The effect of parasitism by the mermithid Romanomermis culicivorax on the dry weight and hemolymph soluble protein content of three species of mosquitoes

The dry weight, hemolymph soluble protein composition, and content of three species of mosquitoes, Culex pipiens, Aedes taeniorhynchus, and Anopheles quadrimaculatus were examined to determine the effects of parasitism by the mermithid nematode Romanomermis culicivorax. The dry weights of infected fourth-stage larvae of all three species were significantly lower than controls. The differences in weight found between infected early and late C. pipiens and A. quadrimaculatus larvae were attributed to the weight of the parasite itself. This difference was not noticeable in A. taeniorhynchus larvae. Hemolymph proteins were severely depleted in all three mosquito species during parasitism by R. culicivorax. Analysis of protein composition by PAGE showed that these depletions were accompanied by a reduction in the number of proteins. Differences between protein composition concentrations were evident between early and late fourthstage control larvae of C. pipiens and A. quadrimaculatus. The concentration of some low-molecular-weight proteins (below 68,000) remained constant between infected and control samples of all three mosquito species.     

Hemolymph pH of the larvae of three species of mosquitoes,and the effect of Romanomermis culicivorax parasitism on the blood pH of Culex pipiens

The hemolymph pHs of late fourth-instar Culex pipiens, Aedes taeniorhynchus, and Toxorhynchites amboinensis were measured with pH-sensitive glass microelectrodes and were slightly alkaline; pH 7.51, 7.62, and 7.37, respectively. The hemolymph pH remained relatively constant during the development of C. pipiens larvae through the third and early and late fourth instars. The hemolymph pH in C. pipiens larvae parasitized with the mermithid nematode Romanomermis culicivorax was unaltered. These measurements provide an approximate pH (ca 7.4) which is normal for the hemolymph of larval mosquitoes, and should be useful for further development of a culture medium for R. culicivorax.     

The effect of nematode parasitism on the osmolality and major cation concentration in the haemolymph of three larval mosquito species

Parasitism by a mermithid nematode, Romanomermis culicivorax, causes severe depletion of haemolymph carbohydrates and proteins in mosquito larvae. We undertook a study to determine if haemolymph osmolality and cation concentrations were affected also by mermithid parasitism. The haemolymph osmolality of R. culicivorax-infected and control Aedes taeniorhynchus and Culex pipiens fourth-instar larvae was not significantly different. However, the haemolymph osmolality decreased significantly in infected Anopheles quadrimaculatus. Each mosquito species demonstrated significant alterations in the haemolymph concentration of at least one cation when infected although the cation concentrations affected differed for each species. The changes observed were statistically significant but the magnitude of change was not great. Overall, despite the severe nutritional burden of the mermithid nematode, these species of mosquito larvae can continue to maintain osmoregulation.     

Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis

Hemolymph glucose, alkaline phosphatase, lactic dehydrogenase, and creatine phosphokinase in Biomphalaria glabrata infected with Angiostrongylus costaricensis were significantly higher on day 27 postinfection (PI) than in uninfected snails. Hemolymph total calcium from infected snails was less on days 6, 12, and 27 PI than that from controls. Total hemolymph protein was similar for controls and infected animals during the entire study. Throughout the study the mean number of amoebocytes/mm³ hemolymph from infected snails was significantly less than that for controls. Mean total wet weights of digestive gland and foot muscle from infected and uninfected snails was similar throughout the study. Mean μg glycogen/mg wet weight of digestive gland from infected snails was significantly greater on days 24, 27, and 28 PI than that from controls. Mean μg glycogen/mg wet weight of foot muscle from infected snails was significantly reduced between days 12 and 28 PI from that of uninfected snails. It is suggested that hemolymph glucose and digestive gland glycogen in infected snails are augmented by glycogen breakdown in the foot muscle of parasitized animals. Elevations in hemolymph enzymes are due to tissue destruction by larvae emerging from the foot muscle of infected snails. Parasite-induced derangements in shell metabolism underlie observed changes in hemolymph calcium in infected snails.     

