Natural variation in Pristionchus pacificus dauer formation reveals cross-preference rather than self-preference of nematode dauer pheromones

Many free-living nematodes, including the laboratory model organisms Caenorhabditis elegans and Pristionchus pacificus, have a choice between direct and indirect development, representing an important case of phenotypic plasticity. Under harsh environmental conditions, these nematodes form dauer larvae, which arrest development, show high resistance to environmental stress and constitute a dispersal stage. Pristionchus pacificus occurs in a strong association with scarab beetles in the wild and remains in the dauer stage on the living beetle. Here, we explored the circumstances under which P. pacificus enters and exits the dauer stage by using a natural variation approach. The analysis of survival, recovery and fitness after dauer exit of eight P. pacificus strains revealed that dauer larvae can survive for up to 1 year under experimental conditions. In a second experiment, we isolated dauer pheromones from 16 P. pacificus strains, and tested for natural variation in pheromone production and sensitivity in cross-reactivity assays. Surprisingly, 13 of the 16 strains produce a pheromone that induces the highest dauer formation in individuals of other genotypes. These results argue against a simple adaptation model for natural variation in dauer formation and suggest that strains may have evolved to induce dauer formation precociously in other strains in order to reduce the fitness of these strains. We therefore discuss intraspecific competition among genotypes as a previously unconsidered aspect of dauer formation.     

Mass Spectrometry–Driven Discovery of Neuropeptides Mediating Nictation Behavior of Nematodes

Neuropeptides regulate animal physiology and behavior, making them widely studied targets of functional genetics research. While the field often relies on differential -omics approaches to build hypotheses, no such method exists for neuropeptidomics. It would nonetheless be valuable for studying behaviors suspected to be regulated by neuropeptides, especially when little information is otherwise available. This includes nictation, a phoretic strategy of Caenorhabditis elegans dauers that parallels host-finding strategies of infective juveniles of many pathogenic nematodes. We here developed a targeted peptidomics method for the model organism C. elegans and show that 161 quantified neuropeptides are more abundant in its dauer stage compared with L3 juveniles. Many of these have orthologs in the commercially relevant pathogenic nematode Steinernema carpocapsae, in whose infective juveniles, we identified 126 neuropeptides in total. Through further behavioral genetics experiments, we identify flp-7 and flp-11 as novel regulators of nictation. Our work advances knowledge on the genetics of nictation behavior and adds comparative neuropeptidomics as a tool to functional genetics workflows.     

The importance of being regular: Caenorhabditis elegans and Pristionchus pacificus defecation mutants are hypersusceptible to bacterial pathogens

Bacterial pathogens have shaped the evolution and survival of organisms throughout history, but little is known about the evolution of virulence mechanisms and the counteracting defence strategies of host species. The nematode model organisms, Caenorhabditis elegans and Pristionchus pacificus, feed on a wealth of bacteria in their natural soil environment, some of which can cause mortality. Previously, we have shown that these nematodes differ in their susceptibility to a range of human and insect pathogenic bacteria, with P. pacificus showing extreme resistance compared with C. elegans. Here, we isolated 400 strains of Bacillus from soil samples and fed their spores to both nematodes. Spores of six Bacillus strains were found to kill C. elegans but not P. pacificus. While the majority of Bacillus strains are benign to nematodes, observed pathogenicity is restricted to either the spore or the vegetative stage. We used the rapid C. elegans killer strain (Bacillus sp. 142) to conduct a screen for hypersusceptible P. pacificus mutants. Two P. pacificus mutants with severe muscle defects and an extended defecation cycle that die rapidly on Bacillus spores were isolated. These genes were identified to be homologous to C. elegans, unc-22 and unc-13. To test whether a similar relationship between defecation and bacterial pathogenesis exists in C. elegans, we used five known defecation mutants. Quantification of the defecation cycle in mutants also revealed a severe effect on survival in C. elegans. Thus, intestinal peristalsis is critical to nematode health and contributes significantly to survival when fed Gram-positive bacteria.     

Interaction Between Neoaplectana carpocapsae and a Granulosis Virus of the Armyworm Pseudaletia unipuncta

Neoaplectaua carpocapsae developed and reproduced in armyworm hosts infected with a granulosis virus (GV). Macerated tissues of dauer juveniles from GV-infecled hosts had sufficient GV to infect 1st and 2nd instar armyworms. Electron-microscope examination of dauer juveniles and adult female nematodes confirmed the presence of GV in the lumen of the intestine. No GV was observed in other tissues of the nematode.     

