Occurrence of Anopheles hermsi (Diptera: Culicidae) in Arizona and Colorado.

Historically, malaria was a significant cause of morbidity and mortality throughout the western United States, and Anopheles freeborni Aitken was thought to be the vector west of the Continental Divide. In 1989, Anopheles hermsi Barr & Guptavanij was described and subsequently found to be an effective laboratory vector of Plasmodium. The adults of these two species are morphologically indistinguishable, and therefore polymerase chain reaction was used to analyze the DNA from 48 mosquitoes collected in Arizona and Colorado (identified morphologically as An. freeborni). All specimens were identified as An. hermsi. This was the first report of An. hermsi in Arizona and Colorado and indicated that this Anopheles species historically may have been a malaria vector in these two western states.

Transmission of vesicular stomatitis virus from infected to noninfected black flies co-feeding on nonviremic deer mice.

Vesicular stomatitis is an economically important arboviral disease of livestock. Viremia is absent in infected mammalian hosts, and the mechanism by which insects become infected with the causative agents, vesicular stomatitis viruses, remains unknown. Because infected and noninfected insects potentially feed on the same host in nature, infected and noninfected black flies were allowed to feed on the same host. Viremia was not detected in the host after infection by a black fly bite, but because noninfected black flies acquired the virus while co-feeding on the same host with infected black flies, it is concluded that a viremic host is not necessary for an insect to be infected with the virus. Thus co-feeding is a mechanism of infection for an insect-transmitted virus.

Laboratory vector competence of black flies (Diptera:Simuliidae) for the Indiana serotype of vesicular stomatitis virus.

In previous experiments we have demonstrated that colonized and wild black flies are competent laboratory vectors of different Mexican and Western USA isolates of vesicular stomatitis virus, serotype New Jersey (VSV-NJ). We have recently demonstrated biological VSV-NJ transmission by black flies using animal models. In the study described here, we tested the vector competence of colonized and wild black flies for the vesicular stomatitis virus, serotype Indiana (VSV-IN). A 1998 equine isolate was used. After a 10 day incubation period, saliva from experimentally infected Simulium vittatum and S. notatum was individually collected and tested for the presence of infectious virus. Virus was detected in the saliva of both species following oral infection, indicating that they are competent laboratory vectors of VSV-IN. In addition, the results suggest that the black fly gut may exert evolutionary pressures on the virus.

Bite transmission of vesicular stomatitis virus (New Jersey serotype) to laboratory mice by Simulium vittatum (Diptera: Simuliidae).

Laboratory-reared female black flies (Simulium vittatum Zetterstedt) were infected experimentally with a 1997 vesicular stomatitis virus New Jersey serotype isolate and allowed to feed on susceptible laboratory mice. All mice exposed to black fly bite seroconverted by day 21 after infection, an indication of virus transmission. In addition, viral RNA was detected in the spleen of several mice. These findings are consistent with the hypothesis that black flies are involved in VSV-NJ transmission during epizootics in the western USA and represent the 1st confirmed example of biological transmission of an arbovirus by a member of the Simuliidae using an animal model.

Anticoagulant activity in salivary gland extracts of black flies (Diptera: Simuliidae).

Anticoagulant activity was determined in salivary gland extracts from four species of black flies, i.e., Simulium vittatum Zetterstedt, Simulium argus Williston, Simulium metallicum Bellardi, and Simulium ochraceum Walker. Inhibition of coagulation factor Xa occurred among all four, whereas thrombin inhibition was detected in S. argus and S. vittatum only. Both bovine and human alpha-thrombins were inhibited with the highest activity occurring with S. argus salivary gland extracts. Factor Xa inhibition was highest in S. ochraceum, an anthropophilic species and vector of Onchocerca volvulus, and lowest in S. vittatum, a primiparous autogenous species that is also zoophilic. Total soluble salivary gland extract protein also varied among the four species with the highest concentration measured in S. ochraceum and the lowest in S. vittatum. A positive correlation was observed between the amount of soluble protein and percentage of inhibition of factor Xa for the four species.

