Phased infectivity in Heterorhabditis megidis: the effects of infection density in the parental host and filial generation

Entomopathogenic nematodes can develop through two or more generations in the cadavers of killed insect hosts. Non-feeding infective juveniles from each generation emerge and may spend prolonged periods searching for a new host. The infectivity of the infective juveniles of Heterorhabditis megidis varies with time after emergence and may not reach a maximum until several weeks have passed. 'Phased infectivity' hypotheses propose that this pattern is adaptive, tending to reduce competition in new hosts. Here we provide further evidence that infectivity is phased in H. megidis. In addition, we show that the basic pattern is modified by infection density in the parental host and by filial generation. Two general patterns were observed: first, infective juveniles that developed under the least crowded conditions (F(1) infective juveniles produced in hosts infected with 16 parent nematodes) reached maximum infectivity after only 15 days, compared to 27 or 39 days for infective juveniles that developed under more crowded conditions (F(1) produced in hosts infected with 103 or 424 parent nematodes or F(2) infective juveniles). Second, infective juveniles had lower infectivity overall when produced under the most crowded conditions (F(2) versus F(1); highest versus lowest infection density). We propose that while lower overall infectivity is a necessary consequence of limited resource availability during infective juvenile development, the difference in the timing of peak infectivity reflects a shift in the fitness gains associated with being maximally infective either 'early' or 'late'. F(1) infective juveniles emerge several days before F(2) infective juveniles, and we suggest that filial generation and infection density in the parental host function as indicators of the potential risk of competition within new hosts.     

昆虫病原线虫感染寄主行为研究进展

昆虫病原线虫斯氏属Steinernema和异小杆属Heterorhabditids线虫是新型的生物杀虫剂。其感染期幼虫是惟一能够侵染寄主昆虫的虫态。这类线虫感染寄主的行为分为寻找寄主、识别寄主和侵染寄主。文章综述昆虫病原线虫感染寄主昆虫的行为以及在感染寄主过程中的影响因素。     

A novel ascaroside controls the parasitic life cycle of the entomopathogenic nematode Heterorhabditis bacteriophora

Entomopathogenic nematodes survive in the soil as stress-resistant infective juveniles that seek out and infect insect hosts. Upon sensing internal host cues, the infective juveniles regurgitate bacterial pathogens from their gut that ultimately kill the host. Inside the host, the nematode develops into a reproductive adult and multiplies until unknown cues trigger the accumulation of infective juveniles. Here, we show that the entomopathogenic nematode Heterorhabditis bacteriophora uses a small-molecule pheromone to control infective juvenile development. The pheromone is structurally related to the dauer pheromone ascarosides that the free-living nematode Caenorhabditis elegans uses to control its development. However, none of the C. elegans ascarosides are effective in H. bacteriophora, suggesting that there is a high degree of species specificity. Our report is the first to show that ascarosides are important regulators of development in a parasitic nematode species. An understanding of chemical signaling in parasitic nematodes may enable the development of chemical tools to control these species.     

Infection success in novel hosts: an experimental and phylogenetic study of Drosophila-parasitic nematodes

Surprisingly little is known about what determines a parasite's host range, which is essential in enabling us to predict the fate of novel infections. In this study, we evaluate the importance of both host and parasite phylogeny in determining the ability of parasites to infect novel host species. Using experimental lab assays, we infected 24 taxonomically diverse species of Drosophila flies (Diptera: Drosophilidae) with five different nematode species (Tylenchida: Allantonematidae: Howardula, Parasitylenchus), and measured parasite infection success, growth, and effects on female host fecundity (i.e., virulence). These nematodes are obligate parasites of mushroom-feeding Drosophila, particularly quinaria and testacca group species, often with severe fitness consequences on their hosts. We show that the potential host ranges of the nematodes are much larger than their actual ranges, even for parasites with only one known host species in nature. Novel hosts that are distantly related from the native host are much less likely to be infected, but among more closely related hosts, there is much variation in susceptibility. Potential host ranges differ greatly between the related parasite species. All nematode species that successfully infected novel hosts produced infective juveniles in these hosts. Most novel infections did not result in significant reductions in the fecundity of female hosts, with one exception: the host specialist Parasitylenchus nearcticus sterilized all quinaria group hosts, only one of which is a host in nature. The large potential host ranges of these parasites, in combination with the high potential for host colonization due to shared mushroom breeding sites, explain the widespread host switching observed in comparisons of nematode and Drosophila phylogenies.     

