At temperate latitudes, Culex (Diptera: Culicidae) mosquitoes typically overwinter as adult females in reproductive arrest and also may serve as reservoir hosts for arboviruses when cold temperatures arrest viral replication. To evaluate their role in the persistence of West Nile virus (WNV) in the Sacramento Valley of California, the induction and termination of diapause were investigated for members of the Culex pipiens (L.) complex, Culex tarsalis Coquillett, and Culex stigmatosoma Dyar under field, seminatural, and experimental conditions. All Culex spp. remained vagile throughout winter, enabling the collection of 3,174 females and 1,706 males from diverse habitats during the winters of 2010–2012. Overwintering strategies included both quiescence and diapause. In addition, Cx. pipiens form molestus Forskäl females remained reproductively active in both underground and aboveground habitats. Some blood-fed, gravid, and parous Cx. tarsalis and Cx. pipiens complex females were collected throughout the winter period. Under both field and experimental conditions, Cx. tarsalis and Cx. stigmatosoma females exposed to autumnal conditions arrested primary follicular maturation at previtellogenic stage I, with primary to secondary follicular ratios <1.5 (indicative of a hormonally induced diapause). In contrast, most Cx. pipiens complex females did not enter reproductive diapause and ovarian follicles matured to ≥stage I–II (host-seeking arrest) or were found in various stages of degeneration. Diapause was initiated in the majority of Cx. tarsalis and Cx. stigmatosoma females by mid-late October and was terminated after the winter solstice, but host-seeking seemed limited by temperature. An accrual of 97.52 ± 30.7 and 162.85 ± 79.3 degree-days after the winter solstice was estimated to be necessary for diapause termination in Cx. tarsalis under field and seminatural conditions, respectively. An increase in the proportion of blood-fed Culex females in resting collections occurred concurrently with diapause termination in field populations based on ovarian morphometrics. WNV RNA was detected in one pool of 18 males and in a single blood-fed female Cx. tarsalis collected during winter. Therefore, both vertically and horizontally infected Culex females may persist through winter and possibly transmit WNV after diapause termination in late winter or early spring in the Sacramento Valley of California.

SUMMARYDetection and assessment of varroa infestations in honey bee colonies are important for successful beekeeping. We examined the use of inert dusts to dislodge mites from adult honey bees that were isolated from their nest. Six dusts (powdered sugar, fine sugar, wheat flour, talcum powder, corn starch and baking soda) were evaluated for their ability to dislodge mites from adult bees collected in jars. We obtained the highest recovery rate with powdered sugar (92.9 ± 5.5%) and talcum powder (84.0 ± 5.6%). We also examined mite survival after recovery with inert dusts and compared it to mite survival after recovery from brood. After 24 h mite survival was significantly greater when mites were recovered with corn starch, powdered sugar, and from brood (F = 22.88, d.f. = 6,35, P <0.0001). Finally, ether and powdered sugar were compared as tools for detecting and assessing the degree of infestation. Powdered sugar did not differ from ether in detecting or assessing low (1-5 mites per sample) infestation levels (F = 2.81, d.f. = 1,49, P = 0.1). At medium (6-30 mites per sample) and high (> 30 mites per sample) infestation levels, more mites were recovered with powdered sugar (medium: F = 14.28, d.f. = 1,29, P = 0.008; high: F = 6.34, d.f. = 1,17, P = 0.023).

West Nile virus (WNV) has been a major public health concern in North America since 1999, when the first outbreak in the Western Hemisphere occurred in New York City. As a result of this ongoing disease outbreak, management of mosquitoes that vector WNV throughout the United States and Canada has necessitated using insecticides in areas where they traditionally have not been used or have been used less frequently. This has resulted in concerns by the public about the risks from insecticide use. The objective of this study was to use reasonable worst-case risk assessment methodologies to evaluate human-health risks for WNV and the insecticides most commonly used to control adult mosquitoes. We evaluated documented health effects from WNV infection and determined potential population risks based on reported frequencies. We determined potential acute (1-day) and subchronic (90-day) multiroute residential exposures from each insecticide for several human subgroups during a WNV disease outbreak scenario. We then compared potential insecticide exposures to toxicologic and regulatory effect levels. Risk quotients (RQs, the ratio of exposure to toxicologic effect) were < 1.0 for all subgroups. Acute RQs ranged from 0.0004 to 0.4726, and subchronic RQs ranged from 0.00014 to 0.2074. Results from our risk assessment and the current weight of scientific evidence indicate that human-health risks from residential exposure to mosquito insecticides are low and are not likely to exceed levels of concern. Further, our results indicate that, based on human-health criteria, the risks from WNV exceed the risks from exposure to mosquito insecticides.

Culex (Diptera: Culicidae) mosquitoes, the primary summer vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), also may serve as overwintering reservoir hosts. Detection of WN viral RNA from larvae hatched from eggs deposited by infected females during late summer and fall may provide evidence for the vertical passage of WNV to overwintering cohorts. To determine whether vertical transmission to the overwintering generation occurs in populations of Culex mosquitoes throughout California, larvae from naturally infected females were tested by family for WN viral RNA by real-time quantitative reverse transcription-polymerase chain reaction during August through October 2011. Viral RNA was detected in 34 of 934 Culex tarsalis Coquillett and Cx. pipiens complex females that laid viable egg rafts. From these egg rafts, Þrst-instar larvae from nine families tested positive, yielding an overall Þeld vertical transmission rate of 26% (n ϭ 34). To determine whether the WNV may be lost transtadially during development to the adult stage, Þrst-instar larvae and adult progeny from experimentally infected Cx. pipiens complex females were assessed for the presence and quantity of WN viral RNA. Most (Ϸ75%) WNV infections were lost from positive families during larval development to the adult stage. In Þeld and laboratory studies, only infected mothers with mean cycle threshold scores Յ20 vertically transmitted WNV to larval progeny, adult progeny, or both. In summary, vertical transmission of WNV was detected repeatedly in naturally infected Culex mosquitoes collected throughout California during late summer and fall, with females having high titered infections capable of passing WNV onto their progeny destined for overwintering.

Surveillance systems for West Nile virus (WNV) combine several methods to determine the location and timing of viral amplification. The value of each surveillance method must be measured against its efficiency and costs to optimize integrated vector management and suppress WNV transmission to the human population. Here we extend previous comparisons of WNV surveillance methods by equitably comparing the most common methods after standardization on the basis of spatial sampling density and costs, and by estimating optimal levels of sampling effort for mosquito traps and sentinel chicken flocks. In general, testing for evidence of viral RNA in mosquitoes and public-reported dead birds resulted in detection of WNV approximately 2-5 weeks earlier than serological monitoring of sentinel chickens at equal spatial sampling density. For a fixed cost, testing of dead birds reported by the public was found to be the most cost effective of the methods, yielding the highest number of positive results per $1000. Increased spatial density of mosquito trapping was associated with more precise estimates of WNV infection prevalence in mosquitoes. Our findings also suