Is there life after parasitism? Survival, longevity, and oogenesis in Acheta domesticus (Orthoptera: Gryllidae) infected with the hairworm, Paragordius varius (Phylum: Nematomorpha).

The costs parasites impose on hosts can lead to reductions in survival and fecundity, but few studies have evaluated the impacts after infection. Hairworms are parasites of terrestrial arthropods that are free-living in aquatic systems as adults. As parasitic juveniles, hairworms acquire nutrients from their definitive hosts, shifting resources away from host development to parasite growth. However, until now, only one study has examined survivorship of naturally infected hosts with hairworms. Using a different hairworm and host system, we conducted experimental infections to examine growth, survivorship, and egg production in virgin female Acheta domesticus infected with the hairworm, Paragordius varius. We found that infected crickets grew significantly less during hairworm development compared to sham-infected control crickets. After releasing their worms, infected crickets survived for 73 ± 32 days but had significantly shorter life spans by an average of 13 days compared to sham-infected control crickets. However, we found that 50% of previously infected crickets produced eggs after releasing their worms. Taken together, these observations suggest that female crickets infected with hairworms may experience less mortality than previous anecdotal evidence suggests. Finally, we discuss the definition of parasitoid and how it relates to nematomorphs, and we suggest that more field and laboratory research is required before suggesting hairworms are parasitoids.

Responses of intended and unintended receivers to a novel sexual signal suggest clandestine communication.

Inadvertent cues can be refined into signals through coevolution between signalers and receivers, yet the earliest steps in this process remain elusive. In Hawaiian populations of the Pacific field cricket, a new morph producing a novel and incredibly variable song (purring) has spread across islands. Here we characterize the current sexual and natural selection landscape acting on the novel signal by (1) determining fitness advantages of purring through attraction to mates and protection from a prominent deadly natural enemy, and (2) testing alternative hypotheses about the strength and form of selection acting on the novel signal. In field studies, female crickets respond positively to purrs, but eavesdropping parasitoid flies do not, suggesting purring may allow private communication among crickets. Contrary to the sensory bias and preference for novelty hypotheses, preference functions (selective pressure) are nearly flat, driven by extreme inter-individual variation in function shape. Our study offers a rare empirical test of the roles of natural and sexual selection in the earliest stages of signal evolution.

Molecular Identification of Oxyspirura Petrowi Intermediate Hosts by Nested PCR Using Internal Transcribed Spacer 1 (ITS1).

Recently, the heteroxenous eyeworm, Oxyspirura petrowi, has gained attention due to its prevalence in the declining game bird, Northern bobwhite (Colinus virginianus), but the intermediate hosts of many nematodes remain unknown. However, identifying the intermediate host of O. petrowi with traditional techniques would be difficult and time-consuming, especially considering there are more than 80 potential orthopteran hosts just in Texas. To screen a large number of samples quickly and effectively, primers for nested PCR (nPCR) were developed using the internal transcribed spacer 1 (ITS1) region. Then the nPCR was used to identify which of the 35 species collected from the Order Orthoptera were potential intermediate hosts of O. petrowi. With this technique, 18 potential intermediate hosts were identified. Later, we collected live specimens of species that tested positive to confirm the presence of larvae, but larvae were not found in the live specimens, nor in the extra tissue of the species that had tested positive for O. petrowi DNA. Despite this, this study demonstrated that nPCR is more sensitive than traditional techniques and can be a valuable tool in determining the intermediate hosts of parasites.

Life-cycle of Oxyspirura petrowi (Spirurida: Thelaziidae), an eyeworm of the northern bobwhite quail (Colinus virginianus).

