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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.

Entomopathogenic nematodes, includingHeterorhabditisandSteinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (PhotorhabdusandXenorhabdus,respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in entomopathogenic nematodes has been impeded by limited genetic tractability, inconsistent growthin vitro, variable cryopreservation, and low mating efficiency. We obtained the recently describedSteinernema hermaphroditumstrain CS34 and optimized itsin vitrogrowth, with a rapid generation time on a lawn of its native symbiotic bacteriaXenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously,S. hermaphroditumisolated from insect hosts was described as first-generation hermaphroditic and second-generation gonochoristic. We discovered that CS34, when grownin vitro,produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens inS. hermaphroditumthat 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 thatS. hermaphroditumCS34 is the first consistently hermaphroditic entomopathogenic nematode and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.

Microbial symbiotic interactions, mediated by small molecule signaling, drive physiological processes of higher order systems. Metabolic analytic technologies advancements provide new avenues to examine how chemical ecology, or conversion of existing biomass to new forms, changes over a symbiotic lifecycle. We examine such processes using the tripartite relationship between nematode host Steinernema carpocapsae, its obligate mutualist bacterium, Xenorhabdus nematophila, and the insects they infect together. We integrate trophic, metabolomics, and gene regulation analyses to understand insect biomass conversion to nematode or bacterium biomass. Trophic analysis established bacteria as the primary insect consumers, with nematodes at trophic position 4.37, indicating consumption of bacteria and likely other nematodes. Significant, discrete metabolic phases were distinguishable from each other, indicating the insect chemical environment changes reproducibly during bioconversion. Tricarboxylic acid cycle components and amino acids were significantly affected throughout infection. These findings contribute to an ongoing understanding of how symbiont associations shape chemical environments.TeaserEntomopathogenic nematodes act as an apex predator in some ecosystems through altering chemical environments of their prey.

Xenorhabdus nematophila bacteria are mutualists of Steinernema 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 carries X. 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 virulent X. nematophila have a selective advantage and accumulate in aging IJs in advance of exposure to insect hosts in which this phenotype is necessary.

Steinernema carpocapsae is parasitic to insects and mutualistic to bacterial symbionts Xenorhabdus nematophila, therefore is valuable to study naturally-occurring microbial symbiosis. As genetic tools in Xenorhabdus bacteria proved to be powerful to reveal the mechanisms underlying bacteria-host interactions, the lack of genetic tools on Steinernema nematodes hampered the study in the molecular pathways regulating the host side of the symbiotic conversation. Here we present our attempts to establish a CRISPR-Cas9 based approach to introduce mutations in S. carpocapsae genes predicted to encode collagens in the nematode cuticle. We are presenting our progress in genome-editing of S. carpocapsae and current efforts aiming to develop CRISPR-Cas9 co-conversion markers in S. carpocapsae by introducing mutations that cause distinctive and heritable phenotypes. Our CRISPR-Cas9 based tools will open a new revenue to study molecular pathways in host-microbes signaling particularly in naturally occurring parasitic and mutualistic symbiosis.

The construction of hydraulic structures such as abutments and piers in natural waterways contributes to the contraction of river channels and the upstream water level rise. The maximum increase in the water level (afflux) is considered to be a major flood risk after a new bridge is built, and the accurate estimation of it is always an important work in designing a bridge construction. However, systematic studies on the prediction of the backwater effects of piers considering the blockage ratios within 10% are rarely reported. In this paper, a 2D hydrodynamic model was numerically simulated to investigate the effect of blockage ratios in the range of 2% - 9% on the amount and location of afflux. The distribution of depth-averaged flow velocities and water level changes around bridge piers was also discussed. Results indicate that the development and attenuation of backwater effects along the river channel falls naturally into 4 regions, and as the blockage ratio gets greater, afflux increases with its location moving farther from the zone strongly influenced by the bridge piers. It can be concluded that a blockage ratio of more than 7% should be one of the key concerns to river engineers in evaluating the capacity of river channels in plains to carry flood flows. On this basis, this article presented a parametric optimization formula based on blockage ratio and Froude number to estimate afflux of rectangle bridge piers with relatively low blockage ratios on plan rivers, and to provide effective assistance in hydraulic calculation of bridge design.

