Isolations of Jamestown Canyon virus (Bunyaviridae: Orthobunyavirus) from field-collected mosquitoes (Diptera: Culicidae) in Connecticut, USA: a ten-year analysis, 1997-2006

Jamestown Canyon virus (JCV) (Bunyaviridae: Orthobunyavirus) is a mosquito-borne zoonosis belonging to the California serogroup. It has a wide geographic distribution, occurring throughout much of temperate North America. White-tailed deer, Odocoileus virginianus are the principal amplification hosts, and boreal Aedes and Ochlerotatus mosquitoes are the primary vectors. A 10-year study was undertaken to identify potential mosquito vectors in Connecticut, quantify seasonal prevalence rates of infection, and define the geographic distribution of JCV in the state as a function of land use and white-tailed deer populations, which have increased substantially over this period. Jamestown Canyon virus was isolated from 22 mosquito species. Five of them, Ochlerotatus canadensis, Oc. cantator, Anopheles punctipennis, Coquillettidia perturbans, and Oc. abserratus were incriminated as the most likely vectors, based on yearly isolation frequencies and the spatial geographic distribution of infected mosquitoes. Jamestown Canyon virus was isolated from Oc. canadensis more consistently and from a greater range of collection sites than any other species. Frequent virus isolations were also made from Aedes cinereus, Aedes vexans, and Oc. sticticus, and new North American isolation records were established for Anopheles walkeri, Culex restuans, Culiseta morsitans, Oc. sticticus, Oc. taeniorhynchus, and Psorophora ferox. Other species from which JCV was isolated included C. melanura, Oc. aurifer, Oc. communis, Oc. excrucians, Oc. provocans, Oc. sollicitans, Oc. stimulans, Oc. triseriatus, and Oc. trivittatus. Jamestown Canyon virus was widely distributed throughout Connecticut and found to consistently circulate in a diverse array of mosquito vectors. Infected mosquitoes were collected from June through September, and peak infection rates paralleled mosquito abundance from mid-June through mid-July. Infection rates in mosquitoes were consistent from year to year, and overall virus activity was directly related to local mosquito abundance. Infected mosquitoes were equally distributed throughout the state, irrespective of land use, and infection rates were not directly associated with the abundance of white-tailed deer, possibly because of their saturation throughout the region.     

Vector competence of California mosquitoes for California encephalitis and California encephalitis-like viruses.

Mosquitoes collected from coastal, inland valley, and alpine locations in California were evaluated for their experimental vector competence for two viruses in the California serogroup (Bunyaviridae:Bunyavirus). Aedes squamiger, a coastal salt marsh mosquito, was an efficient vector of a California encephalitis (CE)-like virus isolated from its habitat (89% of the pledget-fed females became infected and 61% transmitted virus). Aedes dorsalis, a coastal mosquito, and Ae. melanimon, an inland valley mosquito, were competent vectors of prototype CE virus (98% and 100% of the pledget-fed females became infected and 56% and 30%, respectively, transmitted virus). Aedes squamiger and Ae. dorsalis transmitted both viruses vertically to one or more of 20 of their progeny. Culiseta inornata was susceptible to infection with both viruses, but 5% or less transmitted virus perorally. Alpine mosquitoes, Ae. cataphylla, Ae. increpitus, and Ae. tahoensis, became infected with both CE and CE-like viruses, but 3% or less transmitted virus. All species of mosquitoes were more efficient vectors of both viruses following intrathoracic inoculation than following pledget feeding, suggesting the presence of mesenteronal barriers.     

Host preferences of Aedes trivittatus (Diptera: Culicidae) in central Iowa.

