Vertical transmission of West Nile Virus by three California Culex (Diptera: Culicidae) species

Three California Culex species previously identified as efficient laboratory vectors of West Nile (WN) virus were tested for their capability to vertically transmit WN virus. Wild-caught Culex pipiens pipiens L., Culex pipiens quinquefasciatus Say, and two populations of Culex tarsalis Coquillett females were inoculated intrathoracically with 10(2.7 +/- 0.1) plaque-forming units of WN virus. F1 progeny were reared at 18 degrees C and subsequently tested as adults for infectious virus on Vero cell culture. Virus was not detected in 197 pools comprising 4,884 Cx. p. pipiens. The minimum filial infection rate (MFIR) for Cx. p. quinquefasciatus was approximately 3.0/1,000 for 665 progeny tested in 28 pools. There was no virus detected in 102 pools of 2,453 progeny from Cx. tarsalis collected in Riverside County. The MFIR for Cx. tarsalis collected in Yolo County was approximately 6.9/1,000 for 2,165 progeny tested in 86 pools. Mosquito progeny infected vertically during the fall could potentially serve as a mechanism for WN virus to overwinter and initiate horizontal transmission the following spring.     

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.     

Extrinsic incubation periods for horizontal and vertical transmission of West Nile virus by Culex pipiens pipiens (Diptera: Culicidae)

Culex pipiens pipiens L. (Diptera: Culicidae), infected per os from a membrane feeder, transmitted West Nile virus (family Flaviviridae, genus Flavivirus, WNV) at 26 degrees C horizontally during feeding to hamsters and suckling mice and vertically to F1 progeny during egg deposition. Horizontal transmission rates increased with extrinsic incubation, with 75-100% of the females transmitting on days 16 through 25 postinfection (pi). No females deposited eggs infected with WNV after the first bloodmeal on 3-8 d pi. Females vertically transmitted WNV during egg laying after their second, third, and fourth bloodmeals on days 13-33 pi. The vertical transmission rate was 4.7%. The vertical minimal infection rate was 0.52 infected F1 specimens/1,000 specimens tested from females feeding during their second and later bloodmeals on hamsters or suckling mice. The sequence of horizontal and vertical transmission is reported. A female may transmit WNV 1) horizontally to a host during feeding and subsequently vertically to her offspring during egg laying, 2) vertically to her offspring during oviposition without prior horizontal transmission to a host, and 3) horizontally to a host without vertically transmitting the virus. These two means of transmission by Cx. p. pipiens contribute to the relatively high minimum infection rates that are reached in late summer and to the survival of virus during winter and initiation of amplification in the spring in the northeastern United States.     

Swedish Culex torrentium and Cx. pipiens (Diptera: Culicidae) as experimental vectors of Ockelbo virus

The ability of the sibling species Culex pipiens (L.) and Culex torrentium (Martini) from central Sweden to transmit Ockelbo (OCK) virus was determined. Both species became infected after ingesting OCK virus from a viremic chicken; they transmitted this virus to chickens after 21-28 d of extrinsic incubation. In Cx. torrentium, infection rates were 90% or higher, and all 10 refeeding mosquitoes transmitted virus after feeding on chickens with a viremia of at least 10(3.0) plaque-forming units (PFU)/ml. In contrast, only 1 of 28 (4%) of the Cx. pipiens that ingested blood containing 10(3.0-3.9) PFU/ml became infected, and none of 16 refeeding mosquitoes transmitted virus. However, 98 of 184 (53%) of the Cx. pipiens that ingested a blood meal containing at least 10(6.0) PFU/ml became infected. Transmission rates in Cx. pipiens increased with increasing virus concentration in the blood meal to a maximum of 37% in mosquitoes that ingested greater than 10(8.0) PFU/ml. Based on these data, both Cx. pipiens and Cx. torrentium are capable of transmitting OCK virus in an enzootic cycle involving birds as hosts. However, Cx. torrentium appears to be physiologically a more efficient vector than Cx. pipiens.     