Effects of Insecticides on Movement,Nictation, and Infectivity of Steinernema carpocapsae

Movement, nictation, and infectivity of Steinernema carpocapsae strain All were compared for ensheathed (EnJ) and desheathed (DeJ) infective juveniles exposed to the insecticides acephate, dichlorvos, methomyl, oxamyl, or permethrin. Nematode response to various solutions included normal sinusoidal movement, uncoordinated motion, twitching, convulsion or formation of a pretzel shape, an inactive "S" posture with fine twitching, or a quiescent straight posture. The DeJ displayed these movements at lower concentrations of each insecticide than did EnJ. In petri dish bioassays, insecticide-treated EnJ caused generally lower mortality in the common cutworm, Spodoptera litura, than did EnJ alone but caused greater insect mortality than did insecticides alone. Nematode response to chemicals was more clearly demonstrated by nictating behavior than by the movement bioassay. Nictation of DeJ was suppressed by the test chemicals at low concentrations, except for acephate and permethrin. Nictating EnJ or DeJ, regardless of chemical treatment, killed host insects faster than did non-nictating juveniles. Insecticides that enhance nictating behavior at certain concentrations may be used for mixed applications with nematodes.     

Hormone signaling and phenotypic plasticity in nematode development and evolution

Phenotypic plasticity refers to the ability of an organism to adopt different phenotypes depending on environmental conditions. In animals and plants, the progression of juvenile development and the formation of dormant stages are often associated with phenotypic plasticity, indicating the importance of phenotypic plasticity for life-history theory. Phenotypic plasticity has long been emphasized as?a crucial principle in ecology and as facilitator of phenotypic evolution. In nematodes, several examples of phenotypic plasticity have been studied at the genetic and developmental level. In addition, the influence of different environmental factors has been investigated under laboratory conditions. These studies have provided detailed insight into the molecular basis of phenotypic plasticity and its?ecological and evolutionary implications. Here, we review recent studies on the formation of dauer larvae in Caenorhabditis elegans, the evolution of nematode parasitism and the generation of a novel feeding trait in Pristionchus pacificus. These examples reveal a conserved and co-opted role of an endocrine signaling module involving the steroid hormone dafachronic acid. We will discuss how hormone signaling might facilitate life-history and morphological evolution.     

Evolution of Host Search Strategies in Entomopathogenic Nematodes

There is interspecific variation in infective juvenile behavior within the entomopathogenic nematode genus Steinernema. This variation is consistent with use of different foraging strategies along a continuum between ambush and cruise foraging. To address questions about the evolution of foraging strategy, behavioral and morphological characters were mapped onto a phylogeny of Steinernema. Three species, all in the same clade, were classified as ambushers based on standing bout duration and host-finding ability. One clade of six species were all cruisers based on both host-finding and lack of standing behavior. All species in the ambusher clade had a high rate of jumping, all species in the cruiser clade had no jumping, and most intermediate foragers exhibited some level of jumping. Response to volatile and contact host cues was variable, even within a foraging strategy. Infective juveniles in the ambusher clade were all in the smallest size category, species in the cruiser clade were in the largest size categories, and intermediate foragers tended to be more intermediate in size. We hypothesize that the ancestral Steinernema species was an intermediate forager and that ambush and cruise foraging both evolved at least once in the genus.     

Infection behavior of the rhabditid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum.

Infection behavior of the rhabditid nematode Phasmarhabditis hermaphrodita to the grey garden slug Deroceras reticulatum was studied. The dauer (enduring or nonaging) juveniles of P. hermaphrodita invade D. reticulatum within 8-16 hr following external exposure, with the posterior mantle region containing the shell cavity serving as the main portal of entry. The dauer juveniles can recover, multiply, and produce new dauer juveniles in the slug and slug feces homogenates, but not in the soil extract. These results demonstrate that P. hermaphrodita is a facultative parasite of the slug and can complete its life cycle under nonparasitic conditions associated with the host. Although the juvenile and adult nematodes can kill the slug if injected into the shell cavity of the host, only the dauer juvenile can serve as an infective stage in the natural environment.     

Optimal parasite infection strategies: a state-dependent approach

Many macroparasites spend a crucial phase of their life-cycle as free-living infective stages. Despite their importance, however, little theoretical work has considered how evolution may shape the behaviour of these stages. Here, we develop what we believe to be the first stochastic dynamic programming model of parasite life-history strategies to investigate how a trade-off between resource depletion and host encounter rate may shape the optimal infection strategy of a macroparasite. The optimal strategy depends strongly on the probability of host contact and, depending on the relative costs and benefits, macroparasites should adopt either a passive 'ambushing' (sit-and-wait) strategy, an active 'cruising' strategy or a mixed strategy with an initial cruising phase, followed by a switch to ambushing when energy reserves fall to a threshold level. Under no circumstances does the model predict ambush-then-cruise. We use our model to help interpret previously published data on entomopathogenic nematode foraging behaviour, showing how this framework could facilitate our understanding of macroparasite behaviour during this key stage of the life-cycle.     

