Polymerase chain reaction assay identifies Culex nigripalpus: part of an assay for molecular identification of the common Culex (Culex) mosquitoes of the eastern United States

Nucleotide sequence information on internal transcribed spacer (ITS) 1 and ITS 2 regions of the nuclear ribosomal DNA multigene family was used to develop a polymerase chain reaction assay that identifies Culex nigripalpus Theobald. The assay uses species-specific forward and reverse primers for Cx. nigripalpus and can be used along with previously described primers to distinguish among 4 common taxa of Culex (Culex) of the eastern USA with a single thermal cycler program. The assay distinguishes among the 4 taxa Cx. nigripalpus, Cx. restuans Theobald, Cx. salinarius Coquillett, and members of the Cx. pipiens Linnaeus complex. This assay may be used to verify the morphological identification of individual specimens of Culex or to confirm the species composition of mosquito pools.     

Host feeding patterns of Culex mosquitoes and West Nile virus transmission, northeastern United States

To evaluate the role of Culex mosquitoes as enzootic and epidemic vectors for WNV, we identified the source of vertebrate blood by polymerase chain reaction amplification and sequencing portions of the cytochrome b gene of mitochondrial DNA. All Cx. restuans and 93% of Cx. pipiens acquired blood from avian hosts; Cx. salinarius fed frequently on both mammals (53%) and birds (36%). Mixed-blood meals were detected in 11% and 4% of Cx. salinarius and Cx. pipiens, respectively. American robin was the most common source of vertebrate blood for Cx. pipiens (38%) and Cx. restuans (37%). American crow represented <1% of the blood meals in Cx. pipiens and none in Cx. restuans. Human-derived blood meals were identified from 2 Cx. salinarius and 1 Cx. pipiens. Results suggest that Cx. salinarius is an important bridge vector to humans, while Cx. pipiens and Cx. restuans are more efficient enzootic vectors in the northeastern United States.     

Stormwater ponds, constructed wetlands, and other best management practices as potential breeding sites for West Nile virus vectors in Delaware during 2004

We performed longitudinal surveys of mosquito larval abundance (mean mosquito larvae per dip) in 87 stormwater ponds and constructed wetland in Delaware from June to September 2004. We analyzed selected water quality factors, water depth, types of vegetation, degree of shade, and level of insect predation in relation to mosquito abundance. The 2004 season was atypical, with most ponds remaining wet for the entire summer. In terms of West Nile virus (WNV) vectors, wetlands predominantly produce Aedes vexans, culex pipiens pipiens, and Culex restuans. Retention ponds generally produced the same species as wetlands, except that Cx. p. pipiens was more abundant than Cx. restuans in retention ponds. Aedes vexans and Culex salinarius were the most abundant species to Conservation Restoration Enhancement Program ponds. Sand filters uniquely produced high numbers of Cx. restuans, Cx. p. pipiens, and Aedes japonicus japonicus, a newly invasive vector species. Site that alternately dried and flooded, mostly detention ponds, forebays of retention ponds, and some wetlands often produced Ae. vexans, an occasional WNV bridge vector species. Overall, seasonal distribution of vectors was bimodal, with peaks occurring during early and late summer. Ponds with shallow sides and heavy shade generally produced an abundance of mosquitoes, unless insect predators were abundant. Bright, sunny ponds with steep sides and little vegetation generally produced the fewest mosquitoes. The associations among mosquito species and selected vegetation types are discussed.     

