First Report of Mermithidae (Enoplea: Mermithida) Parasitizing Adult Stable Flies in Japan.

Mermithidae is a family of nematodes that parasitize a wide range of invertebrates worldwide. Herein, we report nematodes that were unexpectedly found in three of 486 adult stable flies (Stomoxys calcitrans) captured from three farms (F1, F2, and F3) in different regions of Gifu Prefecture, Japan. We aimed to characterize these nematodes both at the morphological and molecular level. Morphological studies revealed that the nematodes were juveniles of Mermithidae. Phylogenetic analysis based on 18S and 28S rDNA indicated that the mermithids from farms F1 and F2 could be categorized into the same cluster as Ovomermis sinensis and Hexamermis sp., whereas the mermithid from farm F3 clustered with Amphimermis sp. Additionally, these mermithids could be categorized within the same clusters as related mermithids detected in Japan that parasitize various arthropod orders. Our findings suggest that these stable flies may have been parasitized by mermithids already present in the region and that genetically distinct species of mermithids occur across Japan. To the best of our knowledge, this is the first report of mermithids parasitizing adult stable flies in Japan.

Parasites of firebugs in Austria with focus on the "micro"-diversity of the cosmopolitan trypanosomatid Leptomonas pyrrhocoris.

In this work, we investigated parasites of the firebug Pyrrhocoris apterus in Austria and demonstrated that in addition to the extensively studied Leptomonas pyrrhocoris, it can also be infected by Blastocrithidia sp. and by a mermithid, which for the first time has been characterized using molecular methods. This diversity can be explained by the gregarious lifestyle, as well as the coprophagous and cannibalistic behavior of the insect hosts that makes them susceptible to various parasites. In addition, we showed no tight association of the L. pyrrhocoris haplotypes and geographical locations (at least, considering the relatively small scale of locations in Austria) implying that the natural populations of L. pyrrhocoris are mixed due to the mobility of their firebug hosts.

Agamermis sp. (Nematoda: Mermithidae) parasitizing Armadillidium vulgare (Crustacea: Isopoda) in Argentina.

The Mermithidae is a family of nematodes parasitic in many kinds of insects, spiders, leeches, crustaceans and other invertebrates throughout the world. While conducting an assay with entomopathogenic nematodes, we found Armadillidium vulgare (Crustacea: Isopoda) individuals to be infected with Agamermis sp., marking the fourth known discovery of a mermithid infection in the order Isopoda. In this work, we contribute with an 18S rDNA sequence of the isolated nematode and the morphological and morphometrical characterization of the juveniles.

Parasite load effects on sex ratio, size, survival and mating fitness of Heleidomermis magnapapula in Culicoides sonorensis.

Heleidomermis magnapapula parasitizes the blood-feeding midge Culicoides sonorensis. Most (84%) single mermithid infective second stage juveniles (J2) developed into adult females, while parasitism by multiple J2 yielded 97% male adults. Nematodes emerged from the midge larval host as adults and mated immediately; females were ovoviviparous. Host larvae were exposed to nematode J2 and examined intact microscopically to score initial parasite load. Midge hosts were reared individually. Premature midge death, nematode survival within the host, and emerging adult nematode sex ratio and size as a function of load and host size were all tracked. Higher nematode loads produced smaller adult nematode males. The higher loads also increased and accelerated premature host death. Emergence of > 7-9 adult nematode males was rare, but up to 19 tiny males emerged from a single host. Larger midges supported higher parasite loads and a larger total volume of emerged nematode biomass. Virgin adult nematode males then were paired with females of variable, known sizes (volume) and held to determine size effects on fertility (egg hatch), and male survival (longevity). Tested adult males ranged in size from 0.0025 - 0.0334 mm3 and females from 0.0121 - 0.1110 mm3. Logistic regression indicated female nematode fertility was positively influenced by male nematode size, while nematode load and female nematode size had no significant effect. While fertility was reduced statistically in smaller males, even some of the smallest male and female individuals could be fertile. Findings are related to field studies in this system.

Behavior of Aedes albifasciatus (Diptera: Culicidae) larvae from eggs with different dormancy times and its relationship with parasitism by Strelkovimermis spiculatus (Nematoda: Mermithidae).

