Autogeny in Ochlerotatus vigilax (Diptera: Culicidae) from southeast Queensland, Australia

Field and laboratory investigations were undertaken to determine the level of expression of autogeny in the mosquito Ochlerotatus vigilax (Skuse) from southeast Queensland, Australia, and whether there was evidence of seasonal variation. At two field sites in southeast Queensland, Wellington Point and Donnybrook, autogeny rates were determined on six occasions between January 2001 and January 2002. The autogeny rate varied between 71 and 100% at Wellington Point and between 63 and 100% at Donnybrook. Autogenous fecundity ranged from 17 to 63 eggs per female at Wellington Point and from 13 to 88 eggs per female at Donnybrook. Positive relationships were found between adult body size (indicated by wing length), autogeny rate, and fecundity. A laboratory study was conducted to investigate the influence of larval nutrition and adult diet (water versus sucrose) on the expression of autogeny. The autogeny rate at a low-diet treatment was between 73 and 90% when sucrose was withheld from females and 100% when sucrose was provided. All high-diet females were autogenous. Autogenous egg development required 80 +/- 6 h from emergence at 27 degrees C. We conclude that autogeny rates are consistently high in Oc. vigilax from the southeast Queensland region.     

Experimental infection and transmission of Barmah Forest virus by Aedes vigilax (Diptera: Culicidae)

Aedes vigilax (Skuse) mosquitoes colonized from Townsville, Queensland, Australia, were fed on blood containing Barmah Forest virus (BF) isolated from the same species. The colony was susceptible to infection, with an ID50 of 10(2.6) CCID50 per mosquito. Infection and transmission rates for mosquitoes fed 10(3.5) CCID50 virus per mosquito varied from 58 to 100% and 36 to 100%, respectively, between days 3 and 13 after infection. Titers in infected mosquitoes were high by 5 d after infection and did not vary substantially thereafter. Virus transmission to suckling mice occurred from 3 d after infection. This work establishes that Ae. vigilax is a competent vector of BF.     

Laboratory transmission of La Crosse virus by Ochlerotatus j. japonicus (Diptera: Culicidae)

Ochlerotatus j. japonicus, a recent introduction to the United States, was studied to determine its capability to serve as a vector of La Crosse (LAC) virus. A field-collectedpopulation of Ochlerotatus triseriatus, the primary vector of LAC virus, was similarly tested for comparison. After Oc. j. japonicus ingested virus fromhamsters with viremias of 10(3.6-5.4) plaque-forming units (PFU)/ml of blood, its estimated transmission rates were 35-88%. These rates were slightly lower than, though similar to, those for Oc. triseriatus, 75-100%. Viral titers in Oc. j. japonicus peaked at approximately 10(5.5) PFU/ mosquito about 7 d after ingesting blood meal in which the concentration of LAC virus was PFU/ml of blood; virus had disseminated from the midgut in 100% (8/8) of these specimens. These data, combined with the close association between the habitats of Oc. j. japonicus and Oc. triseriatus and the reported expansion of the range of this newly discovered species in the eastern United States, indicate that Oc. j. japonicus could function as an additional vector of LAC virus.     

Larval diet and the vector competence of Culex annulirostris (Diptera: Culicidae) for Murray Valley encephalitis virus

Culex annulirostris Skuse larvae were reared on two different amounts of powdered dog chow and yeast, low intake (2.8 micrograms/larva) and high intake (8.4 micrograms/larva), to produce adults with mean wing lengths of 3.25 and 3.7 mm, respectively. The resulting adults were fed different dosages of Murray Valley encephalitis virus and after 10 d extrinsic incubation at 28 degrees C, the infection rate, transmission rate, salivary gland, and body titers were determined. There were no significant differences in any of these parameters.     

