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As an entomopathogenic nematode (EPN), Steinernema hermaphroditum parasitizes insect hosts and harbors symbiotic Xenorhabdus griffinae bacteria. In contrast to other Steinernematids, S. hermaphroditum has hermaphroditic genetics, offering the experimental scope found in Caenorhabditis elegans. To enable biological analysis of S. hermaphroditum, we have assembled and analyzed its reference genome. This genome assembly has five chromosomal scaffolds and 83 unassigned scaffolds totaling 90.7 Mb, with 19,426 protein-coding genes having a BUSCO completeness of 88.0%. Its autosomes show higher densities of strongly conserved genes in their centers, as in C. elegans, but repetitive elements are evenly distributed along all chromosomes, rather than with higher arm densities as in C. elegans. Either when comparing protein motif frequencies between nematode species or when analyzing gene family expansions during nematode evolution, we observed two categories of genes preferentially associated with the origin of Steinernema or S. hermaphroditum: orthologs of venom genes in S. carpocapsae or S. feltiae; and some types of chemosensory G protein-coupled receptors, despite the tendency of parasitic nematodes to have reduced numbers of chemosensory genes. Three-quarters of venom orthologs occurred in gene clusters, with the larger clusters comprising functionally diverse pathogenicity islands rather than paralogous repeats of a single venom gene. While assembling the genome of S. hermaphroditum, we coassembled bacterial genomes, finding sequence data for not only the known symbiont, X. griffinae, but also for eight other bacterial genera. All eight genera have previously been observed to be associated with Steinernema species or the EPN Heterorhabditis, and may constitute a “second bacterial circle” of EPNs. The genome assemblies of S. hermaphroditum and its associated bacteria will enable use of these organisms as a model system for both entomopathogenicity and symbiosis.
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DiI staining of sensory neurons in the entomopathogenic nematode Steinernema hermaphroditum
Steinernema hermaphroditum entomopathogenic nematodes (EPN) and their Xenorhabdus griffiniae symbiotic bacteria have recently been shown to be a genetically tractable system for the study of both parasitic and mutualistic symbiosis. In their infective juvenile (IJ) stage, EPNs search for insect hosts to invade and quickly kill them with the help of the symbiotic bacteria they contain. The mechanisms behind these behaviors have not been well characterized, including how the nematodes sense their insect hosts. In the well-studied free‑living soil nematode Caenorhabditis elegans, ciliated amphid neurons enable the worms to sense their environment, including chemosensation. Some of these neurons have also been shown to control the decision to develop as a stress-resistant dauer larva, analogous to the infective juveniles of EPNs, or to exit from dauer and resume larval development. In C. elegans and other nematodes, dye-filling with DiI is an easy and efficient method to label these neurons. We developed a protocol for DiI staining of S. hermaphroditum sensory neurons. Using this method, we could identify neurons positionally analogous to the C. elegans amphid neurons ASI, ADL, ASK, ASJ, as well as inner labial neurons IL1 and IL2. Similar to findings in other EPNs, we also found that the IJs of S. hermaphroditum are dye-filling resistant.
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- Award ID(s):
- 2128267
- PAR ID:
- 10333661
- Date Published:
- Journal Name:
- microPublication biology
- ISSN:
- 2578-9430
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Entomopathogenic nematodes (EPNs), including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in EPNs has been impeded by limited genetic tractability, inconsistent growth in vitro, variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as producing hermaphrodites in the first generation. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic EPN and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.more » « less
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Steinernema entomopathogenic nematodes form specific, obligate symbiotic associations with gram-negative, gammaproteobacteria members of the Xenorhabdus genus. Together, the nematodes and symbiotic bacteria infect and kill insects, utilize the nutrient-rich cadaver for reproduction, and then reassociate, the bacteria colonizing the nematodes’ anterior intestines before the nematodes leave the cadaver to search for new prey. In addition to their use in biocontrol of insect pests, these nematode-bacteria pairs are highly tractable experimental laboratory models for animal-microbe symbiosis and parasitism research. One advantageous feature of entomopathogenic nematode model systems is that the nematodes are optically transparent, which facilitates direct observation of nematode-associated bacteria throughout the lifecycle. In this work, green- and red-fluorescently labeled X. griffiniae HGB2511 bacteria were created and associated with their S. hermaphroditum symbiotic nematode partners and observed using fluorescence microscopy. As expected, the fluorescent bacteria were visible as a colonizing cluster in the lumen of the anterior intestinal caecum of the infective stage of the nematode. These tools allow detailed observations of X. griffiniae localization and interactions with its nematode and insect host tissues throughout their lifecycles.more » « less
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Symbiosis, the beneficial interactions between two organisms, is a ubiquitous feature of all life on Earth, including associations between animals and bacteria. However, the specific molecular and cellular mechanisms which underlie the diverse partnerships formed between animals and bacteria are still being explored. Entomopathogenic nematodes transport bacteria between insect hosts, together they kill the insect, and the bacteria consume the insect and serve as food source for the nematodes. These nematodes, including those in the Steinernema genus, are effective laboratory models for studying the molecular mechanisms of symbiosis because of the natural partnership they form with Xenorhabdus bacteria and their straightforward husbandry. Steinernema hermaphroditum nematodes and their Xenorhabdus griffiniae symbiotic bacteria are being developed as a genetic model pair for studying symbiosis. Our goal in this project was to begin to identify bacterial genes that may be important for symbiotic interactions with the nematode host. Towards this end, we adapted and optimized a protocol for delivery and insertion of a lacZ-promoter-probe transposon for use in the S. hermaphroditum symbiont, X. griffiniae HGB2511 (Cao et al., 2022). We assessed the frequencies at which we obtained exconjugants, metabolic auxotrophic mutants, and active promoter-lacZ fusions. Our data indicate that the Tn10 transposon inserted relatively randomly based on the finding that 4.7% of the mutants exhibited an auxotrophic phenotype. Promoter-fusions with the transposon-encoded lacZ, which resulted in expression of β-galactosidase activity, occurred in 47% of the strains. To our knowledge, this is the first mutagenesis protocol generated for this bacterial species, and will facilitate the implementation of large scale screens for symbiosis and other phenotypes of interest in X. griffiniae.more » « less
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Abstract Theory predicts that organisms should diversify their offspring when faced with a stressful environment. This prediction has received empirical support across diverse groups of organisms and stressors. For example, when encountered byCaenorhabditis elegansduring early development, food limitation (a common environmental stressor) induces the nematodes to arrest in a developmental stage called dauer and to increase their propensity to outcross when they are subsequently provided with food and enabled to develop to maturity. Here we tested whether food limitation first encountered during late development/early adulthood can also induce increased outcrossing propensity inC. elegans. Previously well‐fedC. elegansincreased their propensity to outcross when challenged with food limitation during the final larval stage of development and into early adulthood, relative to continuously well‐fed (control) nematodes. Our results thus support previous research demonstrating that the stress of food limitation can induce increased outcrossing propensity inC. elegans. Furthermore, our results expand on previous work by showing that food limitation can still increase outcrossing propensity even when it is not encountered until late development, and this can occur independently of the developmental and gene expression changes associated with dauer.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.17912/micropub.biology.000516