Aedine mosquitoes go through unfavorable periods as dormant eggs. However, extended dormancy times lead to a depletion of egg reserves, which might be partially compensated by changes in larval-feeding behavior. Changes in larval behavior may also be induced by parasitism by mermithids probably as a strategy to reduce the impact of the parasite on the host. The most abundant floodwater mosquito in southern South America is Aedes albifasciatus (Diptera: Culicidae), a species naturally parasitized by Strelkovimermis spiculatus (Nematoda: Mermithidae). This study aimed to evaluate the behavior of fourth-instar larvae of Ae. albifasciatus from eggs with 2, 4, and 6 months of dormancy, parasitized or not by S. spiculatus. To achieve this, eight categories of behavior were defined, and then, each individual was observed for 5 min, and its behaviors and their duration were recorded. The behaviors with the highest percentage of observation time were still (50.1%) and wriggle swimming (33.6%), while those with the lowest percentage of observation time were those involving active foraging (less than 8%). A great variability in the behaviors was observed among individuals within the same treatment. The type and duration of each behavior did not vary in relation to egg dormancy time or parasitism, but parasitism affected the level of larval activity. Parasitized larvae performed, on average, fewer behaviors than non-parasitized larvae. This result supports the hypothesis that S. spiculatus parasitism affects the behavior of Ae. albifasciatus larvae by reducing their level of activity.

The general architecture of black fly-parasite interactions: Parasitism in lotic systems at a continental scale.

We examined the general architecture of interactions between stream-dwelling larval black flies (Diptera: Simuliidae) and their common parasites in 1736 collections across North America. Mermithid nematodes (family Mermithidae), microsporidia (phylum Microsporidia), and the fungus Coelomycidium simulii Debaisieux (phylum Blastocladiomycota) infected larval black flies. We found similar continental distributions for these three parasite taxa across North America. At least one of these taxa was represented in 42.2% of all black fly collections. Species interactions in ecological networks typically imply that each link between species is equally important. By employing quantitative measures of host susceptibilities and parasite dependencies, we provide a more complete structure for host-parasite networks. The distribution of parasite dependencies and host susceptibilities were right-skewed, with low values indicating that most dependencies (parasites) and susceptibilities (hosts) were weak. Although regression analysis between host frequency and parasite incidence were highly significant, frequency analysis suggested that the distributions of parasites differ significantly among the four most common and closely related (same subgenus) species of hosts. A highly significant pattern of nestedness in our bipartite host-parasite network indicated that specialized parasites (i.e., those that interact with few host species) tend to occur as subsets of the most common hosts.

Susceptibility of the floodwater mosquito Aedes albifasciatus from eggs of different dormancy times to the nematode parasite Strelkovimermis spiculatus.

This study aimed to evaluate the effects of egg dormancy times on susceptibility of larvae of the floodwater mosquito Aedes albifasciatus (Diptera: Culicidae) to parasitism by their natural enemy Strelkovimermis spiculatus (Nematoda: Mermithidae) and on their life history traits. Aedes albifasciatus eggs stored for 2, 4, 6, 8 and 10 months were hatched, and the larvae either exposed to S. spiculatus (treatment group) or not exposed (control group). Egg dormancy time had a negative effect on the retention of parasites, but no effect on the prevalence and intensity of parasitism or the melanization of nematodes. The survival to adulthood of control individuals decreased as dormancy time increased, whereas that of exposed individuals that remained uninfected was constant and low. A trend towards increasing development times with longer dormancy times was detected in the control group, but not in the exposed noninfected group. The results suggest nonconsumptive effects of parasites in exposed but not infected larvae from eggs with short dormancy times. In contrast, the relatively low fitness of larvae from eggs with long dormancy times regardless of their contact with the nematodes may be the result of the nutritional deprivation during the egg stage.

Let's go swimming: mermithid-infected earwigs exhibit positive hydrotaxis.

Certain species of parasites have the apparent ability to alter the behaviour of their host in order to facilitate the completion of their own life cycle. While documented in hairworms (phylum Nematomorpha), the ability for mermithid parasites (from the sister phylum Nematoda) to force hosts to enter water remains more enigmatic. Here, we present the first experimental evidence in a laboratory setting that an insect which normally never enters open water (the European earwig Forficula auricularia) will readily enter the water when infected with a mermithid nematode (Mermis nigrescens). Only adult mermithids appear capable of inducing this polarising shift in behaviour, with mermithid length being a very strong predictor of whether their host enters water. However, mermithid length was only weakly associated with how long it took an earwig to enter water following the beginning of a trial. Considering the evidence presented here and its alignment with a proteomic investigation on the same host-parasite system, this study provides strong evidence for adaptive behavioural manipulation and a foundational system for further behavioural and mechanistic exploration.