BACKGROUND: Oxyspirura petrowi (Spirurida: Thelaziidae), a heteroxenous nematode of birds across the USA, may play a role in the decline of the northern bobwhite (Colinus virginianus) in the Rolling Plains Ecoregion of West Texas. Previous molecular studies suggest that crickets, grasshoppers and cockroaches serve as potential intermediate hosts of O. petrowi, although a complete study on the life-cycle of this nematode has not been conducted thus far. Consequently, this study aims to improve our understanding of the O. petrowi life-cycle by experimentally infecting house crickets (Acheta domesticus) with O. petrowi eggs, feeding infected crickets to bobwhite and assessing the life-cycle of this nematode in both the definitive and intermediate hosts. METHODS: Oxyspirura petrowi eggs were collected from gravid worms recovered from wild bobwhite and fed to house crickets. The development of O. petrowi within crickets was monitored by dissection of crickets at specified intervals. When infective larvae were found inside crickets, parasite-free pen-raised bobwhite were fed four infected crickets each. The maturation of O. petrowi in bobwhite was monitored through fecal floats and bobwhite necropsies at specified intervals. RESULTS: In this study, we were able to infect both crickets (n = 45) and bobwhite (n = 25) with O. petrowi at a rate of 96%. We successfully replicated and monitored the complete O. petrowi life-cycle in vivo, recovering embryonated O. petrowi eggs from the feces of bobwhite 51 days after consumption of infected crickets. All life-cycle stages of O. petrowi were confirmed in both the house cricket and the bobwhite using morphological and molecular techniques. CONCLUSIONS: This study provides a better understanding of the infection mechanism and life-cycle of O. petrowi by tracking the developmental progress within both the intermediate and definitive host. To our knowledge, this study is the first to fully monitor the complete life-cycle of O. petrowi and may allow for better estimates into the potential for future epizootics of O. petrowi in bobwhite. Finally, this study provides a model for experimental infection that may be used in research examining the effects of O. petrowi infection in bobwhite.

A parasitological evaluation of edible insects and their role in the transmission of parasitic diseases to humans and animals.

From 1 January 2018 came into force Regulation (EU) 2015/2238 of the European Parliament and of the Council of 25 November 2015, introducing the concept of "novel foods", including insects and their parts. One of the most commonly used species of insects are: mealworms (Tenebrio molitor), house crickets (Acheta domesticus), cockroaches (Blattodea) and migratory locusts (Locusta migrans). In this context, the unfathomable issue is the role of edible insects in transmitting parasitic diseases that can cause significant losses in their breeding and may pose a threat to humans and animals. The aim of this study was to identify and evaluate the developmental forms of parasites colonizing edible insects in household farms and pet stores in Central Europe and to determine the potential risk of parasitic infections for humans and animals. The experimental material comprised samples of live insects (imagines) from 300 household farms and pet stores, including 75 mealworm farms, 75 house cricket farms, 75 Madagascar hissing cockroach farms and 75 migrating locust farms. Parasites were detected in 244 (81.33%) out of 300 (100%) examined insect farms. In 206 (68.67%) of the cases, the identified parasites were pathogenic for insects only; in 106 (35.33%) cases, parasites were potentially parasitic for animals; and in 91 (30.33%) cases, parasites were potentially pathogenic for humans. Edible insects are an underestimated reservoir of human and animal parasites. Our research indicates the important role of these insects in the epidemiology of parasites pathogenic to vertebrates. Conducted parasitological examination suggests that edible insects may be the most important parasite vector for domestic insectivorous animals. According to our studies the future research should focus on the need for constant monitoring of studied insect farms for pathogens, thus increasing food and feed safety.

Identification of two species of Binema Travassos 1925 (Oxyurida: Travassosinematidae) and Cameronia arecoensis Marchissio and Miralles 1987 (Oxyurida: Thelastomatidae) based on morphological and 18S rRNA partial sequence.

Oxyurid nematodes parasitizing the mole cricket Neoscapteriscus vicinus were isolated in the framework of sampling fields of mole-crickets from the pampean region, in Argentina. In this work, molecular characterization of the 18S rRNA partial sequence of nematodes belonging to the families Thelastomatidae (Cameronia arecoensis Marchissio and Miralles 1987) and Travassosinematidae (Binema korsakowi Sergiev 1923 and Binema klossae, Marchissio and Miralles 1993) were carried out. This is the first world report of sequences belonging to B. klossae and C. arecoensis and first Argentinian report of B. korsakowi sequence. Also, morphological and morphometric features of B. klossae, B. korsakowi and C. arecoensis from Argentinian populations are reported.

Sand crickets (Gryllus firmus) have low susceptibility to entomopathogenic nematodes and their pathogenic bacteria.

