Dissemination of western equine encephalomyelitis virus in the potential vector,Culex pipiens pallens

Two western equine encephalomyelitis virus (WEEV) strains have been isolated in China. Our previous studies have verified that the mosquito Culex pipiens pallens Coquillett (Diptera: Culicidae) infected with WEEV was capable of transmitting this arbovirus, but it was not clear how the sequential multiplication and spread of virus occurred within the mosquito. In this study, we observed the distribution of WEEV antigen in orally‐infected Cx. p. pallens by immunohistochemistry in order to better understand the initial infection, dissemination, and transmission of WEEV in the potential vector. Orally‐infected WEEV dissemination varied within the different tissues of Cx. p. pallens, with virus antigen consistently observed in the salivary glands, foregut, midgut epithelial cells, Malpighian tubules, hindgut, and ovarian follicles of some individuals after various days of extrinsic incubation. We suggest that Cx. p. pallens, the potential vector of WEEV, has the ability to harbor the virus through the alimentary system, and the midgut epithelial cell may be the initial site of WEEV replication after ingestion of a viremic blood meal.     

New evidence for the potential role of Culex pipiens mosquitoes in the transmission cycle of West Nile virus in Tunisia

Physiological and molecular characteristics of natural populations of Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) were investigated to elucidate how this species is potentially involved in the transmission of West Nile virus in Tunisia. A total of 215 Cx. pipiens females from 11 breeding habitats were analysed in the laboratory to estimate autogeny and stenogamy rates. They were tested individually for the locus CQ11 to distinguish between the two Cx. pipiens forms, pipiens and molestus. All tested Cx. pipiens populations were stenogamous. Females from underground breeding sites were all autogeneous, whereas females from above‐ground habitats were mostly anautogeneous. Of all the females tested, 59.7% were identified as pipiens, 22.4% as molestus, and 17.9% as hybrid pipiens/molestus. Furthermore, both Cx. pipiens forms and their hybrids were found to co‐occur in sympatry in all sites. The results of this study represent the first evidence that both Cx. pipiens forms and their hybrids are present in Tunisia. Because hybrids able to act as bridge vectors are present in all studied habitats, Tunisia can be considered to have a high degree of receptivity for the establishment of West Nile virus zoonotic cycles.     

Microsatellite loci from the northern house mosquito (Culex pipiens), a principal vector of West Nile virus in North America

Microsatellites were isolated and characterized in the northern house mosquito, Culex pipiens, a widespread pest species and important vector of diseases such as West Nile virus. An enrichment protocol yielded 150 positive clones. We designed primers to amplify 17 unique (GT)_n microsatellites, eight of which amplified cleanly and were polymorphic. A survey of 29 individuals showed that these loci are highly variable with the number of alleles ranging from seven to 19 and expected heterozygosity ranging from 0.66 to 0.93. These markers will be useful for studies of population structure and intraspecific variation in epidemiological characteristics of Cx. pipiens.     

West Nile virus cluster analysis and vertical transmission in Culex pipiens complex mosquitoes in Sacramento and Yolo Counties,California, 2011

West Nile virus (WNV) is now endemic in California, with annual transmission documented by the statewide surveillance system. Although much is known about the horizontal avian‐mosquito transmission cycle, less is known about vertical transmission under field conditions, which may supplement virus amplification during summer and provide a mechanism to infect overwintering female mosquitoes during fall. The current study identified clusters of WNV‐infected mosquitoes in Sacramento and Yolo Counties, CA, during late summer 2011 and tested field‐captured ovipositing female mosquitoes and their progeny for WNV RNA to estimate the frequency of vertical transmission. Space‐time clustering of WNV‐positive Culex pipiens complex pools was detected in the northern Elk Grove area of Sacramento County between July 18 and September 18, 2011 (5.22 km radius; p<0.001 and RR=7.80). Vertical transmission by WNV‐infected females to egg rafts was 50% and to larvae was 40%. The estimated minimal filial infection rate from WNV‐positive, ovipositing females was 2.0 infected females/1,000. The potential contribution of vertical transmission to WNV maintenance and amplification are discussed.     