Inequalities in body size among mermithid nematodes parasitizing earwigs.

Variation among body sizes of adult parasitic worms determines the relative genetic contribution of individuals to the next generation as it affects the effective parasite population size. Here, we investigate inequalities in body size and how they are affected by intensity of infection in Mermis nigrescens (Mermithidae: Nematoda) parasitizing the European earwig Forficula auricularia in New Zealand. Among a population of pre-adult worms prior to their emergence from the host, we observed only modest inequalities in body length; however, among worms sharing the same individual host, inequalities in body sizes decreased with increasing intensity of infection. Thus, the more worms occurred in a host, the more the second-longest, third-longest and even fourth-longest worms approached the longest worm in body length. This pattern, also known from another mermithid species, suggests that worms sharing the same host may have infected it roughly simultaneously, when the host encountered a clump of eggs in the environment. Thus, the life history and mode of infection of the parasite may explain the modest inequalities in the sizes achieved by pre-adult worms, which are lower than those reported for endoparasitic helminths of vertebrates.

Agamermis (Nematoda: Mermithidae) Infection in South Carolina Agricultural Pests.

Native and invasive stink bugs (Hemiptera: Pentatomidae) and the closely related invasive Megacopta cribraria (Hemiptera: Plataspidae) are agricultural pests in the southeastern United States. Natural enemies, from various phyla, parasitize these pests and contribute to population regulation. We specifically investigated Nematoda infections in pentatomid and plataspid pests in one soybean field in South Carolina in 2015. Nematodes were identified through molecular and morphological methods and assigned to family Mermithidae, genus Agamermis. This study reports mermithid nematode infection in immature M. cribraria for the first time and provides the first mermithid host record for the stink bugs Chinavia hilaris, Euschistus servus, and another Euschistus species, and a grasshopper (Orthoptera: Acrididae) in South Carolina. The same Agamermis species infected all hosts. The broad host range and prevalence suggests that Agamermis may be an important contributor to natural mortality of pentatomid and plataspid pests. Previous mermithid host records for the Pentatomidae and Plataspidae worldwide are summarized. Further work is needed to assess the impact of infection on populations over a broader range of agricultural fields and geographic localities.

First report of a mermithid nematode infecting the invasive Megacopta cribraria (Hemiptera: Plataspidae) in the United States.

Megacopta cribraria (Hemiptera: Plataspidae) has become a pest of soybean, Glycine max (L.), in the United States. While several natural enemies of M. cribraria have been reported, our study is the first to report nematodes beneath the pleural membranes in the abdominal cavities of adults. Morphological and molecular analyses suggest this nematode belongs to the family Mermithidae. This first report of a nematode infection in M. cribraria adds to the current inventory of enemies attacking this insect. Our observations provide a basis for future research to examine the impact of nematodes on M. cribraria mortality and to investigate their capacity to reduce populations.

Culex pipiens affected by joint infection of a mosquito iridescent virus and Strelkovimermis spiculatus.

Dual infections with a mosquito iridescent virus (MIV) and the mermithid nematode, Strelkovimermis spiculatus were recorded in natural Culex pipiens populations around La Plata city, Argentina. S. spiculatus was detected in 82% of samples that were positive for MIV infection. Dissected larvae of Cx. pipiens with patent MIV infection presented 42% infection with S. spiculatus. Larvae of Cx. pipiens exposed to MIV and S. spiculatus under laboratory conditions produced a high joint infection rate (82.5%) while no infection was recorded on larvae exposed to virus suspension only. Field and laboratory results suggest a strong association between S. spiculatus and MIV in natural populations of Cx. pipiens, in which S. spiculatus could be a mode of entry for the virus into the mosquito hemocele.

Host Penetration and Emergence Patterns of the Mosquito-Parasitic Mermithids Romanomermis iyengari and Strelkovimermis spiculatus (Nematoda: Mermithidae).

