Diversity of Wolbachia infections in Sri Lankan mosquitoes with a new record of Wolbachia Supergroup B infecting Aedes aegypti vector populations.

Wolbachia bacteria are common endosymbionts of insects and have recently been applied for controlling arboviral vectors, especially Aedes aegypti mosquito populations. However, several medically important mosquito species in Sri Lanka were present with limited information for the Wolbachia infection status. Therefore, the screening of Wolbachia in indigenous mosquitoes is required prior to a successful application of Wolbachia-based vector control strategy. In this study, screening of 78 mosquito species collected from various parts of the country revealed that 13 species were positive for Wolbachia infection, giving ~ 17% infection frequency of Wolbachia among the Sri Lankan mosquitoes. Twelve Wolbachia-positive mosquito species were selected for downstream Wolbachia strain genotyping using Multi Locus Sequencing Type (MLST), wsp gene, and 16S rRNA gene-based approaches. Results showed that these Wolbachia strains clustered together with the present Wolbachia phylogeny of world mosquito populations with some variations. Almost 90% of the mosquito populations were infected with supergroup B while the remaining were infected with supergroup A. A new record of Wolbachia supergroup B infection in Ae. aegypti, the main vectors of dengue, was highlighted. This finding was further confirmed by real-time qPCR, revealing Wolbachia density variations between Ae. aegypti and Ae. albopictus (p = 0.001), and between males and females (p < 0.05). The evidence of natural Wolbachia infections in Ae. aegypti populations in Sri Lanka is an extremely rare incident that has the potential to be used for arboviral vector control.

Three simple devices for preventing development of Aedes aegypti larvae in water jars.

We developed three types of covers that are easily fabricated from plastic screen locally manufactured in Thailand. The covers were designed to permit normal use of water jars for drinking, utility water uses, and water storage. Tests of the covers in the laboratory and field demonstrated that they could completely prevent successful development of Aedes aegypti (L.) in water jars. Initial experience in a village setting demonstrated that these devices were readily manufactured and used in a community-based program. Village residents adapted the covers to local patterns of water use, collecting rainwater through the screen and adjusting cover use to water depth.

Distribution of dengue and Japanese encephalitis among children in rural and suburban Thai villages.

In the rainy season of 1989, IgG and IgM antibodies against dengue and Japanese encephalitis viruses (measured by enzyme-linked immunoassay [ELISA]) in serum from all primary-school children in two areas of central Thailand were sampled in order to choose a study site for more detailed epidemiological and entomological analysis. Students in three schools in the largely non-agricultural, suburban community of Bang Bua Thong, Nontaburi Province were sampled in late June and July. Of 1,477 children, 33/1,000 had recent dengue infection and 7/1,000 had recent JE infection. The rate of dengue infection in each village influenced the rate in schools, in that the rate of the school could be predicted from the proportion of students coming from each village. This result suggested that most transmission occurred in the residential environment; otherwise, the rate in each village going to a single school would be identical. Serum samples were taken in late August in the agricultural community of Hua Samrong, Chachoengsao Province. Of 748 students in two schools, 95/1,000 had signs of recent dengue infection and 32/1,000 had signs of recent JE infection. Two of 12 villages had significantly less flavivirus infection than some other villages and three villages had significantly more flavivirus infection. The children from one village had a dengue infection rate of 256 per 1,000, which was higher than the national average for the worst year (1987) previously recorded in Thailand. Within Hua Samrong, there was evidence for significant dengue transmission in one of the schools and concentrated transmission in small areas of two of the villages. The younger age group (3-8 years old) had significantly higher risk of infection by either flavivirus than older children. Elevated homes with wooden floors had significantly higher risk of dengue in the largest village. The observations from 1989 describe the epidemiological situation in rapidly developing, rural villages. This stage of development is probably being repeated throughout Southeast Asia as formerly isolated, rural villages become connected by transportation and economy to urban centers. What appears to be a single dengue outbreak based on passive surveillance conducted on a regional basis may actually be a variety of epidemiological situations. The practical implication of this conclusion is that application of a combination of vaccination and vector control should be targeted to higher risk areas in order to increase the likelihood of regional dengue virus eradication.

Effect of size and geographic origin of Aedes aegypti on oral infection with dengue-2 virus.

Differences in larval habitats cause variation in the size of Aedes aegypti (L.) adults. We suspected that such size variation was related to the ease with which the mosquitoes could be infected with dengue virus. Using a rearing procedure that produced three distinct size classes of mosquitoes, we determined the percentage of mosquitoes that developed disseminated dengue-2 infection following oral feeding with a suspension containing 3.3 x 10(7) plaque-forming units/ml. Mosquitoes were reared from eggs deposited by females captured in either of two villages in Chachoengsao Province or in Bangkok, Thailand. More of the larger mosquitoes (10.7%) were infected than the medium (5.6%) or small (5.7%) mosquitoes. Mosquitoes from Bangkok were less easily infected (5.0%) than mosquitoes from either of the two villages (8.5% and 10.7%). These results suggest that quantitative risk assessment of dengue transmission may be very difficult unless inoculation rate is measured directly. Also, control procedures that reduce density of larvae in individual containers may exacerbate dengue transmission by creating larger mosquitoes that are more easily infected.

A mosquito densovirus infecting Aedes aegypti and Aedes albopictus from Thailand.

A previously undescribed mosquito densovirus was detected in colonies of Aedes aegypti and Ae. albopictus from Thailand, using a polymerase chain reaction (PCR)-based assay. Phylogenetic analysis of this virus showed it to be most closely related to ADNV isolated from Russian Ae. aegypti. Both Aedes species were susceptible to oral infection with the Thai-strain virus. Larval mortality for Ae. albopictus was higher (82%) than for Ae. aegypti (51%). Aedes aegypti were able to transmit the virus vertically to a high (58%) proportion of G1 progeny, and the virus was maintained persistently for up to six generations. A PCR survey of adult Ae. aegypti and Ae. albopictus in Thailand indicated that only Ae. aegypti are infected in the field, with an overall prevalence of 44%. Densovirus infection in adult Ae. aegypti showed distinct seasonal variation. The Thai strain densovirus may play a role in structuring Ae. albopictus and Ae. aegypti populations in nature.

A fitness advantage for Aedes aegypti and the viruses it transmits when females feed only on human blood.

Literature on arthropod-borne diseases has traditionally supported the notion that mosquito vectors maintain a feeding duality that includes vertebrate blood meals for egg development and sugar meals from plants for the synthesis of flight and survival energy reserves. Aedes aegypti was found to deviate from that feeding pattern by obtaining a reproductive advantage when feeding only on human blood. Female mosquitoes fed human blood alone had a greater net replacement rate and intrinsic rate of growth during all phases of their reproductive life than conspecifics fed human blood plus sucrose. Feeding frequently on human hosts during each gonotrophic cycle is necessary to avoid death due to starvation and increases exponentially the spread of Ae. aegypti-borne disease. Our results help explain why Ae. aegypti is such an unusually efficient vector of human disease; frequent biting of humans results in a high reproductive rate for vectors as well as the viruses they transmit.

Distribution of container-inhabiting Aedes larvae (Diptera: Culicidae) at a dengue focus in Thailand.

A Thai village with dengue transmission was surveyed for Aedes aegypti (L.) and Ae. albopictus (Skuse) larvae at the end of the rainy season. All containers (1,819) in 186 households were surveyed, recording the presence of larvae, container type, level of water, kind of cover, and location. The number of positive containers was proportional to the total number of containers in a household. Standard water jars (approximately 200 liters) contributed 57% and small water jars (< 100 liters) contributed 16% of the total infested containers. Each of the other 10 container types contributed < or = 10% of the total infested containers and were not statistically different from each other. Containers inside houses were infested significantly more often than were those outdoors, those under elevated houses or roofs, or those in bathrooms. Unexpectedly, standard water jars located outdoors that were covered with commercial metal lids were infested significantly more often than were uncovered jars.

Laboratory demonstration of oviposition by Aedes aegypti (Diptera: Culicidae) in covered water jars.

In Thailand, ceramic jars (135-200 liters) covered by aluminum lids commonly are used to store water for household use. In laboratory cages, gravid female Aedes aegypti (L.) were able to enter and oviposit in a covered water jar. Although the aluminum lid was not a complete barrier to gravid females, it reduced the number of eggs oviposited by 77%. A vertically oriented foam rubber ring which was compressed between the lip of the jar and the flange of the lid effectively sealed the jar against ovipositing mosquitoes.