Animal-microbe symbioses are ubiquitous in nature and scientifically important in diverse areas, including ecology, medicine, and agriculture. Steinernema nematodes and Xenorhabdus bacteria compose an established, successful model system for investigating microbial pathogenesis and mutualism. The bacterium Xenorhabdus nematophila is a species-specific mutualist of insect-infecting Steinernema carpocapsae nematodes. The bacterium colonizes a specialized intestinal pocket within the infective stage of the nematode, which transports the bacteria between insects that are killed and consumed by the pair for reproduction. Current understanding of the interaction between the infective-stage nematode and its bacterial colonizers is based largely on population-level, snapshot time point studies on these organisms. This limitation arises because investigating temporal dynamics of the bacterium within the nematode is impeded by the difficulty of isolating and maintaining individual living nematodes and tracking colonizing bacterial cells over time. To overcome this challenge, we developed a microfluidic system that enables us to spatially isolate and microscopically observe individual, living Steinernema nematodes and monitor the growth and development of the associated X. nematophila bacterial communities—starting from a single cell or a few cells—over weeks. Our data demonstrate, to our knowledge, the first direct, temporal, in vivo visual analysis of a symbiosis system and the application of this system to reveal continuous dynamics of the symbiont population in the living host animal. IMPORTANCE This paper describes an experimental system for directly investigating population dynamics of a symbiotic bacterium, Xenorhabdus nematophila, in its host—the infective stage of the entomopathogenic nematode Steinernema carpocapsae. Tracking individual and groups of bacteria in individual host nematodes over days and weeks yielded insight into dynamic growth and topology changes of symbiotic bacterial populations within infective juvenile nematodes. Our approach for studying symbioses between bacteria and nematodes provides a system to investigate long-term host-microbe interactions in individual nematodes and extrapolate the lessons learned to other bacterium-animal interactions.

This paper describes an experimental system for directly investigating population dynamics of a symbiotic bacterium, Xenorhabdus nematophila, in its host—the infective stage of the entomopathogenic nematode Steinernema carpocapsae. Tracking individual and groups of bacteria in individual host nematodes over days and weeks yielded insight into dynamic growth and topology changes of symbiotic bacterial populations within infective juvenile nematodes. Our approach for studying symbioses between bacteria and nematodes provides a system to investigate long-term host-microbe interactions in individual nematodes and extrapolate the lessons learned to other bacterium-animal interactions., Animal-microbe symbioses are ubiquitous in nature and scientifically important in diverse areas, including ecology, medicine, and agriculture. Steinernema nematodes and Xenorhabdus bacteria compose an established, successful model system for investigating microbial pathogenesis and mutualism. The bacterium Xenorhabdus nematophila is a species-specific mutualist of insect-infecting Steinernema carpocapsae nematodes. The bacterium colonizes a specialized intestinal pocket within the infective stage of the nematode, which transports the bacteria between insects that are killed and consumed by the pair for reproduction. Current understanding of the interaction between the infective-stage nematode and its bacterial colonizers is based largely on population-level, snapshot time point studies on these organisms. This limitation arises because investigating temporal dynamics of the bacterium within the nematode is impeded by the difficulty of isolating and maintaining individual living nematodes and tracking colonizing bacterial cells over time. To overcome this challenge, we developed a microfluidic system that enables us to spatially isolate and microscopically observe individual, living Steinernema nematodes and monitor the growth and development of the associated X. nematophila bacterial communities—starting from a single cell or a few cells—over weeks. Our data demonstrate, to our knowledge, the first direct, temporal, in vivo visual analysis of a symbiosis system and the application of this system to reveal continuous dynamics of the symbiont population in the living host animal. IMPORTANCE This paper describes an experimental system for directly investigating population dynamics of a symbiotic bacterium, Xenorhabdus nematophila, in its host—the infective stage of the entomopathogenic nematode Steinernema carpocapsae. Tracking individual and groups of bacteria in individual host nematodes over days and weeks yielded insight into dynamic growth and topology changes of symbiotic bacterial populations within infective juvenile nematodes. Our approach for studying symbioses between bacteria and nematodes provides a system to investigate long-term host-microbe interactions in individual nematodes and extrapolate the lessons learned to other bacterium-animal interactions.