A two-year evaluation of elevated canopy trapping for Culex mosquitoes and West Nile virus in an operational surveillance program in the northeastern United States

The effectiveness of CO2-baited Centers for Disease Control and Prevention miniature light traps elevated in the tree canopy (approximately 7.6 m) was compared with light traps placed at ground level (approximately1.5 m) and grass-sod infused gravid traps for collecting Culex pipiens, Culex restuans, and Culex salinarius and detecting West Nile virus (WNV) activity in an operational surveillance program that encompassed 12 ecologically diverse sites in Connecticut in 2004 and 2005. More than twice as many Cx. pipiens were collected on average in light traps suspended in the tree canopy than in either light or gravid traps placed at ground level. This difference was generally restricted to those collection sites where markedly greater numbers of Cx. pipiens were collected with all trapping methods but was not associated with site-specific urbanization indices. Culex restuans was not preferentially attracted to light traps suspended in the tree canopy. No differences in the overall abundance of this species were recorded with either of the 2 trapping procedures, but both light traps were more effective than the gravid traps. Culex salinarius was significantly more attracted to ground-based light traps than traps suspended in the tree canopy, while gravid traps were ineffective at all sites regardless of the level of urbanization or any other specific land-use characteristic. CO2-baited light traps placed in the tree canopy were generally superior to ground-based light traps for detecting WNV in Cx. pipiens. West Nile virus-infected females were collected more regularly, and the frequency of infected pools was significantly greater. Twofold higher minimum field infection rates (maximum likelihood estimation [MLE] = 6.7 vs. 3.0 per 1,000 mosquitoes) were also recorded from canopy collections of this species, and virus was detected in canopy-collected females several weeks before it was detected in collections from light traps at ground level. We conclude that the use of CO2-baited light traps placed in the tree canopy for targeted trapping of Cx. pipiens and subsequent detection of WNV are likely to yield better overall results than light traps placed at ground level in this region of the northeastern United States. The virus isolation data obtained from Cx. pipiens collected in gravid traps compared favorably both temporally and spatially with results from canopy trap collections. There were no significant differences in the overall frequency of WNV-infected pools or MLEs for Cx. pipiens, but fewer total WNV isolations were made from Cx. pipiens collected in the gravid traps and virus was detected more infrequently. Results reaffirmed the utility of gravid traps as effective surveillance tools for detection of WNV in Cx. pipiens in the northeastern United States. However, findings also demonstrated that CO2-baited light traps placed in the tree canopy provided more consistent results where weekly detection of virus amplification is a critical objective. The comparative effectiveness of ground- and canopy-based light traps for detection of WNV-infected Cx. restuans and Cx. salinarius was inconclusive owing to the limited number of virus isolations that were made from these species during the 2 years of study. However, WNV virus isolations were made several weeks earlier and more frequently from Cx. restuans collected in traps placed in the canopy rather than at ground level in 2004. Results support the view that ground-based light traps are more effective for detection of WNV in Cx. salinarius.     

The abundance and seasonal distribution of Culex mosquitoes in Iowa during 1995-97

The abundance and seasonal distribution of Culex mosquitoes were monitored by measuring oviposition activity during the summers of 1995, 1996, and 1997. Five species of culicine mosquitoes laid egg rafts in ovitraps. Egg rafts of 4 Culex species-Cx. restuans (54.98%), Cx. pipiens (25.41%), Cx. salinarius (12.18%), and Cx. tarsalis (0.14%)-constituted more than 92% of the total egg rafts collected. Culiseta inornata (0.1%) was the only other species to lay viable egg rafts in the ovitraps. A small percentage (7.19%) of egg rafts did not hatch; thus identification was not possible. Compared with New Jersey light trap data in a nearby area, the abundance of Cx. tarsalis and Cs. inornata was markedly underestimated with ovitraps. These data may also reflect differences in the specific location of the 2 trap sites rather than attractiveness of ovitraps for Cx. tarsalis and Cs. inornata. In general, Cx. restuans oviposition activity began in late May, and it was the dominant Culex species through June. After July 1, the number of egg rafts laid by Cx. restuans decreased continually until the end of the summer. Culex pipiens and Cx. salinarius oviposition began in early June and increased gradually during the summer. Over the course of a summer, there were about twice as many Cx. pipiens as there were Cx. salinarius. A crossover in the number of Cx. pipiens and Cx. restuans egg rafts occurred in late July or early August, depending on the year. By late August each year, Cx. pipiens was the most abundant species. At this time, Cx. restuans and Cx. salinarius populations were similar, but each was about half of the Cx. pipiens population. Differences in the abundance of all 3 species could not be explained by changes in ambient temperature (both minimum and maximum) or relative humidity, either within or among years.     

