature changes. Temperature-dependent modulation of behavior may enable IJs to optimize host seeking in response to changing environmental conditions, and may play a previously unrecognized role in shaping the interactions of both beneficial and harmful parasitic nematodes with their hosts.

Entomopathogenic nematodes (EPNs) have been used in biological control but improvement is needed to realize their full potential for broader application in agriculture. Some improvements have been gained through selective breeding and the isolation of additional species and populations. Having genomic sequences for at least six EPNs opens the possibility of genetic improvement, either by facilitating the selection of candidate genes for hypothesis-driven studies of gene-trait relations or by genomics-assisted breeding for desirable traits. However, the genomic data will be of limited use without a more mechanistic understanding of the genes underlying traits that are important for biological control. Additionally, molecular tools are required to fully translate the genomic resources into further functional studies and better biological control.

BackgroundParasitism is a major ecological niche for a variety of nematodes. Multiple nematode lineages have specialized as pathogens, including deadly parasites of insects that are used in biological control. We have sequenced and analyzed the draft genomes and transcriptomes of the entomopathogenic nematode Steinernema carpocapsae and four congeners (S. scapterisci, S. monticolum, S. feltiae, and S. glaseri).ResultsWe used these genomes to establish phylogenetic relationships, explore gene conservation across species, and identify genes uniquely expanded in insect parasites. Protein domain analysis in Steinernema revealed a striking expansion of numerous putative parasitism genes, including certain protease and protease inhibitor families, as well as fatty acid- and retinol-binding proteins. Stage-specific gene expression of some of these expanded families further supports the notion that they are involved in insect parasitism by Steinernema. We show that sets of novel conserved non-coding regulatory motifs are associated with orthologous genes in Steinernema and Caenorhabditis.ConclusionsWe have identified a set of expanded gene families that are likely to be involved in parasitism. We have also identified a set of non-coding motifs associated with groups of orthologous genes in Steinernema and Caenorhabditis involved in neurogenesis and embryonic development that are likely part of conserved protein-DNA relationships shared between these two genera.

There are two ways to maintain fitness in the face of infection: resistance is a host's ability to reduce microbe load and disease tolerance is the ability of the host to endure the negative health effects of infection. Resistance and disease tolerance should be applicable to any insult to the host and have been explored in depth with regards to infection but have not been examined in the context of cancer. Here, we establish a framework for measuring and separating resistance and disease tolerance to cancer in Drosophila melanogaster. We plot a disease tolerance curve to cancer in wild-type flies and then compare this to natural variants, identifying a line with reduced cancer resistance. Quantitation of these two traits opens an additional dimension for analysis of cancer biology.

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.

Invasive mole cricket species in the genus Scapteriscus have become significant agricultural pests and are continuing to expand their range in North America. Though largely subterranean, adults of some species, such as Scapteriscus borellii Giglio-Tos 1894, are capable of long dispersive flights and phonotaxis to male calling songs to find suitable habitats and mates. Mole crickets in the genus Scapteriscus are known to be attracted to and can be caught by audio lure traps that broadcast synthesized or recorded calling songs. We report improvements in the design and production of electronic controllers for the automation of semipermanent mole cricket trap lures as well as highly portable audio trap collection designs. Using these improved audio lure traps, we collected the first reported individuals of the pest mole cricket S. borellii in California. We describe several characteristic features of the calling song of the California population including that the pulse rate is a function of soil temperature, similar to Florida populations of S. borellii. Further, we show that other calling song characteristics (carrier frequency, intensity, and pulse rate) are significantly different between the populations.

The deep sea is Earth's largest habitat but little is known about the nature of deep-sea parasitism. In contrast to a few characterized cases of bacterial and protistan parasites, the existence and biological significance of deep-sea parasitic fungi is yet to be understood. Here we report the discovery of a fungus-related parasitic microsporidium, Nematocenator marisprofundi n. gen. n. sp. that infects benthic nematodes at methane seeps on the Pacific Ocean floor. This infection is species-specific and has been temporally and spatially stable over 2 years of sampling, indicating an ecologically consistent host-parasite interaction. A high distribution of spores in the reproductive tracts of infected males and females and their absence from host nematodes' intestines suggests a sexual transmission strategy in contrast to the fecal-oral transmission of most microsporidia. N. marisprofundi targets the host's body wall muscles causing cell lysis, and in severe infection even muscle filament degradation. Phylogenetic analyses placed N. marisprofundi in a novel and basal clade not closely related to any described microsporidia clade, suggesting either that microsporidia-nematode parasitism occurred early in microsporidia evolution or that host specialization occurred late in an ancient deep-sea microsporidian lineage. Our findings reveal that methane seeps support complex ecosystems involving interkingdom interactions between bacteria, nematodes, and parasitic fungi and that microsporidia parasitism exists also in the deep-sea biosphere.

