Infection, growth and maintenance of Wolbachia pipientis in clonal and non-clonal Aedes albopictus cell cultures.

Insect cell lines provide useful in vitro models for studying biological systems, including interactions between mosquitoes and obligate intracellular endosymbionts such as Wolbachia pipientis. The Aedes albopictus Aa23 cell line was the first cell line developed to allow examination of Wolbachia infections. However, Wolbachia studies using Aa23 can be complicated by the presence of different cell types in the cell line and the substantial temporal variation in infection level. Two approaches were examined to ameliorate infection variability. In the first approach, multiple Aa23 passaging regimes were tested for an effect on infection variability. Fluorescence in situ hybridization (FISH) staining was used to characterize Wolbachia infection level over time. The results demonstrate an impact of passaging method on Wolbachia infection level, with some methods resulting in loss of infection. None of the passaging methods succeeded in effectively mitigating infection level variation. In a second approach, the clonal C7-10 A. albopictus cell line was infected with Wolbachia from Aa23 cells and Drosophila simulans (Riverside), resulting in cell lines designated C7-10B and C7-10R, respectively. Characterization via FISH staining showed greater stability and uniformity of Wolbachia infection in C7-10R relative to the infection in C7-10B. Characterization of the Aa23, C7-10B and C7-10R lines is discussed as a tool for the study of Wolbachia-host cell interactions.

Effects of larval density on the size of Aedes polynesiensis adults (Diptera: Culicidae).

Replicated cohorts of a Tahitian laboratory strain of Aedes polynesiensis Marks were reared at 3 larval densities with a fixed amount of food. For larvae provided with limiting per capita food (i.e., 400 larvae per pan with 500 mg liver powder) relative to standard rearing conditions (i.e., 200 larvae per pan), mean pupal survival as well as male and female mean adult dry weights were significantly reduced and median developmental times were significantly prolonged. However, excess per capita food did not allow low density cohorts (i.e., 100 larvae per pan) to increase adult production, developmental rate, or adult dry weight compared with cohorts reared under standard rearing conditions. Male and female pupal wet weights, adult dry weights, and adult wing lengths all were correlated for Ae. polynesiensis collected as pupae from natural habitats near Papara Commune, Tahiti. Mean adult dry weights of host-seeking females from the same and a neighboring location did not differ significantly from weights of females emerging from field-collected pupae. The comparison of mean adult dry weight of these adults with adults reared at different densities in the laboratory indicated that field populations develop under food-limited conditions. Aedes polynesiensis responds to intraspecific larval competition by producing small adults over elongated developmental periods. Pupal wet weights, adult dry weights, and adult wing lengths are equally acceptable measures of mosquito size for vector and fecundity studies.

Isolation and genetic characterization of a hantavirus (Bunyaviridae: Hantavirus) from a rodent, Oligoryzomys microtis (Muridae), collected in northeastern Peru.

This paper describes the isolation and partial genetic characterization of a hantavirus from a pygmy rice rat, Oligoryzomys microtis, collected within the urban area of Iquitos, Loreto Department, Peru. The virus, designated HTN-007, exhibited the highest degree of genetic similarity to Rio Mamore virus, which was originally described from the same rodent species in eastern Bolivia. Comparison of small and medium segment nucleotide sequence data from HTN-007 and Rio Mamore virus revealed 87% and 85% sequence identity, respectively. Based on these analyses, HTN-007 appears to be a variant of Rio Mamore virus. As such, it represents the first successful isolation of Rio Mamore virus and the first evidence for the existence of a hantavirus in Peru. Serologic studies done by immunofluorescence on blood samples of 56 O. microtis trapped at the collection site indicated that 21.4% had antibodies to hantavirus. In view of the proximity of this rodent species to humans and the close phylogenetic relationship of Rio Mamore virus to hantaviruses that have been associated with human disease, Rio Mamore virus may be a hantavirus of some public health importance in tropical South America.

Aedes polynesiensis in the Society Islands: environmental correlates of isoenzyme differentiation.