West Nile virus surveillance in mosquitoes in New York State, 2000-2004

A West Nile virus (WNV) surveillance system was created and implemented in New York State (NYS) in 2000 and described previously (White et al. 2001). We examine and evaluate the results of mosquito and virus surveillance for 2000 through 2004 exclusive of New York City. Forty-nine counties submitted 1,095,426 mosquitoes in 35,280 pools for WNV assay. Specimens of 47 species were tested, with Culex species accounting for 47.6% of all pools tested. WNV was detected in 814 pools from 10 species, with combined Culex pipiens/Culex restuans pools accounting for 90.8% of all detections. Pools submitted from gravid traps were 5.7 times more likely to be positive than submissions from carbon dioxide-baited light traps. Most human WNV cases resided in counties that conducted mosquito surveillance. Local health departments' use of mosquito surveillance information often led to an enhanced disease prevention response. In NYS, Cx. pipiens/Cx. restuans groups are most likely vectors of WNV. Future efforts to improve system efficacy are discussed.     

Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus.

To control West Nile virus (WNV), it is necessary to know which mosquitoes are able to transmit this virus. Therefore, we evaluated the WNV vector potential of several North American mosquito species. Culex restuans and Cx. salinarius, two species from which WNV was isolated in New York in 2000, were efficient laboratory vectors. Cx. quinquefasciatus and Cx. nigripalpus from Florida were competent but only moderately efficient vectors. Coquillettidia perturbans was an inefficient laboratory vector. As WNV extends its range, exposure of additional mosquito species may alter its epidemiology.     

A survey of mosquitoes breeding in used tire stockpiles in Connecticut

A larval survey of nine used tire disposal sites in Connecticut, was conducted from June through October 1987. No larvae of Aedes albopictus were uncovered indicating that this mosquito is not yet established in the state. However, eight other mosquito species in four genera were found. The most abundant and widely distributed species found breeding in permanent tire dumps were Ae. atropalpus, Ae. triseriatus and Culex restuans. Culex pipiens was the second most common Culex species followed by Cx. territans and Cx. salinarius. Other species included Anopheles punctipennis and Culiseta melanura. Larvae of Ae. atropalpus and Cx. salinarius were most frequently recovered from tire casings exposed to direct sunlight, while Ae. triseriatus and Cs. melanura predominated in tires shaded by bordering trees and low-lying vegetation. The other mosquito species showed no discernable preference for either habitat type.     

Seasonal abundance of Culex restuans and Culex pipiens in southwestern Virginia through ovitrapping

Ovitrap collections were done to determine the seasonal distribution and ovipositional activity of Culex restuans and Culex pipiens in southwestern Virginia during summers 2002 and 2003. In both years, Cx. restuans was the first species collected and was the dominant species throughout the collecting period. More than 90% of the collections were comprised of Cx. restuans. Two oviposition peaks occurred in both years for Cx. restuans, the first in June and second in the middle of July. Culex pipiens made up less than 7% of the total catch and started to occur in the traps in the middle of the trapping season with a peak occurring in August. Although ovipositional activity began to decline for Cx. restuans, at the end of the trapping period, there was a slight increase in ovipositional activity for Cx. pipiens, but not enough to result in a crossover. A third species, Culex salinarius, was collected in the traps, but the total number of egg rafts accounted for less than 1% of the total collection.     