The entomopathogenic nematode, Steinernema scapterisci, a specialist parasite of crickets, has been successfully used to combat the southern mole cricket, Neoscapteriscus borellii, which is an invasive pest of turf grass. As an entomopathogenic nematode, S. scapterisci causes rapid death of the insects it infects and uses bacteria to facilitate its parasitism. However, our understanding of the relative contributions of the nematode, S. scapterisci, and its bacterial symbiont, Xenorhabdus innexi, to parasitism remains limited. Here we utilized the sand cricket, Gryllus firmus, as a model host to evaluate the contributions of the EPNs S. scapterisci and S. carpocapsae, as well as their symbiotic bacteria, X. innexi and X. nematophila, respectively, to the virulence of the nematode-bacterial complex. We found that G. firmus has reduced susceptibility to infection from both S. scapterisci and the closely related generalist parasite S. carpocapsae, but that S. scapterisci is much more virulent than S. carpocapsae. Further, we found that N. borellii has reduced susceptibility to X. nematophila, and that G. firmus has reduced susceptibility to X. nematophila, X. innexi, and Serratia marcescens, much more so than other insects that have been studied. We found that the reduced susceptibility of G. firmus to bacterial infection is dependent on development, with adults being less susceptible to infection than nymphs. Our data provide evidence that unlike other EPNs, the virulence of S. scapterisci to crickets is dependent on the nematode rather than the bacterial symbiont that it carries and we speculate that S. scapterisci may be evolving independence from X. innexi.

Molecular detection of Oxyspirura larvae in arthropod intermediate hosts.

To determine potential intermediate hosts of Oxyspirura petrowi, a common nematode eyeworm of wild gallinaceous birds, various arthropod species including red harvester ants, beetles, wood cockroaches, crickets, grasshoppers, katydids, and desert termites were screened for the presence of O. petrowi using specific polymerase chain reaction (PCR) primers targeting the internal transcribed spacer 2 region (ITS2) of the eyeworm ribosomal deoxyribonucleic acid (rDNA). This is the first study to investigate the intermediate hosts of O. petrowi utilizing molecular techniques. We determined 38% (13/34) of the cockroaches, 27% (3/11) of the crickets, and 23% (68/289) of the grasshoppers which were positive for O. petrowi. Identifying potential intermediate hosts of O. petrowi is essential to better understanding the epizoology of the eyeworm's transmission mechanics and to controlling infections in wild gallinaceous birds.

Infective Juveniles of the Entomopathogenic Nematode Steinernema scapterisci Are Preferentially Activated by Cricket Tissue.

Entomopathogenic nematodes are a subgroup of insect-parasitic nematodes that are used in biological control as alternatives or supplements to chemical pesticides. Steinernema scapterisci is an unusual member of the entomopathogenic nematode guild for many reasons including that it is promiscuous in its association with bacteria, it can reproduce in the absence of its described bacterial symbiont, and it is known to have a narrow host range. It is a powerful comparative model within the species and could be used to elucidate parasite specialization. Here we describe a new method of efficiently producing large numbers of S. scapterisci infective juveniles (IJs) in house crickets and for quantifying parasitic activation of the IJs upon exposure to host tissue using morphological features. We found that parasite activation is a temporal process with more IJs activating over time. Furthermore, we found that activated IJs secrete a complex mixture of proteins and that S. scapterisci IJs preferentially activate upon exposure to cricket tissue, reaffirming the description of S. scapterisci as a cricket specialist.

Calling Behavior of Male Acheta domesticus Crickets Infected with Paragordius varius (Nematomorpha: Gordiida).