Isoenzyme genetic differentiation of Aedes polynesiensis mosquitoes in Raiatea island, French Polynesia, was evaluated by two models of population structure based on seven gene-enzyme systems: Ak, Est, Got, Gpi, Hk, Mdh and Pgm. The ecological model tested whether genetic differentiation is congruent with habitat differences. The isolation model evaluated whether genetic differentiation is proportional to geographical distribution. The ecological model found no significant differentiation between populations of Ae.polynesiensis from beach and forest ecotopes, whereas the isolation model was consistent with the data. However, incipient speciation is opposed by the source-sink system of population dynamics in such small neighbouring islands, where Ae.polynensiensis extinction is readily followed by reinvasion causing considerable gene flow between island populations.

A polymerase chain reaction assay to determine infection of Aedes polynesiensis by Wuchereria bancrofti.

The sensitivity of a previously described polymerase chain reaction (PCR) assay was improved to detect a single mosquito, infected by as few as 1-2 microfilariae of Wuchereria bancrofti, among 20-50 uninfected mosquitoes. Wild-caught Aedes polynesiensis were used to compare assessment of infection by dissection of individuals with the PCR assay of pools of mosquitoes. The PCR assay was at least as sensitive as dissection for detection of mosquitoes infected with W. bancrofti.

Correlation between colorimetric and protein-precipitation assays of tannins dissolved in treehole water.

There was a significant correlation between colorimetric and protein-precipitation assays of tannins dissolved in treehole water. The protein-precipitation potential of early-season treehole water was extremely low relative to total phenolics measured by colorimetry. The protein-precipitation method is recommended because it directly measures protein binding, the property of dissolved tannins relevant to mosquito development. Furthermore, the procedure is not affected by treehole water color and does not produce dangerous wastes.

Evaluation of entomopathogenic bacteria against Aedes polynesiensis, the vector of lymphatic filariasis in French Polynesia.

Thirteen strains among 3 species of entomopathogenic bacteria were tested against 3 medically important mosquito species in French Polynesia. Two strains of Bacillus thuringiensis were highly toxic to Aedes polynesiensis, Aedes aegypti, and Culex quinquefasciatus. Six of 7 strains of Bacillus sphaericus tested were highly toxic to Cx. quinquefasciatus but not to the Aedes spp. Clostridium bifermentans serovar. malaysia was more toxic to Ae. polynesiensis than to the other 2 species. Entomopathogenic bacteria merit field testing for larval mosquito control in French Polynesia.

Tannins in treehole habitats and their effects on Aedes sierrensis (Diptera: Culicidae) production and parasitism by Lambornella clarki (Ciliophora: Tetrahymenidae).

Tannin concentration, measured as total phenolics, varied widely in natural treehole water throughout the period of development for Aedes sierrensis (Ludlow), the western treehole mosquito. Tannic acid induced a parasitic response in free-living populations of the protozoan Lambornella clarki Corliss & Coats, an effect not entirely explained by acidity. Tannic acid concentration influenced mosquito developmental rates, survival, size, and proportion females more than food level, L. clarki parasite exposure, or any interaction of treatments for Ae. sierrensis populations reared in artificial microcosms exposed to natural conditions.

Effect of tannic acid concentration on development of the western treehole mosquito,Aedes sierrensis (Diptera: Culicidae).

Populations ofAedes sierrensis (Ludlow) completed development in tannic acid solutions over a wide concentration range (i.e., 0-1.0 g/liter) in artificial microcosms exposed to field conditions. The most notable effects of high tannic acid concentration were to slow larval developmental rates and to reduce numbers of adults produced; adult size and sex ratio were minimally affected. Vector potential of the western treehole mosquito is discussed in terms of tannin concentration.

Parasitism of newly-hatched Aedes sierrensis (Diptera: Culicidae) larvae by Lambornella clarki (Ciliophora: Tetrahymenidae) following habitat flooding.