Avian defensive behavior and blood-feeding success of the West Nile vector mosquito, Culex pipiens

Avian defensive behavior against host-seeking arthropods influencestransmission of vector-borne pathogens by affecting mosquitobiting rate, either by preventing vector–host contactor by increasing the rate of multiple host feeding. We exposedhouse sparrows (Passer domesticus L.) and chickens (Gallus gallusdomesticus L.) to Culex pipiens pipiens L. overnight in a largeobservation cage and measured avian defensive behavior ratesand mosquito blood-feeding success. Both bird species exhibiteda range of defensive behaviors, 90% of which were foot stomps,head movements, and wing shakes. Total behavior rates increasedproportionately with mosquito density in both species, increasedafter the first hour of mosquito exposure, and decreased asindividual birds were exposed to mosquitoes multiple times.Mosquito blood-feeding success on house sparrows was high overall(82 ± 5%) and independent of behavior rates. Blood-feedingsuccess on chicks was lower (58 ± 5%) and negativelycorrelated with defensive behavior rate after the first hourof mosquito exposure. Results revealed a higher percentage ofpartial blood meals on chicks (18 ± 3% of all blood mealson chicks) than on house sparrows (4.9 ± 3%). Birds ofboth species ate an average of 9.4 ± 1.2% of mosquitoes,and this percentage was positively correlated with defensivebehavior. High mosquito feeding success on house sparrows supportsits role as a potential amplifying host of West Nile virus.     

Evidence for a population expansion in the West Nile virus vector Culex tarsalis

Population genetic structure of the West Nile Virus vector Culex tarsalis was investigated in 5 states in the western United States using 5 microsatellite loci and a fragment of the mitochondrial reduced form of nicotinamide adenine dinucleotide dehydrogenase 4 (ND4) gene. ND4 sequence analysis revealed a lack of isolation by distance, panmixia across all populations, an excess of rare haplotypes, and a star-like phylogeny. Microsatellites revealed moderate genetic differentiation and isolation by distance, with the largest genetic distance occurring between populations in southern California and New Mexico (F(ST) = 0.146). Clustering analysis and analysis of molecular variance on microsatellite data indicated the presence of 3 broad population clusters. Mismatch distributions and site-frequency spectra derived from mitochondrial ND4 sequences displayed pattern's characteristic of population expansion. Fu and Li's D* and F*, Fu's F(S), and Tajima's D statistics performed on ND4 sequences all revealed significant, negative deviations from mutation-drift equilibrium. Microsatellite-based multilocus heterozygosity tests showed evidence of range expansion in the majority of populations. Our results suggest that C. tarsalis underwent a range expansion across the western United States within the last 375,000-560,000 years, which may have been associated with Pleistocene glaciation events that occurred in the midwestern and western United States between 350,000 and 1 MYA.     

淡色库蚊对四种化学物质的嗅觉反应

在室内条件下通过Y型嗅觉仪测试了淡色库蚊Culexpipiens pallens雌成虫对乳酸、丙酮、氨水、辛醇、正庚酸、对甲酚、间甲酚等7种化学物质的嗅觉反应。结果表明:与对照相比,1和10mg/L氨水,1,10和100mg/L正庚酸,1mg/L辛醇,0.1和1mg/L对甲酚对淡色库蚊雌成虫具有显著的引诱作用。比较各处理的相对引诱率,最高的为10mg/L正庚酸,达50%以上;其他处理相对引诱率均低于40%,最低的为0.1mg/L对甲酚。     

西尼罗病毒的RT-PCR检测与鉴定

建立西尼罗病毒敏感、特异、快速的RT-PCR检测方法用于实验室诊断和流行病学监测。采用一步RT-PCR和套式PCR法对西尼罗病毒感染的乳鼠脑和细胞培养上清进行扩增,并对扩增产物进行序列测定。两种方法均可分别从两种组织中扩增出与预期大小相一致的片段,套式PCR法比一步RT-PCR法更为敏感,该扩增片段与西尼罗病毒埃及Eg101株相应序列的同源性为99％。     

Microsatellite loci for the white-dotted mosquito (Culex restuans), a principal vector of West Nile virus in North America

We characterized the first microsatellite loci in the white-dotted mosquito, Culex restuans, a critical early spring West Nile virus vector. An enrichment protocol yielded 960 positive clones of which we sequenced 300. We designed primers to amplify 29 unique di-, tri- and tetranucleotide microsatellites and chose 17 that amplified consistently across populations and were polymorphic. We developed three multiplex primer combinations for all 17 loci. A survey of 44 individuals revealed two to 20 alleles across loci, and expected heterozygosity ranging from 0.17 to 0.89. These markers will allow examination of the life history of this mysterious early season encephalitis vector.     