It is well established that parasites in the phylum Nematomorpha induce suicide behavior of their insect hosts to bring adult worms to the appropriate habitat for emergence. It is not well established, however, whether other nematomorph-induced behavioral alterations occur before worm emergence. The purpose of our study was to evaluate the effect of the nematomorph Paragordius varius on the calling behavior of the male house cricket Acheta domesticus . We hypothesized that cricket calling, an energetically expensive and risky behavior, would be a potential target for nematomorph-induced behavioral alterations. We assessed if and how infection with P. varius affects A. domesticus calling behavior and whether the presence of wings at time of exposure to P. varius influenced changes in calling behavior. We recorded the calling behavior of male A. domesticus over the course of their infection after exposure to P. various before or after wing development. Additionally, we assessed whether winged crickets were "callers" or "noncallers" before exposure. We found that regardless of cricket developmental stage (or age) at time of infection, infected crickets spent significantly less time calling than their uninfected counterparts but only during the later stages of infection. Developmental stage at infection did affect whether crickets became callers: when infected before wing development significantly more uninfected crickets initiated calling; there was no difference between infected and uninfected crickets when infected as winged adults. Infection was a factor in whether callers stopped calling, with more infected crickets ceasing to call than uninfected crickets. This is the first study to show that infection with nematomorphs affects calling behavior of their insect host. Cricket calling behavior is immensely complex and although it was difficult to elucidate the adaptive nature of these parasite-induced behavioral changes, this study lays the groundwork for future studies to begin teasing out the factors that will help make the determination between side effect of infection or parasite/host adaptation.

Alternative Reproductive Tactics Arising from a Continuous Behavioral Trait: Callers versus Satellites in Field Crickets.

Alternative reproductive tactics may arise when natural enemies use sexual signals to locate the signaler. In field crickets, elevated costs to male calling due to acoustically orienting parasitoid flies create opportunity for an alternative tactic, satellite behavior, where noncalling males intercept females attracted to callers. Although the caller-satellite system in crickets that risk detection by parasitoids resembles distinct behavioral phenotypes, a male's propensity to behave as caller or satellite can be a continuously variable trait over several temporal scales, and an individual may pursue alternate tactics at different times. We modeled a caller-satellite-parasitoid system as a spatially explicit interaction among male and female crickets using individual-based simulation. Males varied in their propensity to call versus behave as a satellite from one night to the next. We varied mortality, density, sex ratio, and female mating behavior, and recorded lifetime number of mates as a function of a male's probability of calling (vs. acting as a satellite) along a gradient in parasitism risk. Frequently, the optimal behavior switched abruptly from being pure caller (call every night) to pure satellite (never call) as parasitism rate increased. However, mixed strategies prevailed even with high parasitism risk under conditions of higher background mortality rate, decreasing density, increasing female-biased sex ratio, and increasing female choosiness. In natural populations, high parasitoid pressure alone would be unlikely to yield fixation of pure satellite behavior.

EVALUATION OF VIRULENCE OF STEINERNEMA CARPOCAPSAE TO EUROPEAN MOLE CRICKET GRYLLOTALPA GRYLOTALPA L.

Common European mole cricket (CEML) Grillotalpa grillotalpa L causes damage to field, vegetable crops, and small fruits growing at commercial plantations and nurseries. Chemical control if insecticides are used in poison bates, soil application or seedling/bulbs treatment is not environmentally friendly. Inundative and innoculative release of CTVL biocontrol agents, in particularly, Entomopathogenic nematode is a reliable alternative to chemical control. At the laboratory study the comparison of the ability of commercial strain (Nemastar) and local Ukrainian isolate of Steinernema carpocapsae in various concentrations to parasite in last instar nymph and adults of G. grillotalpa was investigated. Grillotalpa grillotalpa was found as a susceptible host for both commercial and local strains of S. carpocapsae. The life cycle of S. carpocapsae both strains in the adults of G. grillotapla with concentration 50 IJs per larva has been completed 12-15 days at t=25 C. Two generations of S. carpocapsae were able to develop in mole cricket for both strains. Two strains of S. carpocapsae nematode species tested were pathogenic to adults of G. grillotalpa. The mortalities of G. grillotapla last last instars' larva caused by S. carpocapsae were recorded in every concentration tested at least 20 to 150 IJs per larva. Mean larval mortality ranged from 48% to 95% depending upon nematode strain and rate of application. Larval mortality generally increased with increasing of nematode rates. It was significant for both S. carpocapsae strains (Ukr. Isolate F = 26 > 2,86) and commercial strain (Nemastar F = 102,95 > 2,86). Ukrainian local isolate caused a greater percentage of mortality of G. grillotapla adult than commercial strain of S. carpocapsae tested but interactions between nematode strains, application rates were not significant. This study presents new data on effect of S. carpocapsae isolated for Ukraine to key agricultural polyphagous pest G. grillotalpa susceptibility screening under controlled conditions, as presented in this paper is the first step towards the development of a biological control program of G. grillotaplpa by using of entomopathogenic nematodes.

