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Abstract: Eicosanoids are a class of molecules derived from C20 polyunsaturated fatty acids (PUFAs) that play a vital role in mammalian and insect biological systems, including development, reproduction, and immunity. Recent research has shown that insects have significant but lower levels of C20 PUFAs in circulation in comparison to C18 PUFAs. It has been previously hypothesized in insects that eicosanoids are synthesized from C18 precursors, such as linoleic acid (LA), to produce downstream eicosanoids. In this study, we show that introduction of arachidonic acid (AA) stimulates production of cyclooxygenase, lipoxygenase, and cytochrome P450–derived eicosanoids. Downstream immune readouts showed that LA stimulates phagocytosis by hemocytes, while both LA and AA stimulate increased antimicrobial peptide production when D. melanogaster is exposed to a heat-killed bacterial pathogen. In totality, this work identifies PUFAs that are involved in insect immunity and adds evidence to the notion that Drosophila utilizes immunostimulatory lipid signaling to mitigate bacterial infections. Our understanding of immune signaling in the fly and its analogies to mammalian systems will increase the power and value of Drosophila as a model organism in immune studies.

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.

The emergence of infectious diseases presents a significant global health, economic, and security risk. Climate change can unexpectedly lead to the spread of pathogens, vectors, or hosts into new areas, contributing to the rise of infectious diseases. Surveillance plays a crucial role in monitoring disease trends and implementing control strategies. In this study, we document the first discovery of Heterobilharzia americana, a parasitic schistosome of mammals and its intermediate hosts Galba cubensis and Galba humilis along the banks of the Colorado River in California. We conducted multiple samplings of snails from various locations in the region with a previous history of canine schistosomiasis. Nucleotide sequencing of the multiple regions of the snails’ and parasites’ DNA revealed the coexistence of G. cubensis and G. humilis, both infected with H. americana. Phylogenetic analyses further validate the presence of H. americana in California, suggesting a wider distribution than previously reported. Our findings have implications for public health, veterinary medicine, and biodiversity conservation, contributing to developing effective control strategies to prevent the spread of this emerging infectious disease.

Abstract 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.

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.

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.

Three species of Phasmarhabditis were recovered from 75 nurseries and garden centers in 28 counties in California during fall and winter 2012–2021. A total of 18 mollusk species were recovered, most of them invasive. Nematodes were identified by sequencing the D2-D3 expansion segments of the large subunit (LSU or 28S) rRNA. Based on these surveys, P. californica was the most widespread species (37 isolates, 53.6% recovery); followed by P. hermaphrodita (26 isolates; 37.7% recovery); P. papillosa and a closely related P. papillosa isolate (6 isolates; 8.7% recovery). Nematode isolates were mainly collected from four invasive slugs (Deroceras reticulatum, D. laeve, Arion hortensis agg, Ambigolimax valentianus) and snails (Oxychilus spp. and Discus spp.). Results suggest that P. californica and P. hermaphrodita share an ecological niche in Northern, Central, Coastal, and Southern California, north of Los Angeles County.

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. 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.

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.

Succinea snails are considered to be invasive and pestiferous gastropods to those in the floricultural industry. Their small size makes them difficult to locate within large plant shipments, and their presence on decorative plants can constitute for an entire shipment to be rejected for sale and distribution. Research performed on Succinea snails is limited, especially in terms of effective mitigation strategies. The nematode Phasmarhabditis hermaphrodita is a biological control agent used on pestiferous gastropods throughout some European nations. Here, three strains of Phasmarhabditis from the United States (P. hermaphrodita, P. californica, and P. papillosa) were assessed as biological control agents against Succinea snails in controlled laboratory conditions, along with the molluscicide Sluggo Plus® as a control. All species of Phasmarhabditis applied at 30 IJs/cm2 caused significant mortality compared to the non-treated control and treatment with Sluggo Plus®. P. californica caused 100% mortality 6 days after exposure, while P. hermaphrodita and P. papillosa caused the same mortality rate 7 days after exposure. The molluscicide was unable to cause significant mortality compared to the non-treated control. Additional research with US Phasmarhabditis strains, including their non-target effects and distribution may lead to their being a viable option for biological control against Succinea snails.

Abstract Plectus murrayiP. murrayiP. murrayiCaenorhabditis elegansP. murrayiP. murrayiP. murrayiP. murrayiP. murrayi

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.

Evolutionary interactions between parasitoid wasps and insect hosts have been well studied at the organismal level, but little is known about the molecular mechanisms that insects use to resist wasp parasitism. Here we study the interaction between a braconid wasp (Aphidius ervi) and its pea aphid host (Acyrthosiphon pisum). We first identify variation in resistance to wasp parasitism that can be attributed to aphid genotype. We then use transcriptome sequencing to identify genes in the aphid genome that are differentially expressed at an early stage of parasitism, and we compare these patterns in highly resistant and susceptible aphid host lines. We find that resistant genotypes are upregulating genes involved in carbohydrate metabolism and several key innate immune system genes in response to parasitism, but that this response seems to be weaker in susceptible aphid genotypes. Together, our results provide a first look into the complex molecular mechanisms that underlie aphid resistance to wasp parasitism and contribute to a broader understanding of how resistance mechanisms evolve in natural populations.

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 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.

Abstract 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.

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.

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.

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.

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.

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.

Robert A Adler, Ranjodh S GillEndocrinology and Metabolism, McGuire Veterans Affairs Medical Center, Richmond, VA, USAAbstract: While most older patients with osteoporosis are treated with antiresorptive bisphosphonates such as alendronate, risedronate, ibandronate, and zoledronic acid, such drugs have side effects, remain in bone for extended periods, and lead to poor adherence to chronic treatment. Denosumab is a humanized monoclonal antibody and antiresorptive agent that works by decreasing the activity of the receptor activator of nuclear factor kappa B ligand. In major trials in postmenopausal women, denosumab increased bone mineral density by dual energy x-ray absorptiometry in the spine, hip, and distal third of the radius and decreased vertebral, nonvertebral, and hip fractures. Denosumab is administered by subcutaneous injection every six months, suggesting that adherence may be improved with such therapy. In addition, pharmacokinetic studies measuring bone turnover markers imply that the antiresorptive effect diminishes more quickly over time. Whether these properties will lead to fewer long-term side effects needs to be proven. Denosumab has also been studied in men with prostate cancer treated with androgen deprivation therapy. These men, at high risk for fracture, also have increases in spine, hip, and forearm dual energy x-ray absorptiometry, as well as fewer morphologic vertebral fractures on x-ray. Denosumab is approved for postmenopausal women with osteoporosis in the US and Europe and for men on androgen deprivation therapy in Europe.Keywords: osteoporosis, fracture, denosumab, bisphosphonates, dual energy x-ray absorptiometry, androgen deprivation therapy, osteonecrosis of the jaw

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.

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.

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 Pacific Ocean methane seeps on the Pacific Ocean floor. This infection is species-specific and has been temporally and spatially stable over two 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.

Plectus murrayi is a free-living microbivorous nematode endemic to Antarctic soils. Our draft assembly of its mitogenome was 15,656 bp long, containing 12 protein-coding, eight transfer RNA (tRNA), and two ribosomal RNA (rRNA) genes. Mitophylogenomic analyses extend our understanding of mitochondrial evolution in Nematoda.

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.