Wolbachia infection and expression of cytoplasmic incompatibility in Armigeres subalbatus (Diptera: Culicidae).

Polymerase chain reaction screening revealed that Armigeres subalbatus (Coquillett), a vector of filariasis, was infected with the intracellular bacteria Wolbachia. Laboratory crosses between infected males and uninfected females resulted in less than half the number of offspring than control crosses between uninfected individuals when young (2- to 3-d-old) males were used in the cross. However, imcompatibility was lost when old (14- to 17-d-old) males were used. Field-collected females did not show detectable cytoplasmic incompatibility, and this may be because of the age at which males mate in the field. We used head pigment fluorescence levels to age field males collected from mating swarms, and found that 25-63% of swarming males were older than 13 d. Male age may be one factor influencing the observed low levels of cytoplasmic incompatibility detected in the field.

Distribution and diversity of Wolbachia infections in Southeast Asian mosquitoes (Diptera: Culicidae).

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a variety of reproductive alterations in their hosts, the best known being cytoplasmic incompatability. The Wolbachia pipientis assemblage has been divided into two major groups (A and B) and 12 subgroups. We report herein the first systematic survey of Wolbachia in mosquitoes, and the first survey classifying Wolbachia infections by subgroup. Wolbachia were detected in 28.1% of 89 wild-caught mosquito species, based on a polymerase chain reaction assay using ftsZ and wsp gene primers. Infections were found in all major disease vector genera except Anopheles. Nine of the 12 Wolbachia subgroups were represented. Group B Wolbachia strains showed more phylogenetic concordance with their host taxa than group A strains. Of the 25 positive mosquito species, five were superinfected with group A bacteria strains (AA), eight were superinfected with A and B strains (AB), and one was superinfected with group B strains (BB). The widespread distribution of Wolbachia among mosquito species further supports their potential importance in the genetic control of disease vectors.

Female body size, parity, and malaria infection of Anopheles maculatus (Diptera: Culicidae) in peninsular Malaysia.

The relationship among body size (as indicated by wing length), age (as indicated by parity dissections), and malaria infection were observed in host-seeking Anopheles maculatus Theobald females collected in aboriginal villages of peninsular Malaysia. Both ELISA and salivary gland dissections were used to determine malaria infection. The wings of parous females were significantly longer than those of nulliparous females, suggesting that larger females live longer than smaller ones, and thus have a higher vectorial capacity. Body size differences were not detected between infected parous and uninfected parous females. Females infected with only oocysts were significantly larger than females infected with sporozoites. No correlation was found between the number of oocysts or sporozoites and body size in this small sample.

Female Aedes aegypti (Diptera: Culicidae) in Thailand rarely feed on sugar.

Female Aedes aegypti (L.) of two different body sizes and provided with different diets (20% sucrose, water only, or 20% sucrose + human blood) were marked and released together in a rural Thai village. Recaptured marked and unmarked (wild) adults were tested for fructose by the cold-anthrone reaction. Both released and wild females showed a low frequency of sugar feeding in nature; both small-bodied and large-bodied individuals failed to sugar feed significantly. Marked females released with sugar in their crop utilized this energy source over the following 2-3 d but failed to replenish it. In contrast, about one-third of wild, resting males showed evidence of recent sugar feeding. This indicated that the absence of plant sugar in females was not caused by a shortage of nectar sources in this rural domestic environment. Presumably, the nonutilization of plant sugar as an energy source increases the frequency of blood feeding and, therefore, the vectorial capacity of Ae. aegypti.

Survey of the Anopheles maculatus complex (Diptera: Culicidae) in peninsular Malaysia by analysis of cuticular lipids.

Anopheles maculatus Theobald sensu lato is a species complex now consisting of eight sibling species; An. maculatus is still represented by two cytologically distinct forms; i.e., the widely distributed sensu strictu or B, and E from southern Thailand and adjacent areas in northern Malaysia. Cuticular lipid profiles in conjunction with principal component analysis was used to separate An. maculatus form E from sensu stricto form B in a preliminary survey of the An. maculatus complex at five locations spanning peninsular Malaysia. The relative rank orders, from the areas of the five gas chromatographic peaks used to determine lipid differences for specimens from peninsular Malaysia, matched well with those from cytogenetically identified colony specimens of An. maculatus forms B and E. The two-dimensional principal component pattern of specimens identified as form E was highly clumped, which indicated that very similar cuticular lipids were present within this putative malaria vector. Both forms coexisted in peninsular Malaysia, but form E may be dominant except in the south.