Polymorphic microsatellite loci from the West Nile virus vector Culex tarsalis

Since its introduction in 1999, West Nile virus (WNV) has spread across North America. Culex tarsalis is a highly efficient WNV vector species. Many traits such as virus susceptibility, autogeny and host preference vary geographically and temporally in C. tarsalis. Culex tarsalis genomic libraries were developed and were highly enriched for microsatellite inserts (42–96%). We identified 12 loci that were polymorphic in wild C. tarsalis populations. These microsatellites are the first DNA‐based genetic markers developed for C. tarsalis and will be useful for investigating population structure and constructing genetic maps in this mosquito.     

Wide recognition of Culex pipiens and lack of detection of Culex torrentium through biomolecular differentiation of mosquitoes in the Emilia‐Romagna region,Northern Italy

The Culex pipiens complex includes species with reported differences in vector competence for arthropod‐borne viruses, many of which are of significant importance to human health such as the West Nile virus and the Sindbis virus. This group of mosquitoes is difficult to distinguish morphologically; particularly as adult females. In Europe, the two species of the complex, Culex pipiens Linnaeus 1758 and Culex torrentium Martini 1925, are often found sympatrically. With the aim to characterize the presence and spread of both species in the Emilia‐Romagna region, Northern Italy, mosquitoes of the complex – collected during the West Nile virus surveillance plans – were tested by multiplex real‐time PCR for the detection of the two species Cx. pipiens and Cx. torrentium. A total of 24 165 mosquitoes, collected between 2012 and 2014 from 105 sites, and sorted in 204 pools, were tested. All tested pools were found to be composed of Cx. pipiens, whereas Cx. torrentium was not detected. These results indicate a likely absence of Cx. torrentium mosquitoes within the surveyed territory, whereas Cx. pipiens is widely distributed in the area mentioned. This is in line with previous reports, which describe a pre‐alpine distribution of Cx. torrentium in Italy.     

Preliminary report of knockdown resistance in Culex pipiens pallens and Aedes koreicus from Korea

Pyrethroid insecticides have been effective and powerful for controlling mosquitoes. However, abuse of these insecticides increases the number of resistant mosquitoes. In this study, Culex pipiens pallens and Aedes koreicus were collected from an artificial reservoir in the vicinity of a populated area in Korea, which is also a migratory bird catchment area. To monitor resistance to pyrethroid insecticides in mosquitoes, genomic DNA from the collected mosquitoes was sequenced for the kdr mutation in the voltage‐gated sodium channel (VGSC) gene. As a result, three samples with homozygous resistance (17.6%) and one with heterozygous resistance (5.9%) were found among 17 Cx. pipiens pallens specimens. One of the samples had a unique sequence at the amplified VGSC region. Of the 15 Ae. koreicus, no insecticide resistant individuals were found. In Korea, this is the first report of kdr genetic traits in Ae. koreicus and Cx. pipiens pallens and of a unique VGSC allele in Cx. pipiens pallens. Further investigation is needed to monitor the kdr resistance of these species in Korea and to determine how the unique sequence found in Cx. pipiens pallens is related to insecticide resistance.     

Reconciling West Nile virus with the autophagic pathway

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for recurrent outbreaks of meningitis and encephalitis. Several studies analyzing the interactions of this pathogen with the autophagic pathway have reported opposite results with evidence for and against the upregulation of autophagy in infected cells. In this regard, we have recently reported that minimal genetic changes (single amino acid substitutions) in nonstructural proteins of WNV can modify the ability of the virus to induce autophagic features such as LC3 modification and aggregation in infected cells. We think that these results could help explain some of the previously reported discrepancies. These findings could also aid in deciphering the interactions of this pathogen with the autophagic pathway at the molecular level aimed to develop feasible antiviral strategies to combat this pathogen, and other related flaviviruses.     