Mosquito surveillance for West Nile virus in Connecticut, 2000: isolation from Culex pipiens, Cx. restuans, Cx. salinarius, and Culiseta melanura

Fourteen isolations of West Nile (WN) virus were obtained from four mosquito species (Culex pipiens [5], Cx. restuans [4], Cx. salinarius [2], and Culiseta melanura [3]) in statewide surveillance conducted from June through October 2000. Most isolates were obtained from mosquitoes collected in densely populated residential locales in Fairfield and New Haven counties, where the highest rates of dead crow sightings were reported and where WN virus was detected in 1999. Minimum field infection rates per 1,000 mosquitoes ranged from 0.5 to 1.8 (county based) and from 1.3 to 76.9 (site specific). Cx. restuans appears to be important in initiating WN virus transmission among birds in early summer; Cx. pipiens appears to play a greater role in amplifying virus later in the season. Cs. melanura could be important in the circulation of WN virus among birds in sylvan environments; Cx. salinarius is a suspected vector of WN virus to humans and horses.     

West Nile virus infection in mosquitoes, birds, horses, and humans, Staten Island, New York, 2000

West Nile (WN) virus transmission in the United States during 2000 was most intense on Staten Island, New York, where 10 neurologic illnesses among humans and 2 among horses occurred. WN virus was isolated from Aedes vexans, Culex pipiens, Cx. salinarius, Ochlerotatus triseriatus, and Psorophora ferox, and WN viral RNA was detected in Anopheles punctipennis. An elevated weekly minimum infection rate (MIR) for Cx. pipiens and increased dead bird density were present for 2 weeks before the first human illness occurred. Increasing mosquito MIRs and dead bird densities in an area may be indicators of an increasing risk for human infections. A transmission model is proposed involving Cx. pipiens and Cx. restuans as the primary enzootic and epizootic vectors among birds, Cx. salinarius as the primary bridge vector for humans, and Aedes/Ochlerotatus spp. as bridge vectors for equine infection.     

Vector competence of California mosquitoes for West Nile virus

To identify the mosquito species competent for West Nile virus (WNV) transmission, we evaluated 10 California species that are known vectors of other arboviruses or major pests: Culex tarsalis, Cx. pipiens pipiens, Cx. p. quinquefasciatus, Cx. stigmatosoma, Cx. erythrothorax, Ochlerotatus dorsalis, Oc. melanimon, Oc. sierrensis, Aedes vexans, and Culiseta inornata. All 10 became infected and were able to transmit WNV at some level. Ochlerotatus, Culiseta, and Aedes were low to moderately efficient vectors. They feed primarily on mammals and could play a secondary role in transmission. Oc. sierrensis, a major pest species, and Cx. p. quinquefasciatus from southern California were the least efficient laboratory vectors. Cx. tarsalis, Cx. stigmatosoma, Cx. erythrothorax, and other populations of Cx. pipiens complex were the most efficient laboratory vectors. Culex species are likely to play the primary role in the enzootic maintenance and transmission of WNV in California.     

West Nile virus-infected mosquitoes, Louisiana, 2002

Human cases of West Nile virus (WNV) disease appeared in St. Tammany and Tangipahoa Parishes in southeastern Louisiana in June 2002. Cases peaked during July, then rapidly declined. We conducted mosquito collections from August 3 to August 15 at residences of patients with confirmed and suspected WNV disease to estimate species composition, relative abundance, and WNV infection rates. A total of 31,215 mosquitoes representing 25 species were collected by using primarily gravid traps and CO2-baited light traps. Mosquitoes containing WNV RNA were obtained from 5 of 11 confirmed case sites and from 1 of 3 sites with non-WNV disease. WNV RNA was detected in 9 mosquito pools, including 7 Culex quinquefasciatus, 1 Cx. salinarius, and 1 Coquillettidia perturbans. Mosquito infection rates among sites ranged from 0.8/1,000 to 10.9/1,000. Results suggest that Cx. quinquefasciatus was the primary epizootic/epidemic vector, with other species possibly playing a secondary role.     

