Frank B. Ramberg

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.

Microclimate and Human Factors in the Divergent Ecology of Aedes aegypti along the Arizona, U.S./Sonora, MX Border

This study examined the association of human and environmental factors with the presence of Aedes aegypti, the vector for dengue fever and yellow fever viruses, in a desert region in the southwest United States and northwest Mexico. Sixty-eight sites were longitudinally surveyed along the United States–Mexico border in Tucson, AZ, Nogales, AZ, and Nogales, Sonora during a 3-year period. Aedes aegypti presence or absence at each site was measured three times per year using standard oviposition traps. Maximum and minimum temperature and relative humidity were measured hourly at each site. Field inventories were conducted to measure human housing factors potentially affecting mosquito presence, such as the use of air-conditioning and evaporative coolers, outdoor vegetation cover, and access to piped water. The results showed that Ae. aegypti presence was highly variable across space and time. Aedes aegypti presence was positively associated with highly vegetated areas. Other significant variables included microclimatic differences and access to piped water. This study demonstrates the importance of microclimate and human factors in predicting Ae. aegypti distribution in an arid environment.

Salivary gland apyrase in black flies (Simulium vittatum)

Abstract Apyrase enzyme activity was demonstrated in the salivary glands of a colonized strain of Simulium vittatum . Activity was maximum (8.5 ± 0.7 mU/pair of gland equivalents) at pH 8.0, with ADP as substrate and Ca 2+ as the divalent cation. Activity was minimal in newly emerged females (1.6 ± 0.5 mU/pair of gland equivalents) but increased by 48 h. Activity in male salivary glands was marginally detectable (0.7 ± 0.8 mU/pair of gland equivalents), even 72 h after emergence. When newly emerged females were maintained at 4°C, salivary apyrase activity accumulated at a slow rate. Transferring females to warmer temperatures increased the rate of apyrase accumulation, but 27°C did not yield greater activity than 20°C. Apyrase activity was decreased when females engorged on whole bovine blood or on a simulated blood meal. Activity remained low 6 h after feeding, but increased to prefeeding levels by 48 h. During the second, anautogenous gonotrophic cycle, apyrase activity was not greater than during the first, autogenous gonotrophic cycle. Apyrase activity was not related to long term colonization as total salivary gland apyrase activity and pH profile in wild S. vittatum was not different from colonized S. vittatum .

Laboratory Vector Competence of Black Flies (Diptera: Simuliidæ) for the Indiana Serotype of Vesicular Stomatitis Virus

Abstract: 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.

Human and Environmental Factors Affecting Aedes aegypti Distribution in an Arid Urban Environment

Abstract Aedes aegypti has reappeared in urban communities in the southwestern USA in the 1990s after a 40-year absence. In 2003 and 2004, a systematic survey was conducted throughout metropolitan Tucson, AZ, to identify human and environmental factors associated with Ae. aegypti distribution within an arid urban area. Aedes aegypti presence and abundance were measured monthly using the Centers for Disease Control and Prevention enhanced oviposition traps at sampling sites established in a grid at 3- to 4-km intervals across the city. Sampling occurred in the summer rainy season (July through September), the peak of mosquito activity in the region. Multiple regression analyses were conducted to determine relationships between mosquito density and factors that could influence mosquito distribution. House age was the only factor that showed a consistent significant association with Ae. aegypti abundance in both years: older houses had more mosquito eggs. This is the 1st study of Ae. aegypti distribution at a local level to identify house age as an explanatory factor independent of other human demographic factors. Further research into the reasons why mosquitoes were more abundant around older homes may help inform and refine future vector surveillance and control efforts in the event of a dengue outbreak in the region.

Culicoides mohave (Diptera: Ceratopogonidae): New Occurrence Records and Potential Role in Transmission of Hemorrhagic Disease

Abstract Biting midges of the genus Culicoides are important in the transmission of viral diseases affecting wild and domestic ungulates, including bluetongue (BLU) and epizootic hemorrhagic disease (EHD). The primary known vector for these viruses is C. sonorensis Wirth & Jones, however, it has been speculated that other species of Culicoides may also be involved. One potential candidate is C. mohave, a poorly studied species found in inland desert areas of the southwestern United States. In 2000 and 2001, we collected C. mohave and C. sonorensis at six sites in a previously unsurveyed area in the Sonoran Desert of southwestern Arizona and used PCR to detect nucleic acids associated with BLU and EHD viruses. C. mohave was abundant at two low-elevation sites on the study area, but uncommon or absent elsewhere. C. sonorensis commonly occurred along with C. mohave at one site, but was much less abundant. All C. mohave pools were negative for BLU viral RNA, however, 35% yielded positive results for EHD. All C. sonorensis were negative for both BLU and EHD. Our results suggest that C. mohave is a potential vector of EHD virus in this area, however additional studies are needed to determine its ability to transmit EHD.

Care and maintenance of blackfly colonies

The availability of vector species on a continuous basis in the laboratory is an important prerequisite for both basic and applied aspects of research. Unlike other types of filariasis vectors (particularly mosquitoes), the immature stages of the Simuliidae (blackflies) are found exclusively in riverine or other types of running-water habitats. In the laboratory, duplicating this unusual aspect of the biology of the immatures (especially the larvae) poses a major obstacle to colonization of most important vector species. Thus, sustained propagation of many medically-important taxa under artificial conditions is difficult, if not impossible, at the moment. While several species of Simuliidae have been reared successfully through multiple generations in the laboratory, there are currently no colonies of a natural vector.

Ecdysteroids and oocyte development in the black fly Simulium vittatum

BackgroundOocyte development was studied in the autogenous black fly, Simulium vittatum (Diptera, Nematocera), a vector of Onchocerca volvulus, the causative agent of onchocerciasis.ResultsOocyte growth was nearly linear between adult eclosion and was complete by 72 hours at 21°C. The oocyte became opaque at 14 hours after eclosion indicating the initiation of protein yolk deposition. The accumulation of vitellogenin was measured using SDS-PAGE. The density of the yolk protein bands at about 200 and 65 kDa increased during the first and second days after eclosion. The amount of protein in the 200 kDa band of vitellogenin, determined using densitometry, rapidly increased between 12 and 25 hours after eclosion. Ecdysteroid levels were measured using a competitive ELISA. Ecdysteroid levels increased rapidly and subsequently declined during the first day after eclosion.ConclusionThese data show a correlation between the appearance of vitellogenin in the oocyte, and the rise in ecdysteroids. A possible relationship to molting of the nematode, Onchocerca volvulus, is discussed.

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

Abstract 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.