In mutually beneficial and pathogenic symbiotic associations, microbes must adapt to the host environment for optimal fitness. Both within an individual host and during transmission between hosts, microbes are exposed to temporal and spatial variation in environmental conditions. The phenomenon of phenotypic variation, in which different subpopulations of cells express distinctive and potentially adaptive characteristics, can contribute to microbial adaptation to a lifestyle that includes rapidly changing environments. The environments experienced by a symbiotic microbe during its life history can be erratic or predictable, and each can impact the evolution of adaptive responses. In particular, the predictability of a rhythmic or cyclical series of environments may promote the evolution of signal transduction cascades that allow preadaptive responses to environments that are likely to be encountered in the future, a phenomenon known as adaptive prediction. In this review, we summarize environmental variations known to occur in some well-studied models of symbiosis and how these may contribute to the evolution of microbial population heterogeneity and anticipatory behavior. We provide details about the symbiosis betweenXenorhabdusbacteria andSteinernemanematodes as a model to investigate the concept of environmental adaptation and adaptive prediction in a microbial symbiosis.

Xenorhabdus nematophila bacteria are mutualistic symbionts of Steinernema carpocapsae nematodes and pathogens of insects. The X. nematophila global regulator Lrp controls the expression of many genes involved in both mutualism and pathogenic activities, suggesting a role in the transition between the two host organisms. We previously reported that natural populations of X. nematophila exhibit various levels of Lrp expression and that cells expressing relatively low levels of Lrp are optimized for virulence in the insect Manduca sexta . The adaptive advantage of the high-Lrp-expressing state was not established. Here we used strains engineered to express constitutively high or low levels of Lrp to test the model in which high-Lrp-expressing cells are adapted for mutualistic activities with the nematode host. We demonstrate that high-Lrp cells form more robust biofilms in laboratory media than do low-Lrp cells, which may reflect adherence to host tissues. Also, our data showed that nematodes cultivated with high-Lrp strains are more frequently colonized than are those associated with low-Lrp strains. Taken together, these data support the idea that high-Lrp cells have an advantage in tissue adherence and colonization initiation. Furthermore, our data show that high-Lrp-expressing strains better support nematode reproduction than do their low-Lrp counterparts under both in vitro and in vivo conditions. Our data indicate that heterogeneity of Lrp expression in X. nematophila populations provides diverse cell populations adapted to both pathogenic (low-Lrp) and mutualistic (high-Lrp) states. IMPORTANCE Host-associated bacteria experience fluctuating conditions during both residence within an individual host and transmission between hosts. For bacteria that engage in evolutionarily stable, long-term relationships with particular hosts, these fluctuations provide selective pressure for the emergence of adaptive regulatory mechanisms. Here we present evidence that the bacterium Xenorhabdus nematophila uses various levels of the transcription factor Lrp to optimize its association with its two animal hosts, nematodes and insects, with which it behaves as a mutualist and a pathogen, respectively. Building on our previous finding that relatively low cellular levels of Lrp are optimal for pathogenesis, we demonstrate that, conversely, high levels of Lrp promote mutualistic activities with the Steinernema carpocapsae nematode host. These data suggest that X. nematophila has evolved to utilize phenotypic variation between high- and low-Lrp-expression states to optimize its alternating behaviors as a mutualist and a pathogen.