Romanomermis iyengari and Strelkovimermis spiculatus are mermithid nematodes that parasitize mosquito larvae. We describe host penetration and emergence patterns of Romanomermis iyengari and Strelkovimermis spiculatus in laboratory exposures against Culex pipiens pipiens larvae. The mermithid species differed in host penetration behavior, with R. iyengari juveniles attaching to the host integument before assuming a rigid penetration posture at the lateral thorax (66.7%) or abdominal segments V to VIII (33.3%). Strelkovimermis spiculatus attached first to a host hair in a coiled posture that provided a stable base for penetration, usually through the lateral thorax (83.3%). Superparasitism was reduced by discriminating against previously infected hosts, but R. iyengari's ability to avoid superparasitism declined at a higher inoculum rate. Host emergence was signaled by robust nematode movements that induced aberrant host swimming. Postparasites of R. iyengari usually emerged from the lateral prothorax (93.2%), whereas S. spiculatus emergence was peri-anal. In superparasitized hosts, emergence was initiated by males in R. iyengari and females in S. spiculatus; emergence was otherwise nearly synchronous. Protandry was observed in R. iyengari. The ability of S. spiculatus to sustain an optimal sex ratio suggested superior self-regulation. Mermithid penetration and emergence behaviors and sites may be supplementary clues for identification. Species differences could be useful in developing production and release strategies.

The proof is in the poo: Non-invasive method to detect endoparasitic infection.

Almost every animal trait is strongly associated with parasitic infection or the potential exposure to parasites. Despite this importance, one of the greatest challenges that researchers still face is to accurately determine the status and severity of the endoparasitic infection without killing and dissecting the host. Thus, the precise detection of infection with minimal handling of the individual will improve experimental designs in live animal research. Here, we quantified extracellular DNA from two species of endoparasitic worm that grow within the host body cavity, hairworms (phylum Nematomorpha) and mermithids (phylum Nematoda), from the frass of their insect host, a cave weta (Orthoptera: Rhaphidophoridae) and an earwig (Dermaptera: Forficulidae), respectively. Frass collection was done at two successive time periods, to test if parasitic growth correlated with relative DNA quantity in the frass. We developed and optimized two highly specific TaqMan assays, one for each parasite-specific DNA amplification. We were able to detect infection prevalence with 100% accuracy in individuals identified as infected through post-study dissections. An additional infection in earwigs was detected with the TaqMan assay alone, probably because some worms were either too small or degraded to observe during dissection. No difference in DNA quantity was detected between sampling periods, although future protocols could be refined to support such a trend. This study demonstrates that a noninvasive and minimally stressful method can be used to detect endoparasitic infection with greater accuracy than dissection alone, helping improve protocols for live animal studies.

First record of a mermithid worm (Nematoda, Mermithidae) parasitizing a third instar nymph of Triatoma sordida (Stål, 1859) (Hemiptera, Reduviidae, Triatominae) from Mato Grosso, Brazil.

A juvenile specimen of a mermithid (Nematoda) was found parasitizing a third instar nymph of Triatoma sordida from Mato Grosso, Brazil. This is the first record of mermithid parasitism in a triatomine species. The Mermithidae represents a family of nematodes that are specialized insect parasites. Entomonematodes are one of the highly influential agents regulating the population dynamics of insects. This report introduces the opportunity to think about mermithids as a possible candidate for use as triatomine biological control.

First Record of A Species of Mermithidae Braun, 1883 Infecting a Decapod, Palaemon paludosus (Palaemonidae).

The hemocoel of 26 of 30 (86%) eastern grass shrimps, Palaemon paludosus (Gibbes, 1850) (Decapoda: Palaemonidae), captured during June 2017 from several freshwater lakes near Leesburg and Lake Kissimmee, Florida, was infected by juveniles of a nematode species (Mermithidae sp.). Some infected eastern grass shrimps were preserved whole for histopathology, whereas others were dissected to excise parasitic juvenile nematodes, and still others were kept alive in glass aquaria such that post-parasitic (emerged) juvenile nematodes could be opportunistically observed alive and ultimately preserved. Parasitic and post-parasitic juvenile nematodes had cuticle cross-fibers, cephalic papillae, cup-shaped amphids, a horn-shaped vagina, a trophosome, and a caudal appendage, which collectively diagnosed them as Mermithidae sp. They differed from those of nematomorphs (Nematomorpha) by lacking 4 giant cells anteriorly, tegumental bristles, scale or plate-like areoles, a bifurcate or trifurcate posterior end, and an anus. A phylogenetic analysis of the small subunit rDNA (18S) that included all of the available mermithid sequences corresponding to morphologically diagnosed specimens recovered Mermithidae sp. within the clade of mermithids and sister to Ovomermis sinensis Chen, Jian, and Ren, 1991 . This is the first record of a mermithid infection in a decapod and first report of a mermithid infection in an aquatic crustacean from North America (another mermithid infects a terrestrial isopod there). The high prevalence of infection and the multiple geographic localities harboring infected eastern grass shrimps indicated that these infections were not spurious. Because no other decapod is confirmed as a mermithid host, we suspect that these specimens likely represent a new species with a life cycle worth studying, since none for a mermithid involving a decapod nor a crustacean has been elucidated to date. We also provide a table of all mermithid and nematomorph infections in crustaceans.