广东国境口岸五种蚊种核糖体基因第2内转录间隔区分子鉴定方法研究

[目的]建立国境口岸不同蚊种的核糖体基因第2内转录间隔区（rDNA-ITS2）分子鉴定方法及其系统进化关系。[方法]针对蚊虫的rDNA核酸序列保守性，设计扩增rDNA-ITS2编码区的PCR引物，对广州机场、江门和湛江等国境口岸采集的致倦库蚊、三带喙库蚊、白纹伊蚊、中华按蚊、骚扰阿蚊等成蚊和实验室喂养的蚊幼虫进行PCR扩增和序列测定，并与GenBank中已知蚊虫的rDNA-ITS2进行同源性比较和系统进化分析。[结果]不同蚊虫的rDNA-ITS2扩增片断长度不同，M2引物对致倦库蚊、三带喙库蚊、白纹伊蚊、骚扰阿蚊和中华按蚊的扩增片断分别为447bp-520bp、432bp-438bp、527bp-586bp、439bp-448bp和644bp。序列分析和系统进化关系显示，尖音库蚊组和三带喙库蚊聚类为库蚊属，再与白纹伊蚊和骚扰阿蚊聚类为库蚊亚科，库蚊亚科再与中华按蚊进行聚类，分子进化与蚊虫形态学鉴定的亲缘关系保持一致。[结论]建立的rDNA-ITS2分子鉴别技术可成功地应用于国境口岸范围内成蚊和幼蚊的亚科、属和种的区分和确定系统发育关系。这可以弥补蚊虫形态特征信息量的不足等传统分类系统的缺点，对国境口岸范围外来的或新发现的蚊种的鉴别提供了分子水平的技术依据。     

Polymerase chain reaction assay identifies North American members of the Culex pipiens complex based on nucleotide sequence differences in the acetylcholinesterase gene Ace.2

Nucleotide sequence differences in the acetylcholinesterase gene Ace.2 were used to develop an assay to distinguish among North American mosquitoes of the Culex pipiens complex. Taxon-specific polymerase chain reaction primers based on sequence differences within intron 2 of Ace.2 distinguish among the sibling species Cx. pipiens Linneaus and Cx. quinquefasciatus Say and their F1 hybrids. This assay may be used to confirm the species composition of mosquito pools, identify individual specimens collected in arbovirus surveillance programs and other mosquito studies, and define zones of hybridization.     

江苏省西尼罗病毒病潜在蚊媒库蚊发生情况调查研究

目的了解江苏省西尼罗病毒病潜在蚊媒库蚊的构成及发生消长情况,为及时有效地切断西尼罗病毒病传播途径提供科学依据。方法2004年4~11月在江苏省苏州、镇江和连云港3市的城区和郊区设点,采用人工小时法和帐诱法调查成蚊。结果全省西尼罗河病毒病潜在蚊媒库蚊由淡色库蚊、三带喙库蚊和二带喙库蚊等3种构成;全省库蚊平均发生密度野外高于室内,郊区室内高于城区室内。室内平均密度镇江显著高于其他两市,野外平均密度连云港最高。优势种淡色库蚊的消长为双峰型,次优势种三带喙库蚊的消长为单峰型。结论基本了解了江苏省西尼罗病毒病潜在蚊媒库蚊的群落构成及消长情况,尚需掌握流行病学证据证明确为西尼罗河病毒病蚊媒。     

Commercial mosquito trap and gravid trap oviposition media evaluation, Atlanta, Georgia