Rates of development in male and female Wood Frogs and patterns of parasitism by lung nematodes

Researchers are becoming interested in testing whether investment in growth and/or development trades off against investment in parasite defence. We tested this idea by examining relations between development of Wood Frogs (Rana sylvatica) and susceptibility to lung nematodes (Rhabdias ranae). Male and female frogs reared in outdoor mesocosms were the same length and mass at metamorphosis. However, males metamorphosed sooner than females. Lung nematodes were no more likely to penetrate male versus female metamorphs following controlled exposures, but males had higher intensities of adult female worms and the largest worms per host were, on average, of larger size in male metamorphs. Males that took longer to metamorphose carried higher numbers of worms in their lungs than males that metamorphosed early. In comparison, females that developed faster harboured more worms in their lungs than females that took longer to reach metamorphosis. Our results suggest that variation in susceptibility to lung nematodes is influenced by host sex and possibly also by sex-specific relations with developmental rate. Further, male hosts might prove to be a more important source of infective stages of worms than female hosts.     

Sex ratios and sex-biased infection behaviour in the entomopathogenic nematode genus Steinernema

In experimentally infected insects, the sex ratio of first generation nematodes of five species of Steinernema was female-biased (male proportion 0.35-0.47). There was a similar female bias when the worms developed in vitro (0.37-0.44), indicating that the bias in these species is not due to a lower rate of infection by male infective juveniles (IJs). Experimental conditions influenced the proportion of males establishing in insects, indicating that male and female IJs differ in their behaviour. However, there was no evidence that males are the colonising sex in any species, contrary to what has previously been proposed. Time of emergence from the host in which the nematodes had developed influenced sex ratios in experimental infections. In three species (Steinernema longicaudum, Steinernema glaseri and Steinernema kraussei), early emerged nematodes had a higher proportion of males than those that emerged later, with the reverse trend for Steinernema carpocapsae and Steinernema feltiae. In a more detailed in vitro study of S. longicaudum, the proportion of males was similar whether or not the nematodes passed through the developmentally arrested IJ stage, indicating that the female bias is not due to failure of males to exit this stage. The sex ratio in vitro was independent of survival rate from juvenile to adult, and was female-biased even when all juveniles developed, indicating that the bias is not explained by failure of males to develop to adults. The female-biased sex ratio characteristic of Steinernema populations appears to be present from at least the early juvenile stage. We hypothesise that the observed female bias is the population optimal sex ratio, a response to cycles of local mate competition experienced by nematodes reproducing within insect hosts interspersed with periods of outbreeding with less closely related worms following dispersal.     

Sexual dimorphism in immune response, fat reserves and muscle mass in a sex role reversed spider

In sex role reversed species, females and males adopt behaviors that are not the traditional ones for that animal group. Furthermore, this reversal can translate into physiological differences between the sexes in characteristics such as energetic demands or immune response. Allocosa brasiliensis shows a reversal in the sex roles and sexual size dimorphism that would be expected for spiders. Males are larger than females and are sedentary, while females are the mobile sex that looks for males and initiates courtship. Our objective was to explore the occurrence of sexual dimorphism in immune response, fat content and muscular mass in A. brasiliensis, and relate the results to the reproductive strategies of the species. An encapsulation response was used as an estimate of the immune response. Abdominal fat content and leg muscular mass were quantified and the results were compared between females (N = 19) and males (N = 21). Males showed higher values of the three characteristics as compared to females. Life history divergences between the sexes regarding size, mobility and foraging opportunities could be factors driving these differences in immune response and energy requirements.     