Survival of starved Aedes aegypti (Diptera: Culicidae) in Puerto Rico and Thailand.

Survival of adult Aedes aegypti (L.) was studied in Thailand (1995) and Puerto Rico (1996) during periods of high and low dengue virus transmission. Resting males and females were collected inside houses by aspiration. Females were separated into different cages by their degree of engorgement and ovarian development. Teneral adults were obtained from pupae collected from natural breeding sites. All mosquitoes were given access to water, held at ambient temperature in the shade, and their survival monitored daily. We calculated median survival for each stage to estimate when mosquitoes had to feed again or die. No differences in survival between seasons were observed in Thailand. In Puerto Rico, except for wild males, survival was longer in the cool/dry season than in the hot/rainy season, indicating that mosquitoes may need to feed more frequently during the high than low dengue transmission season. During both study periods and at both sites, blood-engorged females survived as long or longer than mosquitoes in other gonotrophic or developmental stages. Except in Puerto Rico during the cool season, when females had a relatively high probability of surviving 3-4 d without feeding, females needed to feed approximately every other day to avoid death caused by starvation. Our results indicate that in some regions, there are seasonal differences in the length of time female Ae. aegypti can survive without feeding, females with a blood meal can survive for a longer time than those without blood, and teneral males can live longer without food than teneral females.

Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto Rico: blood feeding frequency.

We used a histologic technique to study multiple blood feeding in a single gonotrophic cycle by engorged Aedes aegypti (L.) that were collected weekly for 2 yr from houses in a rural village in Thailand (n = 1,891) and a residential section of San Juan, Puerto Rico (n = 1,675). Overall, mosquitoes from Thailand contained significantly more multiple meals (n = 1,300, 42% double meals, 5% triple meals) than mosquitoes collected in Puerto Rico (n = 1,156, 32% double meals, 2% triple meals). The portion of specimens for which frequency of feeding could not be determined was 31% at both sites. We estimated that on average Ae. aegypti take 0.76 and 0.63 human blood meals per day in Thailand and Puerto Rico, respectively. However, frequency of multiple feeding varied among houses and, in Puerto Rico, the neighborhoods from which mosquitoes were collected. In Thailand 65% of the mosquitoes fed twice on the same day, whereas in Puerto Rico 57% took multiple meals separated by > or = 1 d. At both sites, the majority of engorged specimens were collected inside houses (Thailand 86%, Puerto Rico 95%). The number of blood meals detected was independent of where mosquitoes were collected (inside versus outside of the house) at both sites and the time of day collections were made in Puerto Rico. Feeding rates were slightly higher for mosquitoes collected in the afternoon in Thailand. Temperatures were significantly higher and mosquitoes significantly smaller in Thailand than in Puerto Rico. At both sites female size was negatively associated with temperature. Rates of multiple feeding were associated positively with temperature and negatively with mosquito size in Thailand, but not in Puerto Rico. Multiple feeding during a single gonotrophic cycle is a regular part of Ae. aegypti biology, can vary geographically and under different climate conditions, and may be associated with variation in patterns of dengue virus transmission.

Longitudinal studies of Aedes aegypti (Diptera: Culicidae) in Thailand and Puerto Rico: population dynamics.

Aspiration collections of adult Aedes aegypti (L.) were made weekly from inside and outside of houses for 3 yr in a rural Thai village (n = 9,637 females and n = 11,988 males) and for 2 yr in a residential section of San Juan, Puerto Rico (n = 5,941 females and n = 6,739 males). In Thailand, temperature and rainfall fell into distinct seasonal categories, but only temperature was correlated with fluctuations in female abundance. Average weekly temperature 6 wk before mosquitoes were collected and minimum weekly temperature during the week of collection provided the highest correlations with female abundance. Accounting for annual variation significantly improved Thai models of temperature and mosquito abundance. In Puerto Rico, temperature, but not rainfall, could be categorized into seasonal patterns. Neither was correlated with changes in female abundance. At both sites the vast majority of females were collected inside houses and most contained a blood meal. Most teneral females were collected outside. Wing length--an indicator of female size--and parity, egg development or engorgement status were not correlated, indicating that feeding success and survival were not influenced by female size. At both sites, females fed almost exclusively on human hosts (> or = 96%), a pattern that did not change seasonally. In Puerto Rico more nonhuman blood meals were detected in mosquitoes collected outside than inside houses; no such difference was detected in Thailand. Gut contents of dissected females indicated that females in the Thai population had a younger age distribution and fed more frequently on blood than did Ae. aegypti in Puerto Rico. Our results indicated that aspects of this species' biology can vary significantly from one location to another and 1 yr to the next.