Effects of optic lobe ablation on circadian activity in the mosquito, Culex pipiens pallens

ABSTRACT. Surgical manipulation was used to show that in the mosquito, Culex pipiens pallens , the circadian pacemaker is probably not located in the optic lobes. Mosquitoes deprived of the greater part of their optic lobes still maintained a circadian activity rhythm: in DD with τ= c . 20–23 h, in LL with τ= c . 14–15 h. The operated mosquitoes showed diphasic activity which entrained to LD 16:8 h (at 200 lux), implying the existence of an extraocular photoreceptive pathway.     

The arbovirus vector Culex torrentium is more prevalent than Culex pipiens in northern and central Europe

Two species of arbovirus vector, Culex torrentium and Culex pipiens (Diptera: Culicidae), occur in several European countries, but difficulties in their accurate identification and discrimination have hampered both detailed and large‐scale distribution and abundance studies. Using a molecular identification method, we identified to species 2559 larvae of Cx. pipiens/torrentium collected from 138 sites in 13 European countries ranging from Scandinavia to the Mediterranean coast. In addition, samples of 1712 males of Cx. pipiens/torrentium collected at several sites in the Czech Republic were identified to species based on the morphology of their hypopygia. We found that the two species occur together in large areas of Europe, and that Cx. torrentium dominates in northern Europe and Cx. pipiens dominates south of the Alps. The transition in dominance occurs in central Europe, where both species are roughly equally common. There was a strong correlation between the length of the growing season at different sites and occurrences of the two species. As the growing season increases, the proportion and detection of Cx. torrentium decrease, whereas those of Cx. pipiens increase. The present findings have important consequences for the interpretation of the results of studies on major enzootic and link‐vectors of mosquito‐borne bird‐associated viruses (i.e. Sindbis, West Nile and Usutu viruses), especially in central Europe and Scandinavia.     

食物种类对淡色库蚊幼虫生长发育和药剂敏感性的影响

淡色库蚊Culexpipienspallens是我国的一种常见家蚊 ,它传播流行性乙型脑炎 ,影响人类健康。研究淡色库蚊生长发育和药剂敏感性与食物的关系 ,对改善蚊虫饲养方法与监测蚊虫药剂敏感性具有重要意义。作者研究了不同食物饲养对淡色库蚊幼虫生长发育和药剂敏感性的影响。结果表明 ,与用面粉 +酵母粉饲养的相比 ,用鼠饲料饲养的幼虫 ,生长发育速度平均要快 2d ,对氰戊菊酯的敏感性则无明显差异。说明可用鼠饲料来代替面粉 +酵母粉饲养淡色库蚊幼虫     

West Nile virus may have hitched a ride across the Western United States on Culex tarsalis mosquitoes

West Nile virus spread rapidly from east to west across North America, despite the north‐south migratory flyways of its avian hosts. In this issue, Venkatesan & Rasgon (2010) present new data on the population genetics of Culex tarsalis, the dominant West Nile virus vector in the Western United States, suggesting that patterns of mosquito gene flow may better reflect the virus’s expansion from the Midwest to the Pacific than patterns of bird movement. These findings suggest a more significant role for vector dispersal in arboviral range expansion than has previously been appreciated, and they highlight the value of molecular genetic studies of insect vector populations for understanding epidemiology and disease ecology.     

Culex pipiens and Stegomyia albopicta (= Aedes albopictus) populations as vectors for lineage 1 and 2 West Nile virus in Europe

The emerging disease West Nile fever is caused by West Nile virus (WNV), one of the most widespread arboviruses. This study represents the first test of the vectorial competence of European Culex pipiens Linnaeus 1758 and Stegomyia albopicta (= Aedes albopictus) (both: Diptera: Culicidae) populations for lineage 1 and 2 WNV isolated in Europe. Culex pipiens and S. albopicta populations were susceptible to WNV infection, had disseminated infection, and were capable of transmitting both WNV lineages. This is the first WNV competence assay to maintain mosquito specimens under environmental conditions mimicking the field (day/night) conditions associated with the period of maximum expected WNV activity. The importance of environmental conditions is discussed and the issue of how previous experiments conducted in fixed high temperatures may have overestimated WNV vector competence results with respect to natural environmental conditions is analysed. The information presented should be useful to policymakers and public health authorities for establishing effective WNV surveillance and vector control programmes. This would improve preparedness to prevent future outbreaks.