Host-parasite interactions between Lambornella clarki (Ciliophora: Tetrahymenidae) and its natural host, Aedes sierrensis (Diptera: Culicidae), were studied in newly flooded treeholes in northern California between 1986 and 1989. First instar host larvae hatched within 1 to 4 hr of flooding, while free-living trophonts of L. clarki appeared between 7 and 24 hr. As early as 24 hr after flooding, ciliates initiated the first parasite cycle by forming cuticular cysts on first instar larvae; by 64 hr, cysts were observed on larvae collected from all positive holes during all years. While larvae with as many as 12 cysts were observed, most supported only 1 cyst, and successful infections were established by the entry of a single ciliate into the host's hemocoel. Among treehole populations, the proportion of larvae with L. clarki cysts ranged from 2 to 100% at 48 hr indicating that enzootics and epizootics develop rapidly in newly flooded treeholes. Average attack rates from all holes by year ranged between 17.0 and 44.4%. Ciliates began entering hosts 48 to 72 hr after flooding, but some larvae escaped parasitization by molting to the second instar before ciliates penetrated the cuticle. In some treeholes, opportunistic microorganisms entered larvae with the invading ciliates and killed both the host and parasite.

Regulatory role of parasites: impact on host population shifts with resource availability.

Effects of infections by the ciliate Lambornella clarki on larval populations of its mosquito host Aedes sierrensis were examined in laboratory and field studies. When host populations developed with sufficient food, mortality from parasites was additive and reduced the number of emerging mosquitoes. For food-limited populations, mortality was compensatory or depensatory; emerging adults were as or more abundant with higher average fitness than those from uninfected control populations. When nutrients were scarce, parasitic infections relaxed larval competition and increased per capita food by reducing host abundance. Food limitation altered larval feeding behavior, reducing horizontal transmission and subsequent mortality from parasitism.

Emergence characteristics of Aedes sierrensis (Diptera: Culicidae) from California treeholes with particular reference to parasite loads.

Wing lengths, emergence times, and parasite loads were determined for 8,444 newly emerged adult Aedes sierrensis (Ludlow) collected from 17 treehole emergence traps during a sampling period of 149 d. Peak adult emergence occurred in late spring and early summer, but a small second cohort developed in treeholes retaining water through the dry season. Mosquito production was significantly and positively correlated with the maximum volumes of treeholes. Male and female wing lengths and eclosion times varied significantly among populations from different treeholes; within a population, these variables were highly correlated, suggesting that larvae of both sexes respond similarly to treehole conditions. Egg number (but not longevity) was correlated with adult size for laboratory-maintained females collected from emergence traps. These results suggest variation in the vector potential of adult populations produced from different treeholes. Newly emerged mosquitoes were infected by five parasite species that depend on adult hosts for their dispersal. The mermithid nematode Octomyomermis troglodytis Poinar and Sanders and the ciliate Tetrahymena sp. were very rare in adult populations. Gamontocysts of the ascogregarine Ascogregarina clarki Sanders and Poinar infected the Malpighian tubules of adults from six treeholes. Ectoparasitic larval water mites (near Euthyas) were found on 12% of all adults eclosing from eight treeholes and selectively parasitized female hosts over males. The ciliate Lambornella clarki Corliss and Coats was the most common parasite; it infected males and females from seven treeholes in equal proportions. L. clarki had a significant negative impact on adult populations in that 13% of all females from L. clarki-positive treeholes were infected with ciliates, and infected females are parasitically castrated.

Predator-induced trophic shift of a free-living ciliate: parasitism of mosquito larvae by their prey.

Larvae of the treehole mosquito, Aedes sierrensis, release a waterborne factor that induces morphogenesis of one of their prey, the tetrahymenid ciliate Lambornella clarki. Induced free-living trophonts of L. clarki undergo a synchronous response in which cells divide and transform into parasitic cells (theronts) that encyst on larval predators. Parasitic ciliates penetrate the cuticle, enter the hemocoel, and ultimately kill their predator-host. In nature, this trophic shift can lead to predator extinction and dramatic changes in microbial populations. Facultative parasitism by this polymorphic ciliate may have evolved as an antipredator strategy. The experimentally inducible parasitic response of L. clarki provides a novel model for studying cellular morphogenesis of ciliated protozoa.