Effects of temperature and photoperiod on the infection of two mosquito species by the mermithid Romanomermis culicivorax.

Successful invasion by the mermithid Romanornermis eulicivorax declined linearly from 93.6 to 1.5% in Culex tarsalis and from 73.1 to 1.6% i n Aeries dorsalis larvae exposed in the laboratory at 18, 16, 14, 12, and l 0 C. Larvae of C. tarsalis were more susceptible than those of A. dorsalis at 18 and 16 C, but this relationship was reversed at 12 C. Larval mortality during the 48-h exposure period was due primarily to nematode infection . Photoperiod had no effect on infection.

Growth and Development of Romanomermis culicivorax In Vitro.

Various combinations of vertebrate and invertebrate tissue culture and microbiological media were utilized in an attempt to culture Romanomermis culicivorax (Mermithidae: Nematoda) in vitro. Most media were unsuitable and caused nematodes to become lumpy, vacuolated, and granular. Slow and limited growth and development of internal structures of the nematodes were obtained with variously supplemented Grace's tissue culture and Schneider's Drosophila media. In an enriched Grace's medium, development attained by the nematodes after 3-4 wk was comparable to 4-5-day-old parasites grown in vivo in the mosquito host, Culex pipiens. Two molts were observed in vitro. Maximum dimensions in vitro were 7.0-mm length and 87-mum width at the widest point. The stichosome, stichocytes, and trophosome developed prominently. A filiform tail and highly cuticularized tube persisted throughout the culture period in vitro.

Effects of host size and parasite burden on sex ratio in the mosquito parasite Octomyomermis muspratti.

The ratio of Octomyomermis muspratti to the host mosquito at the time of exposure had little effect on the ratio of male to female parasites that resulted. However, the ratio of males to females increased as the number of parasites/host increased. Hosts with a single nematode produced fewer than 1% males in comparison with hosts with 8 parasites which produced about 40% males; hosts with 10 or more nematodes generally produced more male than female nematodes. Males of O. muspratti usually emerged before females because of the earlier death of multiply-infected mosquitoes. The size of the host at the time of invasion bad no significant influence on nematode sex ratios. Since mating is apparently necessary for reproduction in O. muspratti, the low male to female ratios that occur will be important in developing successful mass production techniques.

Parasitism of Kenyan Mosquito Larvae (Diptera: Culicidae) by Romanomermis culicivorax (Nematoda: Mermithidae).

The ability of Romanomermis culicivorax to infect, develop, and emerge from Kenyan mosquito hosts was evaluated in the laboratory. Host species tested were Aedes aegypti, Ae. dentatus, Ae. hirsutus, Anopheles arabiensis, An. coustani, An. funestus, An. gambiae, An. pharoensis, Culex duttoni, Cu. ethiopicus, Cu. poicilipes, Cu. quinquefasciatus, Cu. tigripes, Cu. univittatus, Coquillettidia metallica, Mansonia africana, Ma. uniformis, Mimomyia splendens, Mi. uniformis, Toxorhyncites brevipalpis, and Uranotaenia balfouri. R. culicivorax penetrated all the host species tested and developed and emerged from most of the hosts. Both penetration and some development, but not nematode emergence, were observed from all instars of Ma. uniformis. T. brevipalpis exhibited signs of resistance in the form of melanization of R. culicivorax within 48 hours of infection in all four instar stages. Nematode melanization, especially in older hosts, was observed in Ae. dentatus, Ae. hirsutus, Cu. duttoni, Cu. tigripes, and Mi. splendens. When melanization occurred, the melanized carcass of the nematode was passed on from instar to instar. The implications for field release of R. culicivorax in Kenya are still good, especially in habitats where different mosquito species occupy the same niche at different times, which would allow for nematode recycling.