Wolbachia在我国蚊虫体内感染组织定位的透射电镜观察和PCR检测

通过透射电镜的超微形态观察和分组织的PCR检测发现 ,在我国尖音库蚊复合组和白纹伊蚊体内感染的Wolbachia株不仅局限于生殖组织内。在淡色库蚊 (Cx .pipienspallens)和骚扰库蚊 (Cx .pipiensmolestus)雌蚊卵巢、中肠和胸部肌肉组织中都有B大组Pip组Wolbachia株的感染 ,而头部没有检测到感染。在白纹伊蚊雌蚊卵巢、中肠组织中存在A大组和B大组Pip组的Wolbachia株双重感染 ,胸部肌肉组织中仅发现B大组Pip组Wolbachia株的单独感染 ,头部没有检测到感染     

Effect of incubation at overwintering temperatures on the replication of West Nile Virus in New York Culex pipiens (Diptera: Culicidae)

We examined the effect of simulated overwintering temperatures on West Nile (WN) virus replication in Culex pipiens L. derived from mosquitoes collected during the autumn 1999 WN epizootic in New York. The WN virus was a strain isolated from a dead crow also collected during this outbreak. Virus was recovered from most mosquitoes held exclusively at 26 degres C. In contrast, none of the mosquitoes held exclusively at the lower temperatures had detectable infections. When mosquitoes were transferred to 26 degrees C after being held at 10 degrees C for 21-42 d, infection and dissemination rates increased with increased incubation at 26 degrees C. Future studies involving the attempted isolation of WN virus from overwintering mosquitoes may benefit from holding the mosquitoes at 26 degrees C before testing for infectious virus.     

Binding of western equine encephalomyelitis virus to brush border fragments isolated from mesenteronal epithelial cells of mosquitoes

Brush border fragments (BBF) were isolated from mesenteronal epithelial cells of mosquitoes that are either susceptible (WS Culex tarsalis) or refractory (WR Cx. tarsalis; Culex pipiens) to peroral infection by western equine encephalomyelitis (WEE) virus. The isolated BBF were combined with radiolabeled WEE virus in a binding assay to compare the amount of virus bound by BBF from susceptible and refractory mosquitoes. BBF and WEE virus were mixed in a microcentrifuge tube, incubated for 1 h and centrifuged at 27,000 X g-30 min to pellet WEE virus bound to BBF. Optimal binding occurred at pH 7.2, 20 degrees C and there was no requirement for divalent cations. BBF isolated from perorally susceptible mosquitoes (WS Cx. tarsalis) bound significantly greater amounts of radiolabeled WEE virus, compared to BBF isolated from refractory mosquitoes (WR Cx. tarsalis; Cx. pipiens), in all experiments. The binding of WEE virus to BBF from WS Cx. tarsalis appears to be specific, based on saturation and competitive binding studies; binding to BBF from WR Cx. tarsalis and Cx. pipiens is nonspecific. Scatchard analysis of the binding data for BBF from WS Cx. tarsalis yields an estimated 1.8-3.5 X 10(6) binding sites per mesenteronal epithelial cell with an affinity constant of (Ka) of 2.2 X 10(11) M-1.     

Behavioral differences of invasive container-dwelling mosquitoes to a native predator

Aquatic prey show behavioral modifications in the presence of predation-risk cues that alleviate their risk from predation. Aedes albopictus (Skuse), Aedes japonicus (Theobald), and Culex pipiens L. are invasive mosquitoes in North America, and their larvae are prey for the native mosquito predator, Toxorhynchites rutilus (Coquillett). Ae. albopictus and Ae. japonicus are recent invaders, whereas Cx. pipiens has been in the United States for >100 yr. In the presence of predation-risk cues from Tx. rutilus larvae, Cx. pipiens larvae increased the time spent resting at the surface (least risky behavior) more than the other prey species. Ae. japonicus larvae increased resting at the surface of the containers more than Ae. albopictus larvae in the presence of predation-risk cues. Cx. pipiens larvae spent more time motionless at the surface even in the absence of predation-risk cues when compared with the other species, indicating that Cx. pipiens larvae are the least vulnerable prey. As compared with the other prey species, Ae. albopictus larvae exhibited more high-risk behaviors both in the presence and absence of predation-risk cues, indicating that they are the most vulnerable prey. Ae. albopictus is the superior competitor; however, predation by Tx. rutilus larvae may prevent competitive exclusion by Ae. albopictus and promote coexistence among the three prey species.     