Experimental transmission of eastern equine encephalitis virus by Ochlerotatus j. japonicus (Diptera: Culicidae)

We evaluated the potential for Ochlerotatus j. japonicus (Theobald), a newly recognized invasive mosquito species in the United States, to transmit eastern equine encephalitis (EEE) virus. Aedes albopictus (Skuse) and Culex pipiens (L.) were similarly tested for comparison. Ochlerotatus j. japonicus and Ae. albopictus became infected and transmitted EEE virus by bite after feeding on young chickens 1 d after they had been inoculated with EEE virus (viremias ranging from 10(7.0-8.7) plaque-forming units [PFU]/ml of blood). No Cx. pipiens (n = 20) had detectable levels of virus 14 d after feeding on an EEE-virus infected chicken with a viremia of 10(8.1) PFU per ml of blood. Depending on the viral titer in the donor chicken, infection rates ranged from 55-100% for Oc. j. japonicus and 93-100% for Ae. albopictus. In these two species, dissemination rates were identical to or nearly identical to infection rates. Depending on the viral titer in the blood meal, estimated transmission rates ranged from 15 to 25% for Oc. j. japonicus and 59-63% for Ae. albopictus. Studies of replication of EEE virus in Oc. j. japonicus showed that there was an "eclipse phase" in the first 4 d after an infectious blood meal, that viral titers peak by day 7 at around 10(5.7) per mosquito, and that virus escaped the mid-gut as soon as 3 d after the infectious blood meal. These data, combined with the opportunistic feeding behavior of Oc. j. japonicus in Asia and the reported expansion of its range in the eastern United States, indicate that it could function as a bridge vector for EEE virus between the enzootic Culiseta melanura (Coquillett)-avian cycle and susceptible mammalian hosts.     

Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus

Australian mosquitoes were evaluated for their ability to become infected with and transmit a Torres Strait strain of Japanese encephalitis virus. Mosquitoes, which were obtained from either laboratory colonies and collected using Centers for Disease Control and Prevention light traps baited with CO2 and octenol or reared from larvae, were infected by feeding on a blood/sucrose solution containing 10(4.5 +/- 0.1) porcine stable-equine kidney (PS-EK) tissue culture infectious dose50/mosquito of the TS3306 virus strain. After 14 d, infection and transmission rates of 100% and 81%, respectively, were obtained for a southeast Queensland strain of Culex annulirostris Skuse, and 93% and 61%, respectively, for a far north Queensland strain. After 13 or more days, infection and transmission rates of > 90% and > or = 50%, respectively, were obtained for southeast Queensland strains of Culex sitiens Wiedemann and Culex quinquefasciatus Say, and a far north Queensland strain of Culex gelidus Theobald. Although infection rates were > 55%, only 17% of Ochlerotatus vigilax (Skuse) and no Cx. quinquefasciatus, collected from far north Queensland, transmitted virus. North Queensland strains of Aedes aegypti L., Ochlerotatus kochi (D?nitz), and Verrallina funerea (Theobald) were relatively refractory to infection. Vertical transmission was not detected among 673 F1 progeny of Oc. vigilax. Results of the current vector competence study, coupled with high field isolation rates, host feeding patterns and widespread distribution, confirm the status of Cx. annulirostris as the major vector of Japanese encephalitis virus in northern Australia. The relative roles of other species in potential Japanese encephalitis virus transmission cycles in northern Australia are discussed.     

Vector competence of Aedes vexans (Diptera: Culicidae) for West Nile virus and potential as an enzootic vector

Vector competence of Aedes vexans (Meigen) and Culex pipiens pipiens L. (Diptera: Culicidae) for West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was compared. Infection rates of both species were similar 14 d after feeding on chickens, with WNV titers ranging from 10(4.2) to 10(8.7) plaque-forming units (PFU)/ml. Median infectious doses and 95% confidence intervals (CI) were 10(6.0(5.8, 63)) and 10(5.7(5.4, 5.9)) PFU for Ae. vexans and Cx. p. pipiens, respectively. WNV transmission was not observed in Ae. vexans that fed on chickens with WNV titers < 10(5.0) PFU/ml, in contrast to a mean (95% CI) transmission rate of 7(2,18)% for Cx. p. pipiens. Mean WNV transmission rates for Ae. vexans and Cx. p. pipiens were 13(7,21)% and 10(5,19)%, respectively, after feeding on chickens with WNV titers of 10(5.3 +/- 0.1) and 10(5.7 +/- 0.1) PFU/ml, and 31(25,37)% and 41(30,53)% after feeding on chickens with WNV titers > or = 10(6.1 +/- 0.1) PFU/ml. Time postinfection (p.i.) significantly influenced WNV transmission by Ae. vexans as indicated by a nearly 10-fold increase in transmission rate between days 7 and 14 p.i. Mean WNV load expectorated with saliva ofAe. vexans was 10(2.4(2.1, 2.7)) PFU, and it was not significantly affected by the titer of chickens on which they originally fed or time p.i. These data indicate that vector competence of the primarily mammalophilic Ae. vexans, which also feeds on birds, approaches that of Cx. p. pipiens for WNV. Because peridomestic mammals, such as cottontail rabbits, squirrels, and chipmunks, develop WNV titers infective for Ae. vexans, this species may play a significant role in WNV enzootic cycles.     

Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus

We evaluated the potential for several North American mosquito species to transmit the newly introduced West Nile (WN) virus. Mosquitoes collected in the New York City metropolitan area during the recent WN virus outbreak, at the Assateague Island Wildlife Refuge, VA, or from established colonies were allowed to feed on chickens infected with WN virus isolated from a crow that died during the 1999 outbreak. These mosquitoes were tested approximately 2 wk later to determine infection, dissemination, and transmission rates. Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus (Theobald) were highly susceptible to infection, and nearly all individuals with a disseminated infection transmitted virus by bite. Culex pipiens L. and Aedes sollicitans (Walker) were moderately susceptible. In contrast, Aedes vexans (Meigen), Aedes aegypti (L.), and Aedes taeniorhynchus (Wiedemann) were relatively refractory to infection, but individual mosquitoes inoculated with WN virus did transmit virus by bite. Infected female Cx. pipiens transmitted WN virus to one of 1,618 F1 progeny, indicating the potential for vertical transmission of this virus. In addition to laboratory vector competence, host-feeding preferences, relative abundance, and season of activity also determine the role that these species could play in transmitting WN virus.     

Culex restuans (Diptera: Culicidae) relative abundance and vector competence for West Nile Virus

The abundance and vector competence of Culex restuans Theobald and Culex pipiens L. were compared to determine the relative importance of these species as West Nile virus (WNV) vectors in the northeastern United States. Abundance was estimated from egg raft collections at 12 sites in Albany, Suffolk, and Richmond counties, New York, during July, August, and September 2002 and 2003. Cx. restuans was more abundant than Cx. pipiens in both urban and rural areas, comprising 86% of 1,623 egg rafts collected. Vector competence for WNV was estimated after feeding on an artificial bloodmeal and in vitro transmission assays. The vector competence of the two species for WNV was similar, but the dynamics of infection seems to be mosquito species dependent. These findings suggest an important role for Cx. restuans in WNV transmission cycles in New York.     

Establishment of Ochlerotatus japonicus (Diptera: Culicidae) in Ontario, Canada

Ochlerotatus (Finlaya) japonicus (Theobold) is newly established in Ontario, Canada. It was first discovered in 2001 during the province-wide West Nile virus mosquito surveillance program implemented by the Ontario Ministry of Health and Long Term Care. Although the numbers of adults trapped in CDC light traps were low in 2001 and 2002, they increased during the 2003 and 2004 surveillance seasons. Oc. japonicus larvae also have been collected in large numbers in the Niagara Peninsula in a variety of natural and artificial containers. The number of health units with records for Oc. japonicus has increased over the 4 yr of surveillance, illustrating the ability of this species to rapidly extend its range. As a potential arboviral bridge vector, its establishment in Ontario requires further study and should be considered a public health concern.     

Vector competence of selected African mosquito (Diptera: Culicidae) species for Rift Valley fever virus

Outbreaks of Rift Valley fever (RVF) in Egypt, Yemen, and Saudi Arabia have indicated the potential for this disease to spread from its enzootic areas in sub-Saharan Africa. Because little is known about the potential for most African mosquito species to transmit RVF virus (family Bunyaviridae, genus Phlebovirus, RVFV), we conducted studies to determine the vector competence of selected African species of mosquitoes for this virus. All eight species tested [Aedes palpalis (Newstead), Aedes mcintoshi Huang, Aedes circumluteolus (Theobald), Aedes calceatus Edwards, Aedes aegypti (L.), Culex antennatus (Becker), Culex pipiens (L.), and Culex quinquefasciatus Say], were susceptible to infection, and all except Ae. calceatus, Ae. aegypti and Cx. quinquefasciatus transmitted RVFV by bite after oral exposure. Estimated transmission rates for mosquitoes that successfully transmitted RVFV by bite ranged from 5% for Ae. mcintoshi to 39% for Ae. palpalis for mosquitoes that fed on a hamster with a viremia > or = 10(8) plaque-forming units of virus/ml. We did not recover RVFV from any of 3,138 progeny of infected female mosquitoes. RVFV is unusual among arboviruses in that it has been isolated in nature from a large number of species and that numerous mosquitoes and other arthropods are able to transmit this virus in the laboratory. The recent introduction and spread of West Nile virus into the Americas and the spread of RVFV to the Arabian Peninsula illustrates the potential for viruses, once enzootic in Africa, to spread to other parts of the world.     

