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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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This content will become publicly available on January 1, 2026
A small-scale bacterial-based liquid Culture Method for Steinernema hermaphroditum
Entomopathogenic nematodes (EPN) infect and kill their insect host with the help of symbiotic bacteria. The only known hermaphroditic (androdiecious) EPN, the clade IV Steinernema hermaphroditum, offers opportunities for exploring both parasitic and mutualistic symbiosis, as well as for evolutionary and developmental studies. Experimental and genetic analysis of this animal is now facilitated through the development of forward and reverse genetic tools and improved culturing techniques. Here, we describe a liquid-culture technique adapted for this worm. The method can be a starting point for the development of large-scale cultivation of the worm and provides a method to generate infective juveniles without an insect host and either with or without its native symbiotic bacteria.
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- Award ID(s):
- 2128267
- PAR ID:
- 10634667
- Publisher / Repository:
- microPublication Biology
- Date Published:
- Journal Name:
- microPublication biology
- Volume:
- 2025
- ISSN:
- 2578-9430
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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