Life table characteristics of Anopheles arabiensis (Diptera: Culicidae) under simulated seasonal conditions

Life tables provide a summary of mortality and reproductive schedules and can help explain why certain species proliferate in particular environments. Life table characteristics of Alopheles arabiensis, under a range of simulated seasonal temperature were determined. F1 progeny of field-collected material were observed at four fluctuating temperatures with means of 17.9, 23.2, 26.1, and 21.4 degrees C, representing winter, spring, summer and autumn temperature profiles. Gonotrophic cycles, egg hatchability rates, mean male and female lifespans, reproductive rate, the intrinsic rate of increase, and generation times were calculated. Immature development was fastest in summer (11 d) compared with winter (32 d), resulting in less robust adults. Mean female lifespans were greater than those of the males and also increased with decreasing temperatures. From the age-specific survivorship curve and the gonotrophic cycles, females were found to lay large numbers of eggs especially during spring and summser. Under simulated winter conditions mosquitoes did not oviposit in the laboratory. It was also determined that summer reared mosquitoes are capable of giving just one infected bite while autumn reared mosquitoes can give two infected bites, increasing their transmission potential. Thus life table data go some way toward explaining late season transmission of malaria in South Africa.     

Enhanced cold and desiccation tolerance in diapausing adults of Culex pipiens, and a role for Hsp70 in response to cold shock but not as a component of the diapause program

Culex pipiens L. reared under diapause-inducing conditions (short daylength; 18 degrees C) were more cold tolerant and desiccation resistant than their nondiapausing counterparts (long daylength; 18 degrees C). Upon cold exposure (-5 degrees C), diapausing mosquitoes reared at 18 degrees C survived nearly twice as long as nondiapausing mosquitoes reared at 18 degrees C and 10 times longer than nondiapausing mosquitoes reared at 25 degrees C. Thus, rearing temperature provided partial protection against low temperature injury in nondiapausing mosquitoes, but maximum resistance to cold was attained by the diapause state. In this species, the supercooling point is not a good indicator of cold tolerance. Both diapausing and nondiapausing females had supercooling points of approximately -16 degrees C, but diapausing as well as nondiapausing females died at temperatures well above the supercooling point, suggesting that low temperature mortality was due to indirect chilling injury. Diapause also conferred greater resistance to desiccation (1.6-2-fold increase in survival) compared with the nondiapause state. The gene encoding a 70-kDa heat shock protein, hsp70, was not up-regulated (i.e., more highly expressed) as a part of the diapause program, nor was it up-regulated by desiccation stress, but it was up-regulated during recovery from cold shock. Cx. pipiens thus differs from a number of other diapausing insect species that are known to developmentally up-regulate hsp70 during diapause.     

Laboratory transmission of Japanese encephalitis, West Nile, and Getah viruses by mosquitoes (Diptera: Culicidae) collected near Camp Greaves, Gyeonggi Province, Republic of Korea 2003

We conducted experimental studies to evaluate mosquitoes captured in Paju County, Gyeonggi Province, Republic of Korea, for their ability to transmit West Nile virus (family Flaviviridae, genus Flavivirus, WNV), Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV), and Getah virus (family Togaviridae, genus Alphavirus, GETV) under laboratory conditions. Both Culex pipiens pallens Coquillett and Culex tritaeniorhynchus Giles were highly susceptible to infection with WNV, with infection rates > 65% when allowed to feed on chickens with viremias of approximately 10(7) plaque-forming units (PFU) of virus/ml blood. In contrast, Cx. tritaeniorhynchus were significantly more susceptible to JEV or GETV (infection rate 100%) than were the Cx. p. pallens (infection rate 3% for JEV and 0% for GETV) captured in the same area when allowed to feed on chickens with viremias of approximately 10(5) PFU of virus/ml blood. The detection of JEV in field-collected Cx. tritaeniorhynchus in Gyeonggi Province in 2000 and the demonstrated ability of this species to transmit this virus support the importance of the continued vaccination of Koreans against JEV and indicate a risk of infection for nonvaccinated individuals.     

Effect of environmental temperature on the ability of Culex pipiens (Diptera: Culicidae) to transmit West Nile virus

