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Summary Xenorhabdus nematophila bacteria are mutualists ofSteinernema carpocapsae nematodes and pathogens of insects.Xenorhabdus nematophila exhibits phenotypic variation between insect virulence (V) and the mutualistic (M) support of nematode reproduction and colonization initiation in the infective juvenile (IJ) stage nematode that carriesX. nematophila between insect hosts. The V and M phenotypes occur reciprocally depending on levels of the transcription factor Lrp: high‐Lrp expressors are M+V− while low‐Lrp expressors are V+M−. We report here that variable (wild type) or fixed high‐Lrp expressors also are optimized, relative to low‐ or no‐Lrp expressors, for colonization of additional nematode stages: juvenile, adult and pre‐transmission infective juvenile (IJ). In contrast, we found that after the bacterial population had undergone outgrowth in mature IJs, the advantage for colonization shifted to low‐Lrp expressors: fixed low‐Lrp expressors (M−V+) and wild type (M+V+) exhibited higher average bacterial CFU per IJ than did high‐Lrp (M+V−) or no‐Lrp (M−V−) strains. Further, the bacterial population becomes increasingly low‐Lrp expressing, based on expression of an Lrp‐dependent fluorescent reporter, as IJs age. These data support a model that virulentX. nematophila have a selective advantage and accumulate in aging IJs in advance of exposure to insect hosts in which this phenotype is necessary. -
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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.
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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.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 Tn 10 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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We report on spectroscopic measurements on the
transition in neutral europium-151 and europium-153 at 459.4 nm. The center of gravity frequencies for the 151 and 153 isotopes, reported for the first time in this paper, to our knowledge, were found to be 652,389,757.16(34) MHz and 652,386,593.2(5) MHz, respectively. The hyperfine coefficients for the state were found to be , and , , which all agree with previously published results except for A(153), which shows a small discrepancy. The isotope shift is found to be 3163.8(6) MHz, which also has a discrepancy with previously published results.