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.     

Bioassays for comparative infectivity of mermithid nematodes (Romanomermis iyengari,Romanomermis culicivorax and Strelkovimermis spiculatus) for culicine mosquito larvae

Two improved bioassays were developed to establish infectivity baselines for selection experiments using mermithid nematode variants. Comparative infectivity of Romanomermis iyengari, Romanomermis culicivorax and Strelkovimermis spiculatus using larvae of three mosquito spp. Aedes sierrensis, Aedes aegypti and Culex pipiens were evaluated with “plate” and “tray” bioassays at selected intensity of infections. Using the “plate” bioassay, single mosquito larvae were immersed in 2 ml of water within individual depressions of 12-well, polystyrene tissue culture plates. One, three, or five preparasitic juveniles (J2) were added to each well. In the “tray” bioassay, polyethylene trays containing 500 ml water and 100 mosquito larvae were exposed to 500 (5:1, nematode:insect host) or 1000 (10:1) J2s. Percentage infection (PINF, infectivity) and intensity of infection (IINF, #nematodes per infected larvae) number were determined only after emergence of post-parasitic J3 juveniles. Under the bioassay conditions, all three species of nematodes resulted in infections in all mosquito hosts, but R. iyengari exhibited better effectiveness in the parasitism of mosquito larvae. The three species of mosquitoes presented high levels of susceptibility to each of the three species of nematodes, but in general Cx. pipiens and Ae. sierrensis were slightly more susceptible than Ae. aegypti. The “plate” bioassay was more efficient in measurement of infectivity of the mermithid species and in establishing baseline characteristics for these mosquito-parasitic nematodes. The “tray” bioassay was an effective bioassay for large cohorts of both infective juveniles and host larvae and, potential for field interactions.     

Strelkovimermis spiculatus n. sp. (Mermithidae: Nematoda) Parasitizing Aedes albifasciatus Mac. (Culicidae: Diptera) in Argentina

Strelkovimermis spiculatus n. sp. (Mermithidae: Nematoda), a parasite of the mosquito Aedes albifasciatus Mac. in Argentina, is described. Diagnostic characters of this species include a ventrally shifted mouth opening and spicules fused at the tips. The mermithid has been reared on Culex pipiens L. larvae in the laboratory.     

Biology of Octomyomermis muspratti,a parasite of mosquitoes as it relates to mass production

The first- and second-instar larvae of Culex pipiens quinquefasciatus were equally susceptible to parasitism by the mermithid nematode Octomyomermis muspratti; the third-instar hosts were significantly less susceptible. The yield of postparasitic nematodes was higher from hosts exposed as second instars then from hosts exposed as first or third instars at comparable levels of parasitism. Mortality of the host prior to emergence of the nematode occurred most frequently among first- and third-instar hosts. About 4% of the infected hosts exposed as first and second instars and 37% of the infected hosts exposed as third instars pupated prior to emergence of the postparasitic nematodes. The exposure of mosquitoes as second instars was the most efficient for mass rearing and resulted in the highest yields of postparasitic O. muspratti.     

Arachidonic and other tissue fatty acids of Culex pipiens reared with various concentrations of dietary arachidonic acid