Arthropod intermediate hosts of Abbreviata antarctica (Nematoda: Physalopteridae) in Australia.

This study examines potential arthropod intermediate hosts for the nematode Abbreviata antarctica. Five species of arthropod (tropical native cricket Teleogryllus oceanicus; native roach Drymoplaneta communis; native termite Nasutitermes exitiosus; and 2 introduced species, the East African roach Naupheta cinerea and the Asian cricket Acheta domesticus) were fed feces from a skink, Tiliqua rugosa, containing embryonated eggs of A. antarctica . The insects were dissected at 4 intervals (1 day, 4 days, 8-16 days, and 25-26 days). Viable third-stage larvae were recovered from cysts on the external wall of the midgut and hindgut of 62.5% nymphal and adult T. oceanicus crickets at 25-26 days post-infection and from a single nymphal A. domesticus cricket. No roaches contained eggs or first-stage larvae after 48 hr, and neither eggs nor larvae were found in termites.

Linking predator-prey interactions with exposure to a trophically transmitted parasite using PCR-based analyses.

Parasite transmission is determined by the rate of contact between a susceptible host and an infective stage and susceptibility to infection given an exposure event. Attempts to measure levels of variation in exposure in natural populations can be especially challenging. The level of exposure to a major class of parasites, trophically transmitted parasites, can be estimated by investigating the host's feeding behaviour. Since the parasites rely on the ingestion of infective intermediate hosts for transmission, the potential for exposure to infection is inherently linked to the definitive host's feeding ecology. Here, we combined epidemiological data and molecular analyses (polymerase chain reaction) of the diet of the definitive host, the white-footed mouse (Peromyscus leucopus), to investigate temporal and individual heterogeneities in exposure to infection. Our results show that the consumption of cricket intermediate hosts accounted for much of the variation in infection; mice that had consumed crickets were four times more likely to become infected than animals that tested negative for cricket DNA. In particular, pregnant female hosts were three times more likely to consume crickets, which corresponded to a threefold increase in infection compared with nonpregnant females. Interestingly, males in breeding condition had a higher rate of infection even though breeding males were just as likely to test positive for cricket consumption as nonbreeding males. These results suggest that while heterogeneity in host diet served as a strong predictor of exposure risk, differential susceptibility to infection may also play a key role, particularly among male hosts. By combining PCR analyses with epidemiological data, we revealed temporal variation in exposure through prey consumption and identified potentially important individual heterogeneities in parasite transmission.

Transmission, host specificity, and seasonal occurrence of Cyrtosomum penneri (Nematoda: Atractidae) in lizards from Florida.

Experimental infections and field-collected lizards were used to investigate issues of transmission, host specificity, and seasonal occurrence in the nematode Cyrtosomum penneri (Cosmocercoidea: Atractidae). Anolis sagrei (87 males, 42 females) were captured from the Florida Southern College campus, Polk County, Florida, from October 2010 to September 2011, and 8,803 C. penneri were collected from their intestines. During the breeding season all sexually mature (SVL ≥ 34 mm) A. sagrei were infected, whereas juvenile lizards (SVL <34 mm) were never infected. Experimental infections, using A. sagrei , found that worms were transferred to new hosts venereally, but not during oral exposures. Mating trials confirmed that worms were consistently transferred between hosts during copulation under natural conditions. Experimental exposures found that land snails and crickets do not serve as transport or intermediate hosts, which supports the idea that C. penneri is transferred only during host copulation. Experimental infections to test host specificity in C. penneri successfully infected A. sagrei , Hemidactylus turcicus , and Sceloporus undulatus , but not Anolis carolinensis or Plestiodon inexpectatus. Overall, this is the first study to fully elucidate the life cycle of any atractid nematode, and we suggest a venereal route of transmission for all atractid worms that infect reptilian hosts. Our findings also have implications for the host's reproductive and behavioral biology, e.g., support for covert or satellite males in the A. sagrei mating system.

Dominance is not always an honest signal of male quality, but females may be able to detect the dishonesty.