Commensal Nematodes in the Glands,Genitalia, and Brood Cells of Bees (Apoidea)

Seven species of bees from the eastern United States, representing four families in the Apoidea, were dissected and examined for nematode associates. Dufour''s glands in females of Halictus ligatus, Augochlora pura mosieri, and Augochlorella gratiosa (Halictidae) from Florida were infested with dauer juveniles of Aduncospiculum halicti (Diplogasteridae). The Dufour''s glands of Colletes thoracicus (Colletidae) females from Maryland were infested with dauer juveniles of a new species of Koerneria sp. (Diplogasteridae), and abdominal glands of females of Andrena alleghaniensis (Andrenidae) from New York were infested with dauer juveniles of another new species of Koerneria. The lateral and median oviducts, Dufour''s glands, and poison sacs in females of Anthophora abrupta (Anthophoridae) from Maryland and Alabama were infested with dauer juveniles of a new species of Bursaphelenchus sp. (Aphelenchoididae). Cross sections of the nematode-infested poison sacs of A. abrupta revealed two types of humoral host defense reactions.     

Pathogenicity, development, and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae under axenic in vivo conditions

Galleria mellonella larvae cultured axenically were treated with axenic dauer juveniles of Heterorhabditis bacteriophora and Steinernema carpocapsae. After 3 days S. carpocapsae had killed all insects, with 9.4 +/- 4.3 nematodes per larva. H. bacteriophora were unable to kill G. mellonella, although 13.3 +/- 6.4 nematodes per Galleria were found in the hemocoel. Invading nematodes of both strains recovered from the dauer stage. H. bacteriophora developed into hermaphrodites with eggs and J1 in the uterus and in the hemolymph of the living insects. Development beyond the J1 stage was not recorded. An injection of supernatants from different Photorhabdus luminescens cultures killed the insects but could not provide nutrients to support a further development. Only the injection of bacterial cells supported production of dauers in the axenic insects. Axenic S. carpocapsae developed to adults and produced offspring. After 3 weeks an average of 5275 nematodes per larva were counted, of which 6.7% were dauer juveniles, 39.2% other juvenile stages, 11.9% males, and 42.2% females. Compared to in vivo reproduction in the presence of the symbiotic bacterium Xenorhabdus nematophilus the dauer juvenile yields were low. Even after 5 weeks the percentage of dauer juveniles did not surpass 10%.     

Effect of Steinernema glaseri-infected host exudates on movement of conspecific infective juveniles

The entomopathogenic nematode's decision to infect a host is paramount because once the decision is made it is irrevocable; nematodes that invade a host either develop and achieve reproductive success, or they die. Entomopathogenic nematodes that have a cruiser foraging behavior, such as Steinernema glaseri, follow host-associated cues to locate insects to infect. Most of the host finding and infection dynamics research has focused on the infective juvenile nematodes' responses to cues from live insects such as host-associated volatiles and host contact cues. Few studies focus on how previously infected hosts influence infective juvenile infection behaviors. We investigated how exudates from nematode-infected hosts affect the behavior of S. glaseri infective juveniles. We hypothesized that the infective juvenile's behavioral response to cadavers would change as the state of a nematode-infected host changes during pathogenesis. We examined the effect of exudates collected from infected hosts on infective juvenile locomotory behavior. We detected no effects on nematode repulsion or attraction from exudates produced within the first 48h post-infection. We observed repulsion from accumulated exudates during the 3-48, 3-72, 3-120, and 3-144h intervals. Repulsion from exudates was observed during the 48-66, 72-90, and 120-138h intervals in experiments evaluating daily exudate emissions. The repellent effect of infected host exudates may result in an infective juvenile discriminating between suitable and unsuitable hosts.     

Pristionchus uniformis,should I stay or should I go? Recent host range expansion in a European nematode

Pristionchus pacificus has been developed as a model system in evolutionary developmental biology, evolutionary ecology, and population genetics. This species has a well-known ecological association with scarab beetles. Generally, Pristionchus nematodes have a necromenic association with their beetle hosts. Arrested dauer larvae invade the insect and wait for the host's death to resume development. Only one Pristionchus species is known to frequently associate with a non-scarab beetle. Pristionchus uniformis has been isolated from the chrysomelid Leptinotarsa decemlineata, also known as the Colorado potato beetle, in Europe and North America, but is also found on scarab beetles. This unusual pattern of association with two unrelated groups of beetles on two continents requires the involvement of geographical and host range expansion events. Here, we characterized a collection of 81 P. uniformis isolates from North America and Europe and from both scarab beetles and L. decemlineata. We used population genetic and phylogenetic analyses of the mitochondrial gene nd2 to reconstruct the genetic history of P. uniformis and its beetle association. Olfactory tests on beetles chemical extracts showed that P. uniformis has a unique chemoattractive profile toward its beetle hosts. Our results provide evidence for host range expansion through host-switching events in Europe where P. uniformis was originally associated with scarab beetles and the nematode's subsequent invasion of North America.     