The vertebrate host preferences of Aedes trivittatus mosquitoes were studied to gain an insight into the possible hosts of trivittatus (TVT) virus (California encephalitis group) in Iowa. Engorged mosquitoes were collected with a Malaise trap and Dry Ice-baited CDC miniature light traps. The origin of mosquito blood meals was determined by the capillary tube precipitin test. Of 600 A. trivittatus blood meals tested, 409 (68.2%) reacted positively with anti-rabbit serum. The incidences of mosquitoes feeding on other vertebrate species ranged from 0.2% to 2.5%. The vertebrate host preferences of A. trivittatus suggest a close association between this mosquito species and the eastern cottontail rabbit (Sylvilagus floridanus) in central Iowa. Furthermore, the results indicate that the eastern cottontail rabbit may be an important host for TVT virus in Iowa.     

Detection of host virus-reactive antibodies in blood meals of naturally engorged mosquitoes

Although serosurvey in human or animals is a useful and straightforward strategy routinely used for public health, it often faces different types of impediments: ethics, beliefs, limitation by animal owners, hazard of access to wild animals. To survey virus circulation, we applied the enzyme-linked immunosorbent assay (ELISA) technique to detect Dengue and Japanese encephalitis (JE) virus-reactive antibodies in blood meals collected from mosquitoes without regard to the potential of mosquito species to be a virus vector. ELISA was performed on mosquito colonies and wild specimens collected from farms and urban areas. Blood meals from Aedes aegypti freshly fed on naturally infected volunteers showed the same levels of dengue immunoglobulin (Ig)G and IgM as the sera directly collected from volunteers. A significant clearance of antibodies during the digestion process started from 13 hours after blood meal, and a negative baseline was reached after 30 hours. The ELISA test performed on wild mosquitoes showed that 37% of Culex quinquefasciatus mosquitoes that engorged on humans in a dengue urban endemic area tested positive for dengue IgG, and in a JE virus-endemic area, 88% of Culex tritaeniorhynchus mosquitoes that engorged on pigs from a large pig farm tested positive for JE virus antibodies versus 11% in a small farm. The main limitation of the ELISA method is the antibody cross-reactivity among flaviviruses; also, sampling strategy should be adjusted to take into account that the actual host from which the blood meal was taken may not be determined. Nevertheless, ELISA performed on recently (1-2 days) engorged mosquito, or any other hematophagous arthropod species, could potentially be used as a "wild phlebotomist" to monitor the prevalence or emergence of a variety of pathogens, with less of the practical, ethical, or risk limitations due to direct blood collection from humans and wild or domestic animals.     

Blood meal size as a factor affecting continued host-seeking by Aedes aegypti (L.)

The effect of ingested blood on the host-seeking response of two strains of Aedes aegypti was examined. Using an olfactometer, females fed partial blood meals were scored for host-seeking behavior within 1 h, and their blood meal sizes were measured chemically immediately afterwards. The suppression of host-seeking within 1 h after a blood meal appears to be caused by abdominal distention from ingested blood. Mosquitoes of either strain were attracted to a host when the blood meal size was less than 2.5 microliter; above this threshold there was a sharp decline in the tendency to respond. Small mosquitoes resulting from a low larval diet had a lower threshold, and were more likely to cease host-seeking after a small blood meal. Multiple feeding within a single gonotrophic cycle may result if mosquitoes take small blood meals which are insufficient to terminate host-seeking. Partial meals and reduced feeding success of mosquitoes can result from defensive host behavior, which in the laboratory rat was shown to increase at high mosquito densities.     

