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Background: Entomopathogenic 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. Results: We 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. Conclusions: IJs 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nematodes are ubiquitous organisms that have a significant global impact on ecosystems, economies, agriculture, and human health. The applied importance of nematodes and the experimental tractability of many species have promoted their use as models in various research areas, including developmental biology, evolutionary biology, ecology, and animal-bacterium interactions. Nematodes are particularly well suited for the investigation of host associations with bacteria because all nematodes have interacted with bacteria during their evolutionary history and engage in a variety of association types. Interactions between nematodes and bacteria can be positive (mutualistic) or negative (pathogenic/parasitic) and may be transient or stably maintained (symbiotic). Furthermore, since many mechanistic aspects of nematode-bacterium interactions are conserved, their study can provide broader insights into other types of associations, including those relevant to human diseases. Recently, genome-scale studies have been applied to diverse nematode-bacterial interactions and have helped reveal mechanisms of communication and exchange between the associated partners. In addition to providing specific information about the system under investigation, these studies also have helped inform our understanding of genome evolution, mutualism, and innate immunity. In this review we discuss the importance and diversity of nematodes, "omics"' studies in nematode-bacterial systems, and the wider implications of the findings. [ABSTRACT FROM AUTHOR]

The authors provide information on entomopathogenic nematodes (EPNs). They clarify the meaning of entomopathogenic in nematology, along with the nematode-arthropod associations. They argue that EPN must rapidly kill their hosts with the help of bacterial partners in order to create future generations. The authors also discuss the history, specialization and classification of EPNs.

Abstract: The nematode Steinernema carpocapsae infects and kills many pest insects in agro-ecosystems and is commonly used in biocontrol of these pests. Growth of the nematodes prior to distribution for biocontrol commonly results in deterioration of traits that are essential for nematode persistence in field applications. To better understand the mechanisms underlying trait deterioration of the efficacy of natural parasitism in entomopathogenic nematodes, we explored the maintenance of fitness related traits including reproductive capacity, heat tolerance, virulence to insects and ‘tail standing’ (formerly called nictation) among laboratory-cultured lines derived from natural, randomly mating populations of S. carpocapsae. Laboratory cultured nematode lines with fitness-related trait values below wild-type levels regained wild-type levels of reproductive and heat tolerance traits when outcrossed with a non-deteriorated line, while virulence and ‘tail standing’ did not deteriorate in our experiments. Crossbreeding two trait-deteriorated lines with each other also resulted in restoration of trait means to wild-type levels in most crossbred lines. Our results implicate inbreeding depression as the primary cause of trait deterioration in the laboratory cultured S. carpocapsae. We further suggest the possibility of creating inbred lines purged of deleterious alleles as founders in commercial nematode growth. [Copyright &y& Elsevier]

Summary: Parasitic nematode species often display highly specialized host-seeking behaviors that reflect their specific host preferences. Many such behaviors are triggered by host odors, but little is known about either the specific olfactory cues that trigger these behaviors or the underlying neural circuits. Heterorhabditis bacteriophora and Steinernema carpocapsae are phylogenetically distant insect-parasitic nematodes whose host-seeking and host-invasion behavior resembles that of some devastating human- and plant-parasitic nematodes. We compare the olfactory responses of Heterorhabditis and Steinernema infective juveniles (IJs) to those of Caenorhabditis elegans dauers, which are analogous life stages []. The broad host range of these parasites results from their ability to respond to the universally produced signal carbon dioxide (CO2), as well as a wide array of odors, including host-specific odors that we identified using thermal desorption-gas chromatography-mass spectroscopy. We find that CO2 is attractive for the parasitic IJs and C. elegans dauers despite being repulsive for C. elegans adults [], and we identify a sensory neuron that mediates CO2 response in both parasitic and free-living species, regardless of whether CO2 is attractive or repulsive. The parasites'' odor response profiles are more similar to each other than to that of C. elegans despite their greater phylogenetic distance, likely reflecting evolutionary convergence to insect parasitism. [Copyright &y& Elsevier]

The article discusses the use of nematodes in high school biology courses. Nematodes or roundworms are key in decomposition and can be used as a pesticide. A team of university researchers and science teachers conducted a laboratory module using insecticidal nematodes to aid students in applying inquiry-based methods and to develop experimental design. Students were encouraged to plan experiments to determine which species of nematode functioned best as a pesticide using various lab protocols.