Females prefer dominant males as mating partners in numerous species. Male dominance rank is considered as an honest signal of male quality, because only healthy males in good condition are thought to be able to win fights with other males. Here, we tested whether activation of the immune system influences the success of males in male-male competition and mating in the field cricket, Gryllus integer. We activated the immune system of males with a nylon monofilament (to mimic a parasitoid larva), and arranged fights between male pairs to assess male dominance and associated mating success. Activation of the immune system with nylon monofilament substantially enhanced the fighting success of males during male-male competition but had no effect on mating success. However, sham-manipulation (a wound only) did not have any effect on fighting success although females mated more often with dominant males. Our study suggests that when male crickets meet an apparent survival threat they may behave more dominantly, probably owing to terminal investment. Male success during male-male competition is not always an honest signal of males' quality, but females may be able to detect this dishonesty.

Survival of larval and cyst stages of gordiids (Nematomorpha) after exposure to freezing.

Hairworms infect terrestrial arthropods and are 1 of the most understudied groups of parasites. Recently, life cycles of 2 gordiids (Paragordius varius and Paragordius obamai) have been domesticated in the laboratory. We tested the viability of laboratory reared and post-frozen larval and cyst stages of the North American gordiid, P. varius , frozen at -80 C for 7 mo, and the viability of field collected and post-frozen cysts of the African (P. obamai) and North American ( P. varius ) gordiid frozen at -20 C for 2 mo. All snails exposed to post-frozen or control P. varius larvae became infected with cysts, and there was no significant difference in prevalence or mean intensity of cysts among control or experimental snail groups. As with larvae, no significant differences were observed in prevalence or mean intensity of emerging worms from crickets infected with post-frozen or control P. obamai or P. varius cysts. All female P. obamai and P. varius worms from control and post-frozen cyst infections laid eggs and larvae hatched from some of these eggs. Survival and cyst formation of P. varius larvae exposed to different combinations of drying and/or freezing temperatures indicated that gordiid larvae have the ability to survive drying and freezing, but survival significantly increases during freezing at lower temperatures. The major contribution of our study is the demonstration that gordiid larval and cyst stages can survive freezing temperatures to infect and develop in the next host.

Olfaction shapes host-parasite interactions in parasitic nematodes.

Many parasitic nematodes actively seek out hosts in which to complete their lifecycles. Olfaction is thought to play an important role in the host-seeking process, with parasites following a chemical trail toward host-associated odors. However, little is known about the olfactory cues that attract parasitic nematodes to hosts or the behavioral responses these cues elicit. Moreover, what little is known focuses on easily obtainable laboratory hosts rather than on natural or other ecologically relevant hosts. Here we investigate the olfactory responses of six diverse species of entomopathogenic nematodes (EPNs) to seven ecologically relevant potential invertebrate hosts, including one known natural host and other potential hosts collected from the environment. We show that EPNs respond differentially to the odor blends emitted by live potential hosts as well as to individual host-derived odorants. In addition, we show that EPNs use the universal host cue CO(2) as well as host-specific odorants for host location, but the relative importance of CO(2) versus host-specific odorants varies for different parasite-host combinations and for different host-seeking behaviors. We also identified host-derived odorants by gas chromatography-mass spectrometry and found that many of these odorants stimulate host-seeking behaviors in a species-specific manner. Taken together, our results demonstrate that parasitic nematodes have evolved specialized olfactory systems that likely contribute to appropriate host selection.

Nematomorph parasites indirectly alter the food web and ecosystem function of streams through behavioural manipulation of their cricket hosts.

Nematomorph parasites manipulate crickets to enter streams where the parasites reproduce. These manipulated crickets become a substantial food subsidy for stream fishes. We used a field experiment to investigate how this subsidy affects the stream community and ecosystem function. When crickets were available, predatory fish ate fewer benthic invertebrates. The resulting release of the benthic invertebrate community from fish predation indirectly decreased the biomass of benthic algae and slightly increased leaf break-down rate. This is the first experimental demonstration that host manipulation by a parasite can reorganise a community and alter ecosystem function. Nematomorphs are common, and many other parasites have dramatic effects on host phenotypes, suggesting that similar effects of parasites on ecosystems might be widespread.