Nematodes compose an abundant and diverse invertebrate phylum with members inhabiting nearly every ecological niche. Panagrellus redivivus (the "microworm") is a free-living nematode frequently used to understand the evolution of developmental and behavioral processes given its phylogenetic distance to Caenorhabditis elegans. Here we report the de novo sequencing of the genome, transcriptome, and small RNAs of P. redivivus. Using a combination of automated gene finders and RNA-seq data, we predict 24,249 genes and 32,676 transcripts. Small RNA analysis revealed 248 microRNA (miRNA) hairpins, of which 63 had orthologs in other species. Fourteen miRNA clusters containing 42 miRNA precursors were found. The RNA interference, dauer development, and programmed cell death pathways are largely conserved. Analysis of protein family domain abundance revealed that P. redivivus has experienced a striking expansion of BTB domain-containing proteins and an unprecedented expansion of the cullin scaffold family of proteins involved in multi-subunit ubiquitin ligases, suggesting proteolytic plasticity and/or tighter regulation of protein turnover. The eukaryotic release factor protein family has also been dramatically expanded and suggests an ongoing evolutionary arms race with viruses and transposons. The P. redivivus genome provides a resource to advance our understanding of nematode evolution and biology and to further elucidate the genomic architecture leading to free-living lineages, taking advantage of the many fascinating features of this worm revealed by comparative studies.

Dishevelled (Dsh or Dvl) is an important signaling protein, playing a key role in Wnt signaling and relaying cellular information for several developmental pathways. Dsh is highly conserved among metazoans and has expanded into a multigene family in most bilaterian lineages, including vertebrates, planarians, and nematodes. These orthologs, where explored, are known to have considerable overlap in function, but evidence for functional specialization continues to mount. We performed a comparative analysis of Dsh across animals to explore protein architecture and identify conserved and divergent features that could provide insight into functional specialization with an emphasis on invertebrates, especially nematodes. We find evidence of dynamic evolution of Dsh, particularly among nematodes, with taxa varying in ortholog number from one to three. We identify a new domain specific to some nematode lineages and find an unexpected nuclear localization signal conserved in many Dsh orthologs. Our findings raise questions of protein evolution in general and provide clues as to how animals have dealt with the complex intricacies of having a protein, such as Dsh, act as a central messenger hub connected to many different and vitally important pathways. We discuss our findings in the context of functional specialization and bring many testable hypotheses to light.

BackgroundThe success of a biological control agent depends on key traits, particularly reproductive potential, environmental tolerance, and ability to be cultured. These traits can deteriorate rapidly when the biological control agent is reared in culture. Trait deterioration under laboratory conditions has been widely documented in the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora (Hb) but the specific mechanisms behind these genetic processes remain unclear. This research investigates the molecular mechanisms of trait deterioration of two experimental lines of Hb, an inbred line (L5M) and its original parental line (OHB). We generated transcriptional profiles of two experimental lines of Hb, identified the differentially expressed genes (DEGs) and validated their differential expression in the deteriorated line.ResultsAn expression profiling study was performed between experimental lines L5M and OHB of Hb with probes for 15,220 ESTs from the Hb transcriptome. Microarray analysis showed 1,185 DEGs comprising of 469 down- and 716 up-regulated genes in trait deteriorated nematodes. Analysis of the DEGs showed that trait deterioration involves massive changes of the transcripts encoding enzymes involved in metabolism, signal transduction, virulence and longevity. We observed a pattern of reduced expression of enzymes related to primary metabolic processes and induced secondary metabolism. Expression of sixteen DEGs in trait deteriorated nematodes was validated by quantitative reverse transcription-PCR (qRT-PCR) which revealed similar expression kinetics for all the genes tested as shown by microarray.ConclusionAs the most closely related major entomopathogen to C. elegans, Hb provides an attractive near-term application for using a model organism to better understand interspecies interactions and to enhance our understanding of the mechanisms underlying trait deterioration in biological control agents. This information could also be used to improve the beneficial traits of biological control agents and better understand fundamental aspects of nematode parasitism and mutualism.

A key aspect of parasitic nematode infection is the nematodes' ability to evade and/or suppress host immunity. This immunomodulatory ability is likely driven by the release of hundreds of excretory/secretory proteins (ESPs) during infection. While ESPs have been shown to display immunosuppressive effects on various hosts, our understanding of the molecular interactions between individual proteins released and host immunity requires further study. We have recently identified a secreted phospholipase A2 (sPLA2) released from the entomopathogenic nematode (EPN) Steinernema carpocapsae we have named Sc-sPLA2. We report that Sc-sPLA2 increased mortality of Drosophila melanogaster infected with Streptococcus pneumoniae and promoted increased bacterial growth. Furthermore, our data showed that Sc-sPLA2 was able to downregulate both Toll and Imd pathwayassociated antimicrobial peptides (AMPs) including drosomycin and defensin, in addition to suppressing phagocytosis in the hemolymph. Sc-sPLA2 was also found to be toxic to D.melanogaster with the severity being both dose- and time-dependent. Collectively, our data highlighted that Sc-sPLA2 possessed both toxic and immunosuppressive capabilities. [ABSTRACT FROM AUTHOR]