Aedes japonicus japonicus (Diptera: Culicidae) from Germany have vector competence for Japan encephalitis virus but are refractory to infection with West Nile virus

The interplay between arthropod-borne (arbo) viruses and their vectors is usually complex and often exert unique relationships. Aedes japonicus japonicus (Hulecoeteomyia japonica or Ochlerotatus japonicus japonicus), an invasive mosquito species with laboratory proven vector competence for a number of emerging viruses has been newly introduced to Germany and is currently expanding its range throughout the country. On the other hand, West Nile virus (WNV), an emerging arbovirus originating from Africa, is already circulating in several European countries and might soon be introduced to Germany. Because newly introduced and rapidly expanding vector species pose a potential risk for public health in Germany, we assessed the vectorial capacity of German Ae. j. japonicus populations for WNV and Japanese encephalitis virus (JEV). The results indicate that German Ae. j. japonicus are susceptible for JEV but are refractory to infection with WNV. Of 67 Ae. j. japonicus females challenged by feeding of WNV-containing blood, none had measurable amounts of WNV-RNA (0 % infection rate) on day 14 post-infection. In contrast, all females challenged with JEV were positive for JEV-RNA (100 % infection rate) on day 14 post-infection. The reason for WNV resistance remains to be determined but is independent from co-infection with other flaviviruses or the presence of endosymbiotic Wolbachia, since we found no evidence for other flavivirus infections within 1,033 tested A. j. japonicus females from the sampling region, nor detectable Wolbachia infection within 30 randomly selected individuals.     

Peritrophic matrix formation and Brugia malayi microfilaria invasion of the midgut of a susceptible vector, Ochlerotatus togoi (Diptera: Culicidae)

The mosquito midgut is the first site that vector-borne pathogens contact during their multiplication, differentiation, or migration from blood meal to other tissues before transmission. After blood feeding, the mosquitoes synthesize a chitinous structure called peritrophic matrix (PM) that envelops the blood meal and separates the food bolus from the midgut epithelium. In this study, a systematic investigation of the PM formation and the interaction of Brugia malayi within the midgut of a susceptible vector, Ochlerotatus togoi, were performed using scanning electron microscopy (SEM). SEM analysis of the midguts dissected at different time points post feeding on a B. malayi-infected blood meal (PIBM) revealed that the PM was formed from 45 min PIBM and gradually thickened and matured during 8–18 h PIBM. The PM degraded from 24 to72 h PIBM, when digestion was completed. The invasion process of the microfilariae was observed between 3 and 4 h PIBM. In the beginning of the process, only sheathed microfilariae interacted with the internal face of the PM by its anterior part, and then the midgut epithelium before entering the hemocoel, after that they exsheathed. Microfilarial sheaths lying within the hemocoel were observed suggesting that they may serve as a decoy to induce the immune systems of the mosquitoes to respond to the antigens on the sheaths, thereby protecting the exsheathed microfilariae. These initial findings would lead to further study on the proteins, chemicals, and factors in the midgut that are involved in the susceptibility of O. togoi as a vector of filariasis.     

Effects of temperature on the transmission of west nile virus by Culex tarsalis (Diptera: Culicidae)

Culex tarsalis Coquillett females were infected with the NY99 strain of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) and then incubated under constant temperatures of 10-30 degrees C. At selected time intervals, transmission was attempted using an in vitro capillary tube assay. The median time from imbibing an infectious bloodmeal until infected females transmitted WNV (median extrinsic incubation period, EIP50) was estimated by probit analysis. By regressing the EIP rate (inverse of EIP50) as a function of temperature from 14 to 30 degrees C, the EIP was estimated to require 109 degree-days (DD) and the point of zero virus development (x-intercept) was estimated to be 14.3 degrees C. The resulting degree-day model showed that the NY99 WNV strain responded to temperature differently than a lineage II strain of WNV from South Africa and approximated our previous estimates for St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). The invading NY99 WNV strain therefore required warm temperatures for efficient transmission. The time for completion of the EIP was estimated monthly from temperatures recorded at Coachella Valley, Los Angeles, and Kern County, California, during the 2004 epidemic year and related to the duration of the Cx. tarsalis gonotrophic cycle and measures of WNV activity. Enzootic WNV activity commenced after temperatures increased, the duration of the EIP decreased, and virus potentially was transmitted in two or less gonotrophic cycles. Temperatures in the United States during the epidemic summers of 2002-2004 indicated that WNV dispersal and resulting epicenters were linked closely to above-average summer temperatures.     