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. [ABSTRACT FROM AUTHOR]

The impact of the host immune environment on parasite transcription and fitness is currently unknown. It is widely held that hookworm infections have an immunomodulatory impact on the host, but whether the converse is true remains unclear. Immunity against adult-stage hookworms is largely mediated by Type 2 immune responses driven by the transcription factor Signal Transducer and Activator of Transcription 6 (STAT6). This study investigated whether serial passage of the rodent hookworm Nippostrongylus brasiliensis in STAT6-deficient mice (STAT6 KO) caused changes in parasites over time. After adaptation to STAT6 KO hosts, N. brasiliensis increased their reproductive output, feeding capacity, energy content, and body size. Using an improved N. brasiliensis genome, we found that these physiological changes corresponded with a dramatic shift in the transcriptional landscape, including increased expression of gene pathways associated with egg production, but a decrease in genes encoding neuropeptides, proteases, SCP/TAPS proteins, and transthyretin-like proteins; the latter three categories have been repeatedly observed in hookworm excreted/secreted proteins (ESPs) implicated in immunosuppression. Although transcriptional changes started to appear in the first generation of passage in STAT6 KO hosts for both immature and mature adult stages, downregulation of the genes putatively involved in immunosuppression was only observed after multiple generations in this immunodeficient environment. When STAT6 KO-adapted N. brasiliensis were reintroduced to a naive WT host after up to 26 generations, this progressive change in host-adaptation corresponded to increased production of inflammatory cytokines by the WT host. Surprisingly, however, this single exposure of STAT6 KO-adapted N. brasiliensis to WT hosts resulted in worms that were morphologically and transcriptionally indistinguishable from WT-adapted parasites. This work uncovers remarkable plasticity in the ability of hookworms to adapt to their hosts, which may present a general feature of parasitic nematodes. Author summary: Hookworms infect 170–440 million people globally. Our goal in this study was to understand how the host immune environment affects hookworm fitness and gene expression. It is well accepted that protective immunity against hookworms depends on mechanisms controlled by the Signal Transducer and Activator of Transcription 6 (STAT6). Therefore, we used mice lacking STAT6 (STAT6 KO) to serve as a permissive environment for survival of the rodent hookworm Nippostrongylus brasiliensis. One passage into STAT6 KO hosts resulted in immediate transcriptional changes, including increased feeding and egg production pathways, corresponding with measured increases in fecundity, survival, body size, and feeding observed after additional passages. It took multiple generations (up to 26) of STAT6 KO hosts to see the reduced transcription of genes thought to mediate evasion of the host immune system. Despite WT hosts having an enhanced immune response to STAT6 KO-adapted N. brasiliensis, this line of worms survived as well as the WT-adapted N. brasiliensis and quickly lost the changes they had acquired during passage through STAT6 KO hosts. Our findings suggest that hookworms dynamically alter their transcriptional profile to suit their immediate host immune environment. [ABSTRACT FROM AUTHOR]

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 hostseeking 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. [ABSTRACT FROM AUTHOR]

The article presents information on the effect of olfaction on the host-parasite interactions in parasitic nematodes of animals and plants. It discusses a study on the host-seeking behaviors of six species of entomopathogenic nematodes in response to seven ecologically relevant potential hosts. Findings conclude that parasitic nematodes possess specialized olfactory systems that contribute to differences in host-seeking behaviors.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Nematodes 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. Results: A 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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Author summary: A central aspect of parasitic nematode success is their ability to modify host biology, including evasion and/or subversion of host immunity. Modulation of host biology and the pathology caused by parasitic nematodes is largely effected through the release of proteins and small molecules. There are hundreds of proteins released by nematodes during an infection and few have been studied in detail. Fatty acid- and retinol-binding proteins (FARs) are a unique protein family, found only in nematodes and some bacteria, and are released during nematode infection. We report that nematode FARs from S. carpocapsae, C. elegans and A. ceylanicum dampen fly immunity decreasing resistance to infection. Mechanistically, this is achieved through modulation of the phenoloxidase cascade and antimicrobial peptide production. Furthermore, FARs alter the availability of lipid immune signaling precursors in vivo and show binding specificity in vitro. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Gryllus firmus sand crickets have low susceptibility to EPN infection. • Sand crickets have low susceptibility to bacterial infections. • Mole crickets have low susceptibility to infection with X. nematophila. • Host-killing by Steinernema scapterisci relies on the nematode. Abstract The entomopathogenic nematode, Steinernema scapterisci , a specialist parasite of crickets, has been successfully used to combat the southern mole cricket, Neoscapteriscus borellii , which is an invasive pest of turf grass. As an entomopathogenic nematode, S. scapterisci causes rapid death of the insects it infects and uses bacteria to facilitate its parasitism. However, our understanding of the relative contributions of the nematode, S. scapterisci , and its bacterial symbiont, Xenorhabdus innexi , to parasitism remains limited. Here we utilized the sand cricket, Gryllus firmus , as a model host to evaluate the contributions of the EPNs S. scapterisci and S. carpocapsae , as well as their symbiotic bacteria, X. innexi and X. nematophila , respectively, to the virulence of the nematode-bacterial complex. We found that G. firmus has reduced susceptibility to infection from both S. scapterisci and the closely related generalist parasite S. carpocapsae , but that S. scapterisci is much more virulent than S. carpocapsae. Further, we found that N. borellii has reduced susceptibility to X. nematophila , and that G. firmus has reduced susceptibility to X. nematophila , X. innexi, and Serratia marcescens , much more so than other insects that have been studied. We found that the reduced susceptibility of G. firmus to bacterial infection is dependent on development, with adults being less susceptible to infection than nymphs. Our data provide evidence that unlike other EPNs, the virulence of S. scapterisci to crickets is dependent on the nematode rather than the bacterial symbiont that it carries and we speculate that S. scapterisci may be evolving independence from X. innexi. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricketspecialist 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. [ABSTRACT FROM AUTHOR]