A Technique for Evaluating Heterodera glycines Development in Susceptible and Resistant Soybean

A technique was developed to evaluate Heterodera glycines development in susceptible and resistant soybean. Roots of 3-day-old soybean were exposed to infective juveniles of H. glyci.nes in sand for 8 hours followed by washing and transfer to hydroponic culture. The cotyledons and apical meristem were removed and plants were maintained under constant light, which resulted in a dwarfed plant system. After 15 or 20 days at 27 C, nematodes were rated for development. Emerged males were sieved from the culture water and females were counted directly from the roots. Nematodes remaining in the roots were rated for development after staining and clearing the tissues. The proportion of nematodes at each stage of development and the frequency of completed molts for each stage were calculated from these data. This technique showed that resistance to H. glycines was stage related and did not affect males and females equally in all resistant hosts. The resistance of plant introduction PI 209332 primarily affected development of third and fourth-stage juveniles; ''Pickett'' mainly affected second and third-stage juveniles, whereas PI 89772 affected all stages. Male development was markedly affected in PI 89772 and ''Pickett'' but not in PI 209332.     

Inbreeding and Hybridizing Cyst Nematodes on Pruned Soybeans in Petri Plates

Inbred nematodes propagated on a selecting host are likely to have homozygous genes of interest for investigating the genetics of host-parasite associations. A technique is presented to inbreed soybean cyst nematodes, by sibling matings at each generation, and to cross inbred lines. Soybean seedlings with severely trimmed cotyledons survive well on 0.8% agar. Eggs from a single female are incubated in water in a microtiter well. Virgin as well as mated females result from inoculation of two juveniles per root. Sibling males from the same source are produced by mass inoculations of eggs. Males are added individually to unmated females. Overall success for fertile females was 14% in 1,368 isolations. Three generations of inbreeding by siblings were achieved using nematodes from two populations that differ in their ability to reproduce on differential soybeans. Hybrids from crosses of the two inbred lines tested on differential hosts showed that the influence of Population 1 (selected and inbred on PI 209332) is greater than that of Population 2 (selected and inbred on PI 89772). Reciprocal crosses suggest that the influence of males is stronger than that of females in determining host specificity of F₁ offspring in these crosses. Our technique is simple and effective for inbreeding and crossing soybean cyst nematodes.     

Mating and Sexual Communication by Steinernema carpocapsae (Nemata: Steinernematidae)

Entomopathogenic nematodes are lethal insect parasites that reproduce exclusively inside their hosts in nature. Infection decisions made by the free-living infective-stage juveniles have an impact on reproductive success, but it is likely that mating decisions are made by adults while inside their host. We investigated sexual communication between male and female adult stages of Steinernema carpocapsae (Rhabditida: Steinernematidae) to assess whether mating is chemically mediated during the adult stage or results from incidental encounters between adults inside the insect host. To assess chemical communication, we measured the behavioral response of adult male S. carpocapsae to several different potential sources of chemical information. Male S. carpocapsae responded to virgin females only and were not influenced by mated conspecific females, conspecific males, or heterospecific females. These results show that species-specific communication takes place between adult entomopathogenic nematodes within the host cadaver just prior to mating.     

Host influences on reproduction and establishment of mouse-adapted Nippostrongylus brasiliensis (Nematoda)

Various influences of the host altered the establishment and reproduction of mouse-adapted Nippostrongylus brasiliensis. Establishment and egg production were greater in mature male than in female mice. Worm fecundity increased similarly in both sexes of mice prior to 41 days of host age. Male mice at 62 days of age or older harbored more N. brasiliensis than did females, but the number of nematodes was similar in younger hosts of both sexes. Crowding of male mice significantly increased the establishment of helminths, but not the production of eggs by N. brasiliensis. Pregnancy of the host significantly increased both establishment of and reproduction by N. brasiliensis. Ligation of the bile duct of the mouse reduced both establishment and fecundity of the nematode after subcutaneous or surgical infection with larvae and adults, respectively.     