Salivary apyrase in African and New World vectors of Plasmodium species and its relationship to malaria transmission.

The salivary gland activities of apyrase, an enzyme that prevents platelet aggregation by eliminating ADP, were compared among five members of the Anopheles gambiae species complex and An. albimanus. Within the An. gambiae group, An. quadriannulatus exhibited the lowest amount of enzyme activity at all pH levels measured. Apyrase activity could be separated into three groups at pH 7.5 and 8.0. The two most anthropophilic species (An. gambiae and An. arabiensis) exhibited higher activity at pH 9.0. Anopheles merus and An. melas, both saltwater taxa, and An. albimanus, a New World species, exhibited peak apyrase activity at pH 8.0. When the effects of divalent cations (Ca++, Mg++) on enzyme activity were compared at pH 8.5, apyrase activity in the presence of Mg++ could be separated into three levels. Anopheles gambiae and An. quadriannulatus exhibited reduced activity in the presence of Mg++. Anopheles arabiensis, An. merus, and An. melas displayed the highest relative levels of activity. Anopheles albimanus, with a Mg:Ca ratio of 0.80, was most similar to An. arabiensis. These biochemical differences suggest that different isoenzymes of apyrase have developed within the genus Anopheles.

Antibody responses of BALB/c mice to salivary antigens of hematophagous black flies (Diptera: Simuliidae).

The humoral antibody responses to salivary antigens of Simulium vittatum Zetterstedt were investigated in a BALB/c mouse laboratory model. Production of antisera was stimulated by intraperitoneal immunization with salivary gland extract or by feeding flies directly on depilated mice. Antibody responses in these two groups of mice were compared by western blotting, thus characterizing "true" salivary immunogens present in salivary gland extract. Immunized mice developed IgG, IgM, and IgE antibodies which recognized several salivary gland components, ranging in molecular weight between 26 and 67 kDa. Sera from bitten mice recognized fewer antigens, indicating that some components of the salivary gland extract were poorly immunogenic or absent from the saliva secreted during blood feeding. Antisera raised against S. vittatum also were used to identify cross-reactive immunogens and allergens in salivary gland extracts from other New World simuliids (Simulium argus Williston, S. metallicum Bellardi, and S. ochraceum Walker). SDS-PAGE protein profiles indicated a high degree of similarity between salivary gland extract of S. vittatum and S. argus, and several cross-reacting antigens were identified by western blotting. In contrast, protein profiles of S. ochraceum and S. metallicum differed from the former species, both qualitatively and quantitatively. Antisera demonstrated a low degree of cross-reactivity against salivary gland extract of S. ochraceum, whereas no cross-reactivity was detected against S. metallicum. These observations were confirmed using a monoclonal antibody raised against S. vittatum salivary gland extract (designated SVSG.1.F10), which showed cross-reactivity against S. argus but failed to recognize salivary gland components of either S. ochraceum or S. metallicum.

Biological transmission of vesicular stomatitis virus (New Jersey) by Simulium vittatum (Diptera: Simuliidae).

Simulium vittatum females were shown to be competent vectors for the New Jersey serotype (VSNJ) of vesicular stomatitis virus (Camp Verde strain). Seventy percent of females infected intrathoracically transmitted infectious virions in their saliva after a 10-d incubation period. When infected with virus per os, 63% of the flies tested were positive at day 10, and 45% of flies infected in this manner also secreted virus in their saliva by day 9 or 10 after infection. When ingested by S. vittatum females, VSNJ virus readily replicated and increased from a mean baseline titer of 1.2 x 10(4) pfu per fly to 3 x 10(4) pfu per fly on day 10. An eclipse phase was demonstrated between approximately 18 and 48 h postinfection. This experimental evidence supports the hypothesis that black flies play a major role in the epizootic transmission of VSNJ. This is also the first confirmed example of biological transmission of an arbovirus by a member of the Simuliidae.