Monitoring the Photorhabdus spp. bacterial load in Heterorhabditis bacteriophora dauer juveniles over different storage times and temperatures: A molecular approach.

Biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora can vary in virulence (quality). The influence of their symbiotic bacteria Photorhabdus spp. inside the infective dauer juvenile (DJ) on DJ quality has not received much attention in the past. The presence of the bacteria in the DJ is crucial for its biocontrol potential. This investigation provides a method to quantify the bacterial load inside the DJ based on a qPCR technique. Information from the genome of Photorhabdus laumondii strain DE2 was used to identify single copy genes with no homology to any other bacterial accessions. One gene (hereby named CG2) was selected for primers design and for further qPCR experiments. Cross-amplification tests with P. thracensis and P. kayaii, also symbionts of H. bacteriophora, were positive, whereas no amplicons were produced for P. temperata or Xenorhabdus nematophila. We tested our qPCR system in DJ populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the virulence was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this factor correlated with the reduction of bacterial DNA on the respective DJ population. We observed that stored DJ kept virulent up to 90 days and thereafter the virulence as well as the amount of bacterial DNA drastically decreased. Storage temperature also influenced the bacterial survival. Inside formulated DJ, the loss of bacterial DNA on the DJ population was accelerated under storage temperatures below 7.5 °C, suggesting that reproduction of the bacterial cells takes place when growth temperature is favorable. The role of bacterial survival inside stored DJ can now be adequately addressed using this molecular quality-control technique.

Enhancing mass production of Heterorhabditis bacteriophora: influence of different bacterial symbionts (Photorhabdus spp.) and inoculum age on dauer juvenile recovery.

The entomopathogenic nematode Heterorhabditis bacteriophora (Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and developmentally arrested, invading host insects. They carry cells of their bacterial symbiont Photorhabdus spp. in the intestine. Once inside the insect´s hemolymph the DJs perceive a food signal, triggering them to exit the DJ stage and regurgitate the Photorhabdus cells into the insect's haemocoel, which kill the host and later provide essential nutrients for nematode reproduction. The exit from the DJ stage is called "recovery". For commercial pest control, nematodes are industrially produced in monoxenic liquid cultures. Artificial media are incubated with Photorhabdus before DJs are added. In absence of the insect's food signal, DJs depend on unknown bacterial food signals to trigger exit of the DJ stage. A synchronized and high DJ recovery determines the success of the industrial in vitro production and can significantly vary between nematode strains, inbred lines and mutants. In this study, fourteen bacterial strains from H. bacteriophora were isolated and identified as P. laumondii, P. kayaii and P. thracensis. Although the influence of bacterial supernatants on the DJ recovery of three inbred lines and two mutants differed significantly, the bacterial impact on recovery has a subordinate role whereas nematode factors have a superior influence. Recovery of inbred lines decreased with age of the DJs. One mutant (M31) had very high recovery in bacterial supernatant and spontaneous recovery in Ringer solution. Another mutant (M88) was recovery defective.

Pheno- and genotyping in vitro dauer juvenile recovery in the nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of the carried symbiotic bacterium of the genus Photorhabdus. Inside the host, the DJs perceive signals from the insect's haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6-67.2% vs 1.6-35.7%). A subset of mutant lines presented high variability of virulence against mealworm (Tenebrio molitor) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM H2O2; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in H. bacteriophora. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs. KEYPOINTS: • Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known. • We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes. • We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.

Population size impacts host-pathogen coevolution.

Ongoing host-pathogen interactions are characterized by rapid coevolutionary changes forcing species to continuously adapt to each other. The interacting species are often defined by finite population sizes. In theory, finite population size limits genetic diversity and compromises the efficiency of selection owing to genetic drift, in turn constraining any rapid coevolutionary responses. To date, however, experimental evidence for such constraints is scarce. The aim of our study was to assess to what extent population size influences the dynamics of host-pathogen coevolution. We used Caenorhabditus elegans and its pathogen Bacillus thuringiensis as a model for experimental coevolution in small and large host populations, as well as in host populations which were periodically forced through a bottleneck. By carefully controlling host population size for 23 host generations, we found that host adaptation was constrained in small populations and to a lesser extent in the bottlenecked populations. As a result, coevolution in large and small populations gave rise to different selection dynamics and produced different patterns of host-pathogen genotype-by-genotype interactions. Our results demonstrate a major influence of host population size on the ability of the antagonists to co-adapt to each other, thereby shaping the dynamics of antagonistic coevolution.

The genomic basis of Red Queen dynamics during rapid reciprocal host-pathogen coevolution.

Red Queen dynamics, involving coevolutionary interactions between species, are ubiquitous, shaping the evolution of diverse biological systems. To date, information on the underlying selection dynamics and the involved genome regions is mainly available for bacteria-phage systems or only one of the antagonists of a eukaryotic host-pathogen interaction. We add to our understanding of these important coevolutionary interactions using an experimental host-pathogen model, which includes the nematode Caenorhabditis elegans and its pathogen Bacillus thuringiensis We combined experimental evolution with time-shift experiments, in which a focal host or pathogen is tested against a coevolved antagonist from the past, present, or future, followed by genomic analysis. We show that (i) coevolution occurs rapidly within few generations, (ii) temporal coadaptation at the phenotypic level is found in parallel across replicate populations, consistent with antagonistic frequency-dependent selection, (iii) genomic changes in the pathogen match the phenotypic pattern and include copy number variations of a toxin-encoding plasmid, and (iv) host genomic changes do not match the phenotypic pattern and likely involve selective responses at more than one locus. By exploring the dynamics of coevolution at the phenotypic and genomic level for both host and pathogen simultaneously, our findings demonstrate a more complex model of the Red Queen, consisting of distinct selective processes acting on the two antagonists during rapid and reciprocal coadaptation.

Travelling at a slug's pace: possible invertebrate vectors of Caenorhabditis nematodes.

BACKGROUND: How do very small animals with limited long-distance dispersal abilities move between locations, especially if they prefer ephemeral micro-habitats that are only available for short periods of time? The free-living model nematode Caenorhabditis elegans and several congeneric taxa appear to be common in such short-lived environments, for example decomposing fruits or other rotting plant material. Dispersal is usually assumed to depend on animal vectors, yet all current data is based on only a limited number of studies. In our project we performed three comprehensive field surveys on possible invertebrate vectors in North German locations containing populations of C. elegans and two related species, especially C. remanei, and combined these screens with an experimental analysis of persistence in one of the vector taxa. RESULTS: Our field survey revealed that Caenorhabditis nematodes are commonly found in slugs, isopods, and chilopods, but are not present in the remaining taxonomic groups examined. Surprisingly, the nematodes were frequently isolated from the intestines of slugs, even if slugs were not collected in close association with suitable substrates for Caenorhabditis proliferation. This suggests that the nematodes are able to enter the slug intestines and persist for certain periods of time. Our experimental analysis confirmed the ability of C. elegans to invade slug intestines and subsequently be excreted alive with the slug feces, although only for short time periods under laboratory conditions. CONCLUSIONS: We conclude that three invertebrate taxonomic groups represent potential vectors of Caenorhabditis nematodes. The nematodes appear to have evolved specific adaptations to enter and persist in the harsh environment of slug intestines, possibly indicating first steps towards a parasitic life-style.