Nippostrongylus brasiliensis: reversibility of reduced-energy status associated with the course of expulsion from the small intestine in rats

Gastrointestinal nematodes require energy for active establishment in the gut against intestinal flow and peristaltic motion. In this study we employed CellTiter-Glo Luminescent Cell Viability Assay to measure the ATP value of individual adult Nippostrongylus brasiliensis during the course of immune-mediated expulsion from the small intestine in rats. The ATP values of adult worms taken from the lumen of the distal small intestine were lower than worms collected from the lumen of the proximal small intestine. Moreover, values from worms in the lumen of the proximal small intestine were lower than those from worms in the mucosa, the preferred site of adult N. brasiliensis. The reduction of ATP values in worms from each region was observed not only at expulsion phase, but also at established phases of the infection suggesting that energy metabolism of the parasites is independent of host immune response. When adult worms with low ATP values on day 12 post-infection were implanted surgically into the small intestine of na?ve rats, the worms re-established in recipients and completely restored the ATP values. Short in vitro culture of adult worms under low oxygen tension resulted in low ATP value in the worms. These results suggested that adult worms were dislodged from their preferred site by intact energy metabolism activity.     

Laboratory infection of blackflies (Simuliidae) and midges (Chironomidae) by the mosquito mermithid, Romanomermis culicivorax

Penetrating infective juveniles of Romanomermis culicivorax usually killed first-stage larvae of Chironomus maturus Johann., Chironomus sp., Simulium damnosum Theo., and S. venustum Say. Nematodes were melanized and died after they entered fourth stage larvae of 2 chironomid species, but no host reaction was evident after entry into fourth-stage blackfly larvae. In contrast, the nematodes initiated development in the latter hosts, which died before the nematodes completed their development.     

Parasitism of Molluscs by Nematodes: Types of Associations and Evolutionary Trends

Although there are no confirmed fossil records of mollusc parasitic nematodes, diverse associations of more than 108 described nematode species with slugs and snails provide a fertile ground for speculation of how mollusc parasitism evolved in nematodes. Current phylogenic resolution suggests that molluscs have been independently acquired as hosts on a number of occasions. However, molluscs are significant as hosts for only two major groups of nematodes: as intermediate hosts for metastrongyloids and as definitive hosts for a number of rhabditids. Of the 61 species of nematodes known to use molluscs as intermediate hosts, 49 belong to Metastrongyloidea (Order Strongylida); of the 47 species of nematodes that use molluscs as definitive hosts, 33 belong to the Order Rhabditida. Recent phylogenetic hypotheses have been unable to resolve whether metastrongyloids are sister taxa to those rhabditids that use molluscs as definitive hosts. Although most rhabditid nematodes have been reported not to kill their mollusc hosts prior to their reproduction, some species are pathogenic. In fact, infective juveniles of Phasmarhabditis hermaphrodita vector a lethal bacterium into the slug host in which they reproduce. This life cycle is remarkably similar to the entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae. Also, the discoveries of Alloionema and Pellioditis in slugs are interesting, as these species have been speculated to represent the ancestral forms of the entomopathogenic nematodes. Development of the infective stage appears to be an important step toward the acquisition of molluscs as definitive hosts, and the association with specific bacteria may have arisen in conjunction with the evolution of necromeny.     

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.     

Correlated evolution between host immunity and parasite life histories in primates and oxyurid parasites

Maturation time is a pivotal life-history trait of parasitic nematodes, determining adult body size, as well as daily and total fecundity. Recent theoretical work has emphasized the influence of prematurational mortality on the optimal values of age and size at maturity in nematodes. Eosinophils are a family of white blood cells often associated with infections by parasitic nematodes. Although the role of eosinophils in nematode resistance is controversial, recent work has suggested that the action of these immune effectors might be limited to the larval stages of the parasite. If eosinophils act on larval survival, one might predict, in line with theoretical models, that nematode species living in hosts with large eosinophil numbers should show reduced age and size at maturity. We tested this prediction using the association between the pinworms (Oxyuridae, Nematoda) and their primate hosts. Pinworms are highly host specific and are expected to be involved in a coevolutionary process with their hosts. We found that the body size of female parasites was negatively correlated with eosinophil concentration, whereas the concentration of two other leucocyte families-neutrophils and lymphocytes-was unrelated to female body size. Egg size of parasites also decreased with host eosinophil concentration, independently of female size. Male body size was unrelated to host immune parameters. Primates with the highest immune defence, therefore, harbour small female pinworms laying small eggs. These results are in agreement with theoretical expectations and suggest that life histories of oxyurid parasites covary with the immune defence of their hosts. Our findings illustrate the potential for host immune defence as a factor driving parasite life-history evolution.     