Chloroform/methanol extracts were prepared from groups of Culex pipiens reared in synthetic dietary media provided with various concentrations of arachidonic acid. Extracts were analyzed by gas-liquid chromatography to determine the fatty acid composition of whole extracts and also of phospholipid and triacylglycerol fractions separated by thin-layer chromatography from the whole extracts. The same extracts were also tested for their ability to support flight of adult C. pipiens reared in basal synthetic diet containing various concentrations of the extracts: this provided a bioassay for the presence of arachidonic acid or related polyunsaturates in the extracted lipid, since adults can fly only if provided, as larvae, with dietary arachidonic or related fatty acids. For comparison, chromatographic and bioassay data obtained from normal stock mosquitoes, reared in crude septic medium, are also presented. All extracts were shown by gas-liquid chromatography to contain some arachidonic acid and other polyunsaturated fatty acids. The proportions of arachidonic acid in extracts from mosquitoes reared in synthetic media were greater the greater the concentration of dietary arachidonic acid provided; and in the bioassay, extracts induced more flight activity in test mosquitoes the higher the dietary arachidonic acid provided for extracted mosquitoes. Extracts from stock-reared mosquitoes were more active in the bioassay than synthetic dietreared extracts, even though gas-liquid chromatography indicated lower proportions of arachidonic acid in stock-reared extract. However, stock-reared extract contained a substantial proportion of gammalinolenic acid, which is flight active for C. pipiens, as well as more linolenic acid and a large amount of linoleic acid, both of which are semi-active for flight; thus, stock-reared extract contained a higher overall proportion of flight-inducing fatty acids. Proportions of polyunsaturates in the phospholipid fractions of extracts from synthetic diet-reared mosquitoes were much greater than in the unfractionated extracts, whereas polyunsaturates were virtually absent from the triacylglycerol fractions, indicating a sequestering of polyunsaturates into phospholipids.     

Age and size at maturity of the mosquito Culex pipiens infected by the microsporidian parasite Vavraia culicis

The costs of parasitism to a host''s reproductive success (RS) often increase with time since infection. For hosts experiencing this type of infection, it is predicted that they will maximize their RS by bringing forward their schedule of reproduction. This is because the costs associated with such a response can be discounted against a reduced future RS due to parasitism. The microsporidian Vavraia culicis is a natural parasite of mosquitoes and one whose costs increase over time as its spores accumulate and damage host tissues. As larvae, male and female Culex pipiens mosquitoes behaved differently towards infection with V. culicis. Infected females pupated earlier than uninfected females and tended to emerge as smaller adults, indicating a cost to their fecundity. However, the age and size at maturity of infected male mosquitoes was no different from uninfected males. The results of this study support theoretical predictions and highlight the potential roles that host gender and density-dependent interactions may have in determining the response of host life-history traits to parasitism.     

Host hemolymph monophenoloxidase activity in parasitized Manduca sexta larvae and evidence for inhibition by wasp polydnavirus

In insects, one of the primary routes of defense against parasites is encapsulation by hemocytes followed by melanization, in which tyrosine and DOPA are converted to melanin via toxic quinone intermediates. This report describes the use of an in vitro radiochemical assay to monitor hemolymph monophenoloxidase (MPO) conversion of [³H]tyrosine to [³H]DOPA, using a method utilized previously for dipteran and mammalian enzymes. Parasitism of fifth instar tobacco hornworm larvae by the braconid wasp Cotesia congregata depresses the rate of hemolymph monophenoloxidase activity in the host. Significant inhibition of hemolymph MPO was detectable in newly parasitized larvae and terminal stage hosts. A similar effect was seen in unparasitized larvae following injection of sucrose-gradient purified wasp polydnavirus (PDV) particles, which are normally injected by the female wasps into the host, suggesting the inhibition may be virally mediated. Hemolymph MPO activity was assayed in vitro 24 h after injection of PDV in vivo, and intercalation of viral DNA by exposure to psoralen and long-wave u.v. light eliminated its inhibitory effect on MPO. Despite inhibition of hemolymph MPO activity by parasitism, host cuticular enzymes appear unimpaired, since cuticular melanization occurs at sites of integumental wounding during emergence of the wasps from the host. Red pigments appear in the dorsal vessel and integument of parasitized and virus-injected larvae; whether these pigmentation changes are related to effects of parasitism on tyrosine metabolism and ommochrome biosynthesis remains to be determined.     