Blood-feeding patterns of Aedes aegypti (Diptera: Culicidae) collected in a rural Thai village.

Aedes aegypti (L.) were collected by aspiration once each week from in and around houses in a rural village in Chachoengsao Province. Thailand, during May 1990 to June 1991. Of the 1,230 specimens analyzed with a sandwich ELISA, 73% reacted to one or more of the seven hosts which we tested. Eighty-eight percent (789/896) of all detectable meals were identified as being from a single host (human). Patent multiple meals (double and triple), of which one was always human, were detected in 7% (66/896) of the specimens. The probability of feeding on a human, either as a single host or in a patent mixed meal, was > 0.90 during 12 of the 14 mo of the study; this probability never dropped below 0.85. The probability of Ae. aegypti taking multiple blood meals during a single gonotrophic cycle varied widely from 0.0 to 1.0. The possibility is discussed, including epidemiological significance, that there are seasonal fluctuations in the frequency of multiple feeding by Ae. aegypti and that multiple feeding increases in concert with seasonal trends for dengue infections of humans in Thailand.

Analysis of survival of young and old Aedes aegypti (Diptera: Culicidac) from Puerto Rico and Thailand.

It generally is assumed that the daily probability of survival of wild adult mosquitoes is independent of age. To test this assumption we conducted mark-release-recapture studies in Puerto Rico and Thailand to determine if estimated daily survival rates between two different age cohorts of the dengue vector Aedes aegypti (L.) were the same. Survivorship was estimated with nonlinear regression analysis using bootstrapping to obtain estimates of errors. Initial recapture success of the younger cohort was greater than the older cohort at both locations. Our analysis revealed a significantly greater survival rate for the younger cohort of females in Puerto Rico, and no significant differences between age cohorts in Thailand. For comparison, a traditional approach for analyzing these type of data, linear regression of log-transformed captures over time (exponential model), was used to calculate the probability of daily survival based on slopes of linear regression lines for recaptured mosquitoes. With this method, the estimated daily survival rate of older females (13-23 d old) was significantly greater than survival of younger ones (3-13 d old) in Puerto Rico and Thailand. In addition, short-range movement of mosquitoes was observed in Puerto Rico; maximum dispersal distance detected was 79 m. Survival rates of adult Ae. aegypti may be age-dependent and nonlinear regression analysis is a sensitive approach for comparing patterns of mosquito survival based on mark, single release, multiple recapture data.

Attractant resting boxes for rapid collection and surveillance of Aedes aegypti (L.) inside houses.

Three different resting station designs made of cardboard covered with black cloth were evaluated for their attractiveness to Aedes aegypti populations resting inside houses in Thailand. Box designs attracted more females (but not males) than an open-panel design. Thirty to 60% of all adult Ae. aegypti resting inside houses were collected by aspirating from 2-4 resting boxes placed inside houses. Tall boxes (90 cm) did not attract more females or males than short boxes (45 cm). Forty-two percent more females (but fewer males) were attracted to boxes when oviposition bowls were placed inside the boxes. Four boxes per house attracted 32% more mosquitoes than two boxes. Aspirating from artificial resting stations is an efficient and rapid method for sampling male and teneral, bloodfed, and gravid female Ae. aegypti resting inside houses.

Effect of mechanical shock on hydrostatic balance and survival of mosquito pupae.

The presence and distribution of ventral air space gas assures that mosquito pupae are positively buoyant and that they float, dive, and ascend in an upright, balanced orientation. Our objective was to test the effects of mechanical shocks of varying magnitude on mosquito pupae representing 3 genera. Forces that disrupt the pupa's buoyancy and/or hydrostatic balance are of a much lower magnitude than those that would cause tissue damage. Once hydrostatic balance and/or buoyancy are compromised, pupae are unable to restore them and eventually drown. This could represent a weak link in the mosquito life cycle. It is possible that mechanical shock or sound-generating devices could be designed that could break this link.