Environmental temperature can affect the ability of mosquitoes to transmit an arbovirus. However, results of various studies indicate that these effects are not consistent among viruses or mosquito species, and there is no information available on the effect of environmental temperature on the ability of North American mosquito species to transmit West Nile (WN) virus. We evaluated the effect of incubation temperature (18, 20, 26, or 30 degrees C) on the ability of Culex pipiens L. derived from specimens collected during the outbreak in New York in 1999 to transmit a strain of WN virus obtained from a crow that died during this outbreak. Although mosquitoes fed on the same viremic chickens, infection rates were directly related to subsequent incubation temperatures. In mosquitoes held at 30 degrees C, virus was recovered from nearly all mosquitoes tested, disseminated infections were detected as early as 4 d after the infectious blood meal, and >90% of all mosquitoes had a disseminated infection 12 or more days after the infectious blood meal. In contrast, for mosquitoes held at 18 degrees C, disseminated infections were not detected until 25 d after the infectious blood meal, and even after 28 d, <30% contained a disseminated infection. Results for mosquitoes held at 20 and 26 degrees C were intermediate for both infection and dissemination rates. The effect of environmental temperature should to be considered when evaluating the vector competence of these mosquitoes and modeling risk of WN virus transmission in nature.     

The potential of Aedes triseriatus (Diptera: Culicidae) as an enzootic vector of West Nile virus

The susceptibility of Aedes triseriatus (Say) (Diptera: Culicidae) to low levels of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was determined and compared with that of Culex pipiens L. to assess the likelihood of its participation in an enzootic cycle involving mammals. Ae. triseriatus and Cx. pipiens were exposed to WNV by feeding on baby chickens with WNV serum titers ranging from 10(4.1 +/- 0.1) to 10(8.6 +/- 0.1) plaque-forming units (PFU)/ml and from 10(4.1 +/- 0.1) to 10(7.0) PFU/ml, respectively. Infection rates and 95% confidence intervals (CIs) of 8% (4, 14) and 25% (15, 38) occurred in Ae. triseriatus and Cx. pipiens after feeding on chickens with WNV titers of 10(4.1 +/- 0.1) PFU/ml and increased to 65% (49, 79) and 100% (72, 100) in Ae. triseriatus and Cx. pipiens after feeding on chickens with titers of 10(7.1 +/- 0.1) PFU/ml. The mean infection rate of Ae. triseriatus ranged from 97% (84, 100) to 100% (79, 100) after feeding on chickens with WNV titers of > or = 10(8.2) PFU/ml. The infectious dose (ID)50 values for Ae. triseriatus and Cx. pipiens were 10(6.5) (6.4, 6.7) and 10(4.9) (4.6, 5.1) PFU/ml, respectively. The combined estimated transmission rate of Ae. triseriatus at 14 and 18 d after feeding on chickens with a mean WNV titer of 10(8.6 +/- 0.1) PFU/ml was 55%. Although Ae. triseriatus is significantly less susceptible to WNV than Cx. pipiens, the susceptibility of Ae. triseriatus to WNV titers < 10(5.0) PFU/ml and its ability to transmit WNV suggest that Ae. triseriatus has the potential to be an enzootic vector among mammalian populations.     

Asymmetrical competition and patterns of abundance of Aedes albopictus and Culex pipiens (Diptera: Culicidae)

We tested for competitive advantage among larvae of Aedes albopictus (Skuse) and Culex pipiens L. in a laboratory experiment and determined the frequency and spatial and temporal patterns of co-occurrence in the field in East St. Louis, IL. In a laboratory competition experiment at multiple combined densities of Ae. albopictus and Cx. pipiens larvae, Ae. albopictus survivorship and developmental times were significantly affected by conspecific densities but not by Cx. pipiens densities. In contrast, Cx. pipiens survivorship and developmental times were significantly affected by both conspecific and Ae. albopictus densities. Per capita rate of increase (r') for Ae. albopictus cohorts declined significantly due to density of conspecifics, but not density of Cx. pipiens. Interspecific competition between Ae. albopictus and Cx. pipiens under these laboratory conditions was strong and asymmetrical, with the effect of Ae. albopictus on Cx. pipiens much stronger than the reverse. In monthly samples from tire sites in East St. Louis, Ae. albopictus was highly seasonal, occurring in relatively low abundance from early May to July and increasing in abundance in August and September. Co-occurrence corresponded to the seasonality of Ae. albopictus, with Cx. pipiens encountering Ae. albopictus in more tires and at higher numbers within a tire, in August and September. Abundance of both species was high in residential areas and was unrelated to overstory cover, total nitrogen, and total phosphorus. Abundance of Ae. albopictus, but not of Cx. pipiens, was positively associated with conductivity. We expect Cx. pipiens to suffer from the effects of interspecific competition in tires in which it encounters Ae. albopictus. Interspecific competition between these species may be of both ecological and medical importance.     

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.     