Population genetics of Ochlerotatus eatoni (Diptera: Culicidae) endemic species to two Macaronesian Islands

Analyses of 11 isoenzyme loci of Ochlerotatus eatoni (Edwards, 1916), endemic to two Macaronesian Islands (Madeira and Tenerife, Canary Islands), revealed substantial genetic structure in the study populations. Samples from sites on the south and north of Madeira displayed a significant reduction of variability compared with those from central Madeira and Tenerife. The Tenerife population exhibited a severe deficit of heterozygosity with similar magnitude across all the loci examined. The complex pattern of variation in Oc. eatoni is because of interplay of breeding structure, genetic drift, and geographical and historical factors. From these findings, we concluded that island colonization by Oc. eatoni was not marked by founder effect.     

Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission

The ability of the invading NY99 strain of West Nile virus (WNV) to elicit an elevated viremia response in California passerine birds was critical for the effective infection of Culex mosquitoes. Of the bird species tested, Western scrub jays, Aphelocoma coerulescens, produced the highest viremia response, followed by house finches, Carpodacus mexicanus, and house sparrows, Passer domesticus. Most likely, few mourning, Zenaidura macroura, or common ground, Columbina passerine, doves and no California quail, Callipepla californica, or chickens would infect blood-feeding Culex mosquitoes. All Western scrub jays and most house finches succumbed to infection. All avian hosts produced a lower viremia response and survived after infection with an endemic strain of St. Louis encephalitis virus. Culex species varied in their susceptibility to infection with both viruses, with Culex stigmatosoma Dyar generally most susceptible, followed by Culex tarsalis Coquillett, and then Culex p. quinquefasciatus Say. Populations within Culex species varied markedly in their susceptibility, perhaps contributing to the focality of WNV amplification. Transmitting female Cx. tarsalis expectorated from six to 3,777 plaque-forming units (PFU) of WNV during transmission trials, thereby exposing avian hosts to a wide range of infectious doses. Highly susceptible house finches and moderately susceptible mourning doves were infected by subcutaneous inoculation with decreasing concentrations of WNV ranging from 15,800 to <0.3 PFU. All birds became infected and produced comparable peak viremias on days 2-3 postinoculation; however, the rise in viremia titer and onset of the acute phase of infection occurred earliest in birds inoculated with the highest doses. WNV virulence in birds seemed critical in establishing elevated viremias necessary to efficiently infect blood feeding Culex mosquitoes.     

Vector competence of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) for dengue virus in the Florida Keys

In 2009-2011, Monroe County in southern Florida experienced locally acquired and traveler-imported focal dengue outbreaks. Aedes aegypti (L.) is the primary vector of dengue virus (DENV) worldwide, is prevalent in Monroe County, and is the suspected vector in Florida. Ae. albopictus (Skuse) is also known to be an important vector of DENV and this species is ubiquitous in Florida; however, it is not yet established in Monroe County. Florida Ae. aegypti (Key West and Stock Island geographic colonies) and Ae. albopictus (Vero Beach geographic colony) were fed blood containing 3.7 Log10 plaque-forming unit equivalents of DENV serotype 1 isolated from a patient involved in the Key West, FL, outbreak in 2010. Mosquitoes were maintained at extrinsic incubation temperatures of 28 or 30 degrees C for an incubation period of 14 d. Vector competence was assessed using rates of infection (percent with virus-positive bodies), dissemination (percent infected with virus-positive legs), and transmission (percent infected with virus-positive saliva). No significant differences were observed in rates of infection or dissemination between Ae. aegypti or Ae. albopictus at either extrinsic incubation temperature. Transmission was observed only at 28 degrees C in both Ae. aegypti (Key West) and Ae. albopictus. The assessment of local mosquito populations for their DENV vector competence is essential and will aid mosquito control operators interested in pinpointing specific vector populations for control. The extent to which vector competence is affected by seasonal changes in temperature is discussed and provides baseline risk assessment data to mosquito control agencies.