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 ≤24 h. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The bacterium X enorhabdus nematophila is a mutualist of entomopathogenic S teinernema 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:: Tn 5, 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:: Tn 5 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. [ABSTRACT FROM AUTHOR]

The grey field slug, Deroceras reticulatum, is an agricultural pest causing damage to a wide variety of crops each year. The nematode Phasmarhabditis hermaphrodita has been shown to effectively kill this slug in field-simulated conditions, leading to its widespread use as a biological control agent in Europe. However, recently discovered isolates of Phasmarhabditis from California have not been tested in a field-simulated environment. The lethality of three local isolates of Phasmarhabditis (P. hermaphrodita, P. californica, & P. papillosa) as well as the molluscicide Sluggo Plus® was assessed on D. reticulatum in a lath house. Remaining leaf area on Canna lilies and slug mortality were recorded after 3 weeks of exposure to treatments. Local isolates efficiently killed D. reticulatum and protection from leaf damage was attained by treatment with P. papillosa. Further experimentation is required to assess plant protection afforded by Phasmarhabditis as plants in some trials may have been in poor health. The three tested Phasmarhabditis isolates are reasonable candidates for biological control within the United States but additional information, particularly on the lethality to non-target gastropods, is needed before an informed decision on their use can be made. [ABSTRACT FROM AUTHOR]

Background: The 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. Results: An 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. Conclusion: As 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. [ABSTRACT FROM AUTHOR]

• O. chongmingensis (Oc) has lower virulence to waxworms than S. carpocapsae (Sc). • Oc is more attracted to infected and freeze-killed insects than uninfected hosts. • Oc grows faster than Sc, including on primary and secondary phase X. nematophila. • The bacterial associate of Oc is antagonistic to the growth of X. nematophila. Entomopathogenic nematodes (EPNs) continue to be explored for their potential usefulness in biological control and pest management programs. As more insect-associated species of nematodes are discovered and described, it is possible that scavengers and kleptoparasites may be mischaracterized as EPNs. If a nematode species is truly an entomopathogen it should display similar infectivity, as well as behaviors and preferences, to those of established EPN species, such as Steinernema carpocapsae. In this study we evaluated dauers of the putative EPN species Oscheius chongmingensis. We examined virulence, odor preferences as a measure of host-seeking behavior, and features of its bacterial symbiont Serratia nematodiphila. We determined that O. chongmingensis behaves more like a scavenger than an EPN. Not only did O. chongmingensis exhibit very poor pathogenicity in Galleria mellonella (wax moth larvae), it also displayed odor (host-seeking) preferences that are contrary to the well-known EPN S. carpocapsae. We also found that the bacterial symbiont of O. chongmingensis was antagonistic to S. carpocapsae ; S. carpocapsae IJs were unable to develop when S. nematodiphila was a primary food source. We conclude that there is insufficient evidence to support the characterization of O. chongmingensis as an EPN; and based on the attributes of its preferences for already-infected or deceased hosts, suggest that this nematode is a scavenger, which may be on an evolutionary trajectory leading to an entomopathogenic lifestyle. [ABSTRACT FROM AUTHOR]