Review: artificial container-breeding mosquitoes and cemeteries: a perfect match

Artificial container-breeding mosquitoes, such as Aedes aegypti, Ae. albopictus, and Culex pipiens, are well-recognized vectors of diseases throughout the world. Cemeteries are considered major sources of mosquitoes and the results of more than 30 studies concerning mosquitoes in cemeteries have been published over the last decade. The characteristics of these environments in regard to the availability of resources for mosquito development were discussed. Also, studies about early detection of Aedes vectors, ecological issues, and mosquito control performed in cemeteries were reviewed. Among 31 mosquito species found breeding in cemeteries from 16 countries, the invasive Ae. aegypti and Ae. albopictus were the most frequent ones. Species of the genus Ochlerotatus, Culex, Toxorhynchites, Culiseta, Armigeres, Lutzia, Uranotaenia, and Tripteroides were also reported. Overall, cemeteries are highly suitable habitats for artificial container-breeding mosquitoes due to the great availability of the different resources that they need (i.e. sugar substances, blood, shelter and water-filled containers). In addition, these places are mostly ideal settings to perform studies in urbanized areas because of high mosquito abundance, heterogeneity of macro- and microhabitats, and an easier access in comparison with private premises. However, the feasibility of a cemetery as a study area must be evaluated in each case considering the objectives of the study and cemetery characteristics.     

Identification of mammalian blood meals in mosquitoes by a multiplexed polymerase chain reaction targeting cytochrome B

To date, no polymerase chain reaction diagnostic technique exists to directly identify mammalian blood meals from mosquitoes by sized DNA fragments following agarose gel electrophoresis. We have developed a vertebrate-specific multiplexed primer set based on mitochondrial cytochrome b to identify the mammalian blood hosts of field-collected mosquitoes. Although designed for the study of African malaria vectors, the application of this tool is not restricted to this disease system. Validation of this diagnostic technique on dried anopheline and culicine field specimens collected in Zambia and Mali demonstrated that blood meals could be identified 2-7 months after collection. Time course experiments showed that host DNA was detectable in frozen mosquito abdomens 24-30 hours post-feeding. Additionally, multiple blood meals from different mammals could be detected in a single mosquito. This diagnostic assay will be a valuable tool for identifying the blood meals of field-collected mosquitoes where people and alternative mammal hosts are present.     

Pathologic changes in the midgut of Culex tarsalis following infection with Western equine encephalomyelitis virus.

Midguts of two strains of the mosquito vector Culex tarsalis were examined by light and electron microscopy following infection with western equine encephalomyelitis virus. Infection of the highly susceptible Knight's Landing strain with high-titered blood meals resulted in pathologic changes in the midgut epithelium after 2-4 days of incubation; lesions included sloughing of epithelial cells into the lumen and necrosis of cells in situ. Infection of Knight's Landing strain mosquitoes with low-titered blood meals and infection of the less susceptible Fort Collins strain with high-titered blood meals did not result in a significant increase in detached luminal cells, with respect to uninfected controls. Sloughing of infected cells into the midgut lumen may contribute to modulation of the mosquito infection. Lesions in the midgut of Cx. tarsalis are inconsistent with traditional views that regarded arbovirus infections of mosquito vectors as non-pathologic. These findings demonstrate that mosquito pathology is not an oddity limited to the previously described interaction between Culiseta melanura and eastern equine encephalomyelitis virus, and suggest that alphaviruses in general may adversely affect their mosquito vectors in nature.     

Detection and monitoring of mosquito flaviviruses in Spain between 2001 and 2005

Between the years 2001 and 2005, a total of 72,895 female mosquitoes were trapped during their season of abundance, and analyzed. They were sorted into 4,723 pools belonging to 20 Culicidae species from the Anopheles, Aedes, Ochlerotatus, Culex, Culiseta, Coquillettidia, and Uranotaenia genera. The aim was to detect arboviral RNA directly from mosquito homogenates for the genera Alphavirus, Flavivirus, and Phlebovirus. The study formed part of general arbovirus transmission research in four of the most important wetlands in Spain; in the provinces of Girona, Barcelona, Tarragona, and Huelva. The mosquitoes were collected using human bait, CO(2) traps, or light traps, and they were pooled according to date of collection, location, and species. No arboviral RNA from known pathogenic arboviruses was found. However, 111 pools tested positive for unknown mosquito Flavivirus, the only genus detected. The Flavivirus sequences identified were different from all known Flavivirus mosquito viruses, but very close to Kamiti River virus or cell fusing agent virus. The maximum likelihood estimation infection rate (MLE) was calculated for all regions and species. Aedes albopictus had the highest MLE at 47.14, followed by Ae. vexans with 43.67 (over the entire area). These species were followed by Culiseta annulata, with 36.00. The most common species, Ochlerotatus caspius and Culex pipiens, had low MLE values-0.94 and 0.38, respectively-over the area as a whole.     