Role of mermithid nematodes in biological control of mosquitoes

Mermithid nematodes have been reported from at least 63 species of mosquitoes from all over the world, but until recently they have received little attention. Such nematodes are prime candidates as biological control agents because they have adapted to the life cycle of the host; are host specific; produce high levels of parasitism; kill the hosts; are easily handled; have a high reproductive potential; are free swimming and can be disseminated easily in the infective stage; and can be used in an inundative or inoculative manner to control mosquitoes. Nevertheless, with the exception of one mermithid species, little is known about factors that influence parasitism in mosquitoes. Also, only one species of mermithid has been successfully mass cultured to date. The exception is the mermithid Reesimermis nielseni. With this mermithid, high levels of parasitism can be obtained in natural populations of mosquitoes at reasonable dosages; preparasitic nematodes can be applied easily by using many of the standard techniques used to apply insecticides; and R. nielseni can establish itself in many of the sites in which it is released. The same potentials exist for mermithids of other aquatic insects.     

Romanomermis culicivorax parasitism and the development, growth, and feeding rates of two mosquito species

The dry weight, development, and feeding rates of Culex pipiens and Toxorhynchites amboinensis larvae infected with the nematode, Romanomermis culicivorax, were measured and gross conversion efficiences were calculated. The weight of C. pipiens larvae infected at two different inoculum levels did not differ significantly from controls until day 6 postinfection (PI). Infected larvae of T. amboinensis were significantly lighter than controls at days 2, 4, and 6 PI. The rate of larval mosquito development was slowed after day 3 in parasitized individuals of both mosquito species. Infection significantly retarded the interval feeding rate of the filter-feeding C. pipiens throughout development. Infected T. amboinensis larvae consumed significantly fewer prey larvae of C. pipiens than controls. Calculation of gross conversion efficiency (GCE) showed that lightly infected C. pipiens larvae had an elevated GCE early in the infection but were less efficient relative to controls after 4 days PI. Lightly parasitized T. amboinensis had a lower total GCE than controls.     

Some effects of mermithid parasitism on the larval blackflies Prosimulium mixtum fuscum and Simulium venustum

Histochemical studies on field-collected specimens of larval blackflies showed no significant differences in endocrine activity between mermithid-infected and uninfected Prosimulium mixtum fuscum larvae of similar body dimensions. In Simulium venustum, mermithid parasitism significantly increased the nuclear DNA/RNA activity of the corpus allatum gland as well as the volume and amount of stored neurosecretory material in the corpus cardiacum gland. A marked decline in glycogen concentration and the amount of fat body tissue was evident in both infected simuliid species. Fat body nucleic acid activity was unaffected by mermithid parasitism. The significance of these effects of mermithid parasitism is discussed with regard to the host-parasite relationship.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

The influence of starvation on mortality,development, and protein content in parasitized and unparasitized Tribolium castaneum

The effects of starvation on mortality, development, and protein content in Nosema whitei-infected and uninfected Tribolium castaneum were investigated. T. castaneum larvae, starved for 4, 6, 8, and 10 days postinfection, showed an increase in larval mortality. Pupal mortality also increased, producing a decrease in adult emergence. Starvation of larvae for 6 days or more delayed development and the average time to adult emergence increased. These effects tended to be more marked in Nosema-infected larvae. No consistent pattern of weight changes was observed in either the uninfected or infected pupae and adults. Infected T. castaneum larvae showed a significant decrease in protein compared to controls. Starvation apparently does not aggravate this condition nor does it have any significant effects on the total hemolymph protein content of uninfected and infected larvae.     

Alterations in carbohydrate and fatty acid levels of Lymantria dispar larvae caused by a microsporidian infection and potential adverse effects on a co-occurring endoparasitoid,Glyptapanteles liparidis