上海市中心城区成蚊种群动态及新变化研究

为全面掌握上海中心城区蚊虫种群动态及新变化,选取城区蚊虫较易出现的公园和绿化区域环境类型（人民广场及人民公园地区）,采用CO：诱捕法进行连续超过220d的成蚊动态监测研究。结果显示,2012年224d共捕捉到蚊虫20826头,包括3属6种;淡色库蚊和白纹伊蚊为上海中心城区优势蚊种（构成比分别为71．8％和19．7％）;不同蚊种季节分布差异性显著,淡色库蚊和白纹伊蚊高峰期分别位于5～6月和7～8月,且整体上淡色库蚊密度高于白纹伊蚊（除8月下旬外,其余均P〈0．05）,三带喙库蚊在7月中下旬显著上升（分别为681和580头／旬）;蚊虫13变化趋势波动幅度较大,在相同的月份或季节亦出现较大日变化差异;将2008～2011年历史监测数据与2012年进行趋势比较,仅个别年份间有显著相关性：不同年份间的旬分布亦存在统计学差异。本研究提示,上海市中心城区的优势蚊种仍为淡色库蚊和白纹伊蚊,该两种蚊种的季节分布与某些文献报道存在差异,主要由上海城区的气候特点决定;上海城区的蚊媒防治工作仍应针对这两种蚊虫的季节分布采取针对性的有效防控措施,此外三带喙库蚊对城市环境的适应性不断提高,为上海城区的蚊媒防治工作提出了新的挑战。     

Oviposition activity patterns and West Nile virus infection rates for members of the Culex pipiens complex at different habitat types within the hybrid zone, Shelby County, TN, 2002 (Diptera: Culicidae)

Oviposition activity and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) infection rates were assessed for members of the Culex pipiens complex from July through December 2002 by using gravid traps placed at four ecologically different sites in the southern portion of the hybrid zone in Shelby County, TN. Molecular assays identified three members of the Cx. pipiens complex: Cx. pipiens pipiens L., Cx. p. quinquefasciatus Say, and Cx. p. pipiens-Cx. p. quinquefasciatus hybrids (hybrids). The Cx. pipiens complex accounted for 90% of mosquitoes collected in gravid traps. All 285 WNV-positive mosquitoes were Culex mosquitoes, and 277 (97%) were Cx. pipiens complex mosquitoes. Infection rates among members of the Cx. pipiens complex were not significantly different. Infection rates were significantly higher at two urban sites than at a rural site, and WNV was not detected at a forested site. At urban sites, abundances of members of the Cx. pipiens complex corresponded to a simple latitude model of the hybrid zone. Cx. p. quinquefasciatus was most abundant (46.4%), followed by hybrids (34.1%) and Cx. p. pipiens (19.5%). The relative abundances at a rural site were reversed with Cx. p. pipiens (48.4%) being most abundant. This demonstrates that spatial habitat variation may profoundly influence the distribution of members of the Cx. pipiens complex within the hybrid zone. Members of the Cx. pipiens complex did not display different oviposition patterns. However, oviposition patterns assessed hourly at urban and rural sites were significantly different. At urban sites, oviposition activity of Cx. pipiens complex mosquitoes was bimodal with an evening peak associated with sunset and a morning peak associated with sunrise. At the rural site, the evening peak was pronounced and the morning peak weak and similar to nighttime activity.     

West Nile virus infection rates in Culex nigripalpus (Diptera: Culicidae) do not reflect transmission rates in Florida

We describe the first documented field transmission of West Nile (WN) virus by a North American mosquito. WN was first detected in northern Florida in 2001. An intensive mosquito trapping and surveillance program was conducted in this region for four nights to assess mosquito transmission of WN. Four mosquito traps, each with a single sentinel chicken, were placed at five different locations on each of four nights. A total of 11,948 mosquitoes was collected, and 14 mosquito pools were found to contain WN, giving a minimum infection rate between 1.08 and 7.54 per 1,000. Only one of the 80 sentinel chickens seroconverted to WN, demonstrating a single mosquito transmission event during the study and a mosquito transmission rate of between 0.8 and 1 per 1,000. Culex nigripalpus Theobald was responsible for WN transmission to the sentinel chicken, although both Cx. nigripalpus and Culex quinquefasciatus Say were found infected with WN. Mosquito transmission rates are reported in this study for the first time for a WN outbreak. This information is essential to determine risk of human and animal infection.