Field trials evaluating the effectiveness of selected gravid trap oviposition media and commercially available mosquito traps were conducted in southern Fulton County (Atlanta), GA, from June 9 to June 18 and June 24 to July 4, 2002, respectively. Total number of mosquitoes and number of each species captured during the tests were compared using a Latin square design. For the gravid trap infusion media, significant differences were found for total number of mosquitoes collected where sod > or = hay > or = hay side-by-side diluted hay > dilute hay side-by-side hay > or = oak > diluted hay. Only Aedes albopictus (oak), Culex quinquefasciatus (sod and both concentrated hay infusions), and Culex restuans (sod) were captured in significantly greater numbers using a particular infusion. Significant differences for the total number of mosquitoes collected were also observed in the commercial mosquito traps such that the gravid trap > ultra violet up-draft > or = Mosquito Magnet Pro > or = omnidirectional Fay-Prince trap with CO2 > up-draft CDC-style with CO2 > or = CDC-style with CO2. Significant differences in numbers collected among traps were noted for several species, including Aedes vexans, Aedes albopictus, Cx. quinquefasciatus, Cx. restuans, and Culex salinarius. Results from these field trap and infusion evaluations can enhance current surveillance efforts, especially for the primary vectors of West Nile virus and other arboviruses.     

Unexpected spatiotemporal abundance of infected Culex restuans suggest a greater role as a West Nile virus vector for this native species

Difficulties in correctly differentiating Culex restuans mosquitoes from Culex pipiens have left the spatiotemporal mechanisms underlying the epidemiology of West Nile virus (WNV) in the northeastern United States largely unresolved. We performed weekly surveys across a natural to urban gradient of sites in central New Jersey (USA) and used a rapid and cheap DNA extraction and a species-specific PCR assay to create single species pools for WNV testing. To assess seasonal trends we combined these results with WNV surveillance records generated from grouped Cx. restuans/Cx. pipiens pools tested in 2011–2012. Cx. restuans was found to be highly abundant within all sites and reached especially high abundance in urban wetland habitats greatly disturbed by human action. In contrast, the seasonal presence of Cx. pipiens was greatest in residential and urban habitats and its presence in natural areas was minimal throughout the season. WNV infection rates in both species were similar but Cx. restuans was consistently found infected first and more frequently, even as early as May, whereas WNV was first detected in Cx. pipiens in late July. WNV activity peaked during the month of August when WNV was commonly isolated from both species. The peak in WNV activity in August observed for both species was consistent with data from 2011 to 2012 when Cx. restuans and Cx. pipiens were grouped, although analyzing single species pools increased overall predicted infection levels. Our results support the preeminence of Cx. restuans as an enzootic vector of WNV and strongly suggest this species has become a “native invasive” exploiting human modified habitats and reaching very high abundance there. Importantly, high infection rates in disturbed wetland sites with high populations of Cx. restuans suggest this species may enable the introduction of WNV to urbanized environments where both Culex contribute to transmission potentiating disease risk.     

Oviposition preferences of Culex restuans and Culex pipiens (Diptera: Culicidae) for selected infusions in oviposition traps and gravid traps

Field studies were conducted in southwestern Virginia to determine the ovipositional preferences of Culex restuans and Culex pipiens by using ovitraps and gravid traps baited with selected infusions. For the ovitrap collections, 4 different infusions (manure, hay, grass, and rabbit chow) were used. Significant differences among infusions were detected on most sample dates for both species. For 3 of the first 4 wk of collections, the manure infusion collected significantly more Cx. restuans than all the other infusions. The hay and grass infusions collected the majority of the egg rafts during weeks 5-9. Cx. pipiens egg rafts were absent from the first 3 wk of collections. Of the remaining 6 wk, 4 showed significant differences in attractiveness of infusions, with the hay and grass infusions preferred by Cx. pipiens. Two infusions, manure and hay, were used for the gravid trap experiment and both Cx. restuans and Cx. pipiens data were combined for analysis. Only the first 2 wk showed significance, with manure being preferred over hay in both weeks. In later collections, the relative attractiveness of the hay infusion increased. A seasonal shift in infusion preference may be related to incubation temperature during preparation of the infusions. New infusions were prepared each week and incubation was done outside. Increased attractiveness of the hay infusion coincided with higher average temperatures in July and August. Hay infusion was very effective for trapping both Cx. pipiens and Cx. restuans in southwestern Virginia and is more convenient to use than manure. However, cool outside temperatures in the early season may interfere with the fermentation process and thus incubation should be done for a longer time or brought indoors.