Infection of Lymantria dispar host larvae by the entomopathogenic microsporidium Vairimorpha sp. has a negative impact on the performance of the endoparasitic braconid Glyptapanteles liparidis. To investigate possible causes for this effect, we studied to what extent nutritional host suitability is altered by the microsporidium. Therefore, we analyzed carbohydrates and fatty acids in host larvae after Vairimorpha infection and/or parasitism by G. liparidis. Trehalose levels were significantly reduced in the hemolymph of infected hosts. After day five post infection, it was detected only in traces. Four to six days later, the glycogen resources were depleted in infected larvae. Parasitism by G. liparidis, on the other hand, led to increased hemolymph trehalose levels during the early endoparasitic phase but to a significant decrease at the end of its larval development. No effect of parasitism on the glycogen content was ascertained. Hemolymph levels of the fatty acids analyzed, such as palmitic, stearic, oleic, linoleic, and linolenic acid, were significantly reduced in microsporidia-infected L. dispar. Vairimorpha sp. develops as an intracellular parasite in the fat body of the host larva and synthesis of trehalose and fatty acids may be disturbed. Moreover, microsporidia may also harness metabolites or energy produced by host cells. We conclude that the microsporidia-induced decrease in hemolymph carbohydrates and fatty acids adversely affects growth and development of parasitoid larvae.     

Amino acids in nectar enhance longevity of female Culex quinquefasciatus mosquitoes

Culex mosquitoes feed on a wide range of nectars consisting of mostly carbohydrates and amino acids, however, little is known about the utilization and effects of these different carbohydrates and their accompanying amino acids on longevity. Culex quinquefasciatus larvae were reared on low- and high-quantity food diets to produce adults that were nutritionally representative of wild-caught and laboratory-reared mosquitoes. Emerging adults reared on low- or high-quantity food diets as larvae were then provided Lantana camara nectar mimics containing mixtures of carbohydrates and amino acids to evaluate effects of nectar amino acids on longevity. Carbohydrates (with or without amino acids) were a critical component of the adult diet, and in their absence, adult mosquitoes died within 3-5 days. The nectar mimic that contained both carbohydrates and amino acids did not increase adult longevity of males originating from either poorly or well-fed larvae. However, females receiving adult diets containing both carbohydrates and amino acids lived 5% longer than females fed adult diets with only sugar.     

Effects of temperature and host age upon the encapsulation of Metaphycus stanleyi and Metaphycus helvolus eggs by brown soft scale Coccus hesperidum

Encapsulation of eggs inserted by Metaphycus stanleyi (Hymenoptera: Encyrtidae) into the brown soft scale Coccus hesperidum (Homoptera: Coccidae) became more frequent as the host matured. This occurred with both laboratory reared and field-collected parasites. After parasitism for 24 hr at 27°C, encapsulation frequency did not differ in hosts reared at 20° or at 27°C, but significantly increased in hosts reared at 33°C. When parasitism and rearing were carried out at the same temperature, the percentage of eggs encapsulated increased from 48.7% at 27°C to 94.1% at 33°C. With M. helvolus, the percentage of eggs encapsulated was considerably higher than with M. stanleyi; e.g., 99.3 vs 48.7%, respectively, at 27°C. At 20° and 27°C, some M. helvolus development occurred in the larvae of brown soft scale but none at 33°C; the adult stages of the host encapsulated all the parasite eggs at these temperatures.     

Prevalence of parasitism and adult survival time of Aedes albifasciatus (Diptera: Culicidae) parasitized by Strelkovimermis spiculatus (Nematoda: Mermithidae)

We described the carryover of Strelkovimermis spiculatus (Poinar and Camino) (Nematoda: Mermithidae) from mosquito larvae, the primary site of maturation, to adults. We analyzed the survival time of male and female Aedes albifasciatus (Macquart) (Diptera: Culicidae) parasitized by S. spiculatus, the time of emergence of nematodes from adult mosquitoes, and the state of parasitism in the same mosquito cohorts during the immature stages. Mosquito larvae with single and multiple parasitism (up to 11 parasites) were observed. The mortality of mosquito larvae and adults was produced in all cases where at least one mermithid emerged. The mortality of S. spiculatus showed an increasing trend in mosquito larvae with larger numbers of nematodes and was higher in larvae parasitized by eight or more nematodes. Maximum survival of parasitized adult females of Ae. albifasciatus was 38 days, while non‐parasitized adult males and females survived 39 and 41 days, respectively. Strelkovimermis spiculatus mortality was observed in Ae. albifasciatus larvae with single or multiple parasitisms. The spread of mermithid parasitism in adult mosquito populations is discussed.