Molecular identification of blood-meal sources in Culiseta melanura and Culiseta morsitans from an endemic focus of eastern equine encephalitis virus in New York

Eastern equine encephalitis (EEE) virus perpetuates in an enzootic cycle involving ornithophilic mosquito vectors, principally Culiseta melanura (Coquillett) and avian amplification hosts. To better understand the role of Cs. melanura and Culiseta morsitans (Theobald) in the epizootiology of EEE virus, we collected blood-fed mosquitoes between 31 May and 15 October 2004 at two sites associated with an EEE virus focus in central New York and identified the source of vertebrate blood by nucleotide sequencing of polymerase chain reaction (PCR) products of the cytochrome b gene. Analysis of 484 Cs. melanura and 122 Cs. morsitans revealed that 94.2% and 86.9%, respectively, acquired blood solely from avian hosts. Blood meals derived exclusively from mammals were detected in 0.8% of Cs. melanura and 1.6% of Cs. morsitans. Individual mosquitoes containing mixed-blood meals from both avian and mammalian hosts were also detected in 5.0% of Cs. melanura and 11.5% of Cs. morsitans. Wood thrush constituted the most common vertebrate host for Cs. melanura (23.6%) and Cs. morsitans (30.9%), followed by American robin, song sparrow, ovenbird, red-eyed vireo, and common yellowthroat. Mammalian-derived blood meals were identified as white-tailed deer, horse, domestic cat, and eastern pipistrelle bat. There were three isolations of EEE virus from Cs. melanura and one from Cs. morsitans. These results suggest that wood thrush and a few other passerine birds may play key roles in supporting EEE virus transmission in the northeast and possibly throughout the geographic range of EEE in North America. The frequency of mammalian feedings also suggests that Cs. melanura and Cs. morsitans may play a role in the transmission of EEE virus to equines, in addition to maintaining enzootic transmission among avian hosts. We report the first isolation of arboviruses from mosquito vectors concomitant with the identifications of their blood meal sources.     

2019—2020年哈密市蚊虫种类分布及携带病毒调查

目的　了解新疆维吾尔自治区哈密市蚊虫种类及病毒携带情况,并鉴定该市尖音库蚊所属亚种。方法　分别于2019、2020年7月中旬,在新疆维吾尔自治区哈密市伊州区、伊吾县、巴里坤县采用诱蚊灯法捕捉蚊虫,鉴定所捕获蚊虫种属及尖音库蚊种属。采用逆转录PCR技术检测捕获蚊虫携带黄病毒属、甲病毒属、布尼亚病毒属、流行性乙型脑炎病毒、辽宁病毒、Tahyna病毒、蜱传脑炎病毒及西尼罗病毒情况。结果　在哈密市伊州区、伊吾县、巴里坤县共捕获蚊虫1 496只,分属于3属3种。尖音库蚊为优势蚊种,占所捕获蚊虫总数的65.91%（986只）;其次为里海伊蚊,占30.55%（457只）;阿拉斯加脉毛蚊最少,仅占3.54%（53只）。所捕获尖音库蚊经雄蚊尾器鉴定为尖音库蚊指名亚种。经逆转录PCR法检测,所捕获蚊虫黄病毒属、甲病毒属、布尼亚病毒属、流行性乙型脑炎病毒、辽宁病毒、Tahyna病毒、蜱传脑炎及西尼罗病毒均为阴性。结论　2019—2020年哈密市存在尖音库蚊、里海伊蚊、阿拉斯加脉毛蚊3种蚊虫,以尖音库蚊为优势蚊种;所捕获尖音库蚊均为指名亚种,未发现蚊类携带相关虫媒病毒。     

Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegypti

Reverse genetics is a powerful tool for understanding gene functions and their interactions in the mosquito innate immunity. We took the transgenic approach, in combination with the RNA interference (RNAi) technique, to elucidate the role of mosquito REL1, a homolog of Drosophila Dorsal, in regulation of Toll immune pathway in the mosquito Aedes aegypti. By transforming the mosquitoes with DeltaREL1-A or a double-stranded RNA construct of REL1 driven by the female fat body-specific vitellogenin (Vg) promoter with the pBac[3xP3-EGFP, afm] vector, we generated two different transgenic mosquito strains, one with overexpressed AaREL1 and the second with AaREL1 knockdown. Both strains had a single copy of the respective transgene, and the expression in both transgenic mosquitoes was highly activated by blood feeding. Vg-DeltaREL1-A transgenic mosquitoes activate Toll immune pathway in the fat body by blood feeding. The overexpression of both isoforms, AaREL1-A and AaREL1-B, in Vg-DeltaREL1-A transgenic mosquitoes resulted in the concomitant activation of Aedes Sp?tzle1A and Serpin-27A, independent of septic injury. The same phenotype was observed in the mosquitoes with RNAi knockdown of an Aedes homolog to Drosophila cactus, an IkappaB inhibitor of Drosophila Toll pathway. The effect of the transgenic RNAi knockdown of AaREL1 on mosquito innate immunity was revealed by increased susceptibility to the entomopathogenic fungus Beauveria bassiana and the reduced induction of Spz1A and Serpin-27A gene expression after fungal challenge. These results have proven that AaREL1 is a key downstream regulator of Toll immune pathway in the mosquito A. aegypti.     

Antioxidative systems defense against oxidative stress induced by blood meal in Aedes aegypti

The release of iron from hemoglobin via the digestion of a blood meal in female mosquitoes can potentially induce oxidative damage and even death. These mosquitoes need an effective antioxidant to prevent this. We carried out this study to determine the antioxidant activities of ferritin, glutathione peroxidase (GPx), glutathione S-transferase (GST) and catalase, and glutathione (GSH). These enzymes had their greatest activity among 4 day old virgin female mosquitoes. Using a single blood feed model, groups of female mosquitoes were tested at 4, 7 and 20 days post-emergence. They were allowed to feed on a hamster for 1 hour. The engorged mosquitoes were collected at 48 and 72 hours after their blood meal. There were no changes in GSH, GPx, GST or catalase levels, but ferritin levels increased markedly (about 2-3 fold) by 48 hours post blood-feed in all mosquito age groups. On repeated blood-feed experiments, mosquitoes aged 4 days were blood fed, once every 3 days and were collected 48 hours after their most recent blood meal. A significant decrease in GSH and GPx activity and a further increase in ferritin, were detected. Ferritin levels were 0.19+/-0.03 and 0.14+/-0.02 ng/microg protein in the repeat and single blood-feed groups, respectively. These results suggest ferritin is an inducible, sensitive defense system protecting against oxidative stress caused by iron derived from blood meals in Aedes aegypti mosquitoes.     

Identification of mosquito blood meals by enzyme-linked immunosorbent assay

A micro enzyme-linked immunosorbent assay for identifying mosquito blood meals is described. Antisera for the assay were prepared in rabbits and guinea pigs and purified by precipitation with cross-reacting blood sera. Positive blood source identifications could be made to the generic level using as little as 58 micrograms of ingested blood serum. The blood meals of 37 of 38 laboratory mosquitoes fed on seven host animals were correctly identified, including a mosquito that contained a double feed.     

Experimental transmission of a microsporidian pathogen from mosquitoes to an alternate copepod host.