Seasonal dynamics of Skrjabinoptera phrynosoma (Nematoda) infection in horned lizards from the Alvord Basin: temporal components of a unique life cycle

The nematode Skrjabinoptera phrynosoma is a stomach parasite of horned lizards in the genus Phrynosoma. This nematode demonstrates a distinctive life cycle wherein entire gravid females harboring infective eggs exit with lizard feces. Pogonomyrmex spp. harvester ants collect these females and feed them to their larvae, which are the only stages of the intermediate host that can become infected. We hypothesized that the seasonal dynamics of nematode abundance within lizard hosts would be correlated with the seasonal availability of suitable intermediate hosts. To describe seasonal variation of nematode population variables and elucidate the timing of critical events in the parasite life cycle, nematodes were collected from both hosts across three collection periods in the ant-and-lizard activity season of 2008 in the Alvord Basin of southeastern Oregon. Among 3 collection periods, and across the activity season, nematodes were harvested from individual Phrynosoma platyrhinos , and the distribution of developmental categories and body lengths of nematodes was analyzed to determine the seasonal change in nematode population composition. Pogonomyrmex spp. ants were collected in pit-fall traps and dissected to determine infection prevalence. The abundance of non-gravid female and juvenile nematodes collected from lizards' stomachs decreased significantly between the early and late collection period, which was likely a consequence of the sequential conversion of these developmental categories to gravid females. The presence of gravid female nematodes peaked in cloacal and fecal collections during mid-season. The body lengths of male nematodes increased as the activity season progressed, perhaps due to growth, but their abundance remained the same. Smaller juvenile nematodes were present in late-season collections from lizards, possibly indicating new acquisitions from infected ants. We propose that once a set population of male nematodes establishes in lizards' stomachs, newly acquired juvenile nematodes develop into non-gravid females that mate, become gravid females, and exit the lizard mid-season. We additionally suggest that the exit of females may be timed with the peak foraging activity of ant intermediate hosts and access to larval ants in the nests. Infection prevalence in the intermediate host was low, with only 1 of 6,000 dissected harvester ants containing a single larval nematode. The temporal dynamics of S. phrynosoma populations within P. platyrhinos at this northern locale is most likely driven by the seasonal availability of harvester ant intermediate hosts.     

Relationship between the successful infection by entomopathogenic nematodes and the host immune response

Reproduction of entomopathogenic nematodes requires that they escape recognition by a host's immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS-PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.     

Evaluation of Morphological Variability in Meloidogyne arenaria

Seven populations, representing cytological race A (triploid, 3n = 51-56) and the two host races (infective and noninfective on peanut) of Meloidogyne arenaria were studied with light microscopy (LM) and scanning electron microscopy (SEM). Characteristics of root-knot nematodes, recently recommended as useful taxonomic traits, were reexamined among these populations, and their variability both within and between populations was ascertained. We found that stylet morphology of females and head and stylet morphologies of males and second-stage juveniles were the most reliable characters for identification. The two host races of M. arenaria could not be distinguished morphologically. Two of the populations could be separated consistently from the remainder but were not sufficiently divergent to be considered new species. These two variant populations were similar; neither produced males in culture, and they differed from the typical populations in female perineal patterns (LM) as well as in cephalic structure (SEM) and tail shape (LM) of second-stage juveniles. In morphometric studies, most characters of the variant populations differed significantly from those of the typical M. arenaria.