Effect of Exsheathment on Motility and Pathogenicity of Two Entomopathogenic Nematode Species

The effect of sheath loss on motility and pathogenicity of the entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae, was examined using both naturally and chemically exsheathed (desheathed) infective juveniles. Exsheathed S. carpocapsae showed increased motility on agar compared to sheathed nematodes. The presence of a host increased motility threefold in all S. carpocapsae treatments. These results suggest that activation of S. carpocapsae host finding may result from sheath loss in addition to host stimuli. Desheathed H. bacteriophora were significantly less motile than the sheathed or exsheathed groups. The decreased motility may be due to adverse effects of the chemical treatment for desheathment. Sheath loss did not affect the pathogenicity of either species.     

Species-specific recognition of beetle cues by the nematode Pristionchus maupasi

SUMMARY The environment has a strong effect on development as is best seen in the various examples of phenotypic plasticity. Besides abiotic factors, the interactions between organisms are part of the adaptive forces shaping the evolution of species. To study how ecology influences development, model organisms have to be investigated in their environmental context. We have recently shown that the nematode Pristionchus pacificus and its relatives are closely associated with scarab beetles with a high degree of species specificity. For example, P. pacificus is associated with the oriental beetle Exomala orientalis in Japan and the northeastern United States, whereas Pristionchus maupasi is primarily isolated from cockchafers of the genus Melolontha in Europe. Here, we investigate how Pristionchus nematodes identify their specific insect hosts by using chemotaxis studies originally established in Caenorhabditis elegans . We observed that P. maupasi is exclusively attracted to phenol, one of the sex attractants of Melolontha beetles, and that attraction was also observed when washes of adult beetles were used instead of pure compounds. Furthermore, P. maupasi chemoattraction to phenol synergizes with plant volatiles such as the green leaf alcohol and linalool, demonstrating that nematodes can integrate distinct chemical senses from multiple trophic levels. In contrast, another cockchafer-associated nematode, Diplogasteriodes magnus , was not strongly attracted to phenol. We conclude that interception of the insect communication system might be a recurring strategy of Pristionchus nematodes but that different nematodes use distinct chemical cues for finding their beetle hosts.     

Steinernema feltiae: ammonia triggers the emergence of their infective juveniles

Entomopathogenic nematodes complete their life cycles inside dead insects. The emergence of new infective juveniles from the cadaver has been attributed (but never demonstrated) to food depletion or to the accumulation of metabolites from the breakdown of the host's tissues. Here we give evidence that emergence is triggered by ammonia, a product of nematode defecation. We found that the emergence of Steinernemafeltiae infective juveniles from Galleriamellonella cadavers was stimulated by a particular level of ammonia. Emergence was delayed when ammonia in the cadaver was decreased and was prompted when increased. These findings will further improve the understanding of the nematode life cycle. Here we speculate that production of infective juveniles can be mediated by ammonia and work in a manner analogous to that of the dauer recovery inhibiting factor (DRIF) in Caenorhabditiselegans.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Distinct patterns of genetic variation in Pristionchus pacificus and Caenorhabditis elegans, two partially selfing nematodes with cosmopolitan distribution

Hermaphroditism has evolved several times independently in nematodes. The model organism Caenorhabditis elegans and Pristionchus pacificus are self-fertile hermaphrodites with rare facultative males. Both species are members of different families: C. elegans belongs to the Rhabditidae and P. pacificus to the Diplogastridae. Also, both species differ in their ecology: C. elegans is a soil-dwelling nematode that is often found in compost heaps. In contrast, field studies in Europe and North America indicate that Pristionchus nematodes are closely associated with scarab beetles. In C. elegans, several recent studies have found low genetic diversity and rare out-crossing events. Little is known about diversity levels and population structure in free-living hermaphroditic nematodes outside the genus Caenorhabditis. Taking a comparative approach, we analyse patterns of molecular diversity and linkage disequilibrium in 18 strains of P. pacificus from eight countries and four continents. Mitochondrial sequence data of P. pacificus isolates reveal a substantially higher genetic diversity on a global scale when compared to C. elegans. A mitochondrial-derived hermaphrodite phylogeny shows little geographic structuring, indicating several worldwide dispersal events. Amplified fragment length polymorphism and single strand conformation polymorphism analyses demonstrate a high degree of genome-wide linkage disequilibrium, which also extends to the mitochondrial genome. Together, these findings indicate distinct patterns of genetic variation of the two species. The low level of genetic diversity observed in C. elegans might reflect a recent human-associated dispersal, whereas the P. pacificus diversity might reflect a long-lasting and ongoing insect association. Thus, despite similar lifestyle characteristics in the laboratory, the reproductive mode of hermaphroditism with rare facultative males can result in distinct genetic variability patterns in different ecological settings.