Meiospores of a microsporidian parasite Amblyospora sp. (Protozoa: Microspora) from larval Aedes cantator mosquitoes were directly infectious to an alternate copepod host, Acanthocyclops vernalis (Arthropoda: Crustacea). Infections ranged from 6.7% to 60.0% in laboratory tests when meiospores and copepods were maintained together for 10-30 days in filtered water from the breeding site or in a balanced salt solution. Pathogen development takes place within host adipose tissue and is fatal to the copepod. The entire developmental sequence of this microsporidian in the copepod is unikaryotic and there is no ultrastructural evidence of a sexual cycle or a restoration of the diploid condition in the alternate host. Single uninucleated spores similar to those previously described for the genus Pyrotheca are formed. Results demonstrate that haploid meiospores of Amblyospora from mosquitoes have the function of transmitting the pathogen to another host and that members of this genus are polymorphic and have at least three distinct developmental cycles, each producing a different spore.     

Preliminary investigations on fauna of mosquitoes (Culicinae) in Lódź and environs

Species composition of mosquitoes (Diptera, Culicinae) was investigated in 2000 year. The mosquitoes were caugh from april till october, twice a month, at 6 stations. Seven species from genus Aedes were found: A. beclemishevi, A. ciprinus, A. cantans, A. flavescens, A. communis A. punctor, and A. vexans. A. cantans was most numerous (32,4). From genus Culex only one soecies was found (Culex pipiens). Furthermoce, Mansonia richardii and Culiseta annulata were caugh. The agresiveness toward men showed by mosquitoes was highest in august.     

Identification of mosquito avian-derived blood meals by polymerase chain reaction-heteroduplex analysis

A polymerase chain reaction (PCR) heteroduplex assay (HDA) was developed to identify avian derived mosquito blood meals to the species level. The assay used primers amplifying a fragment of the cytochrome B gene from vertebrate but not invertebrate species. In Culex tarsalis fed on quail, PCR products derived from the quail cytochrome B gene were detected seven days post-engorgement. In an analysis of wild-caught mosquitoes, 85% of blood-fed mosquitoes produced detectable PCR products. Heteroduplex patterns obtained from bird-derived PCR products were found to permit the unambiguous identification of all species examined. No intraspecific variation in HDA patterns was found. The PCR-HDA was used to characterize blood meals in wild caught Cx. tarsalis. Of the 67 blood meals analyzed, 60% were derived from avian sources. Of the avian blood meals, 65% were derived from a single host, the common grackle.     

Nutritional stress affects mosquito survival and vector competence for West Nile virus

Most anautogenous female mosquitoes ingest plant carbohydrates for flight energy and survival, and they imbibe vertebrate blood for egg development. We evaluated the effect of different sucrose meals following a blood meal containing West Nile virus (WNV) on Culex pipiens pipiens survival, nutritional status, and susceptibility to viral infection and transmission. Ten days after blood feeding, no mosquitoes survived on distilled water, 55% survived on 2% sucrose, 61% on 10 and 20% sucrose meals, and over 70% survived on 40% sucrose. There was a positive correlation between sucrose meal concentration and detectable sugars, glycogen, and lipid in whole-body homogenates. Average sugar values increased from 0 microg per starved mosquito (range 0-1.0 microg) to an average of 392 microg per mosquito fed on 40% sucrose (85-1088 microg). Average glycogen values increased from 0 microg (0-5.7 microg) to an average of 620 microg (118-1421 microg). Average lipid values were identical for mosquitoes in the starved and 2% sucrose series (38 microg) and increased to 172 microg per mosquito fed on 40% sucrose (92-266 microg). Mosquitoes in all sucrose series were equally susceptible to WNV infection (p > 0.5), but mosquitoes with lower nutrient reserves as a result of lower sucrose meals were more likely to orally transmit virus (p < 0.05). We discuss how mosquito nutritional status influences probability of daily survival, susceptibility to infection, and vectorial capacity. We conclude that maintaining C. p. pipiens on standard 10% sucrose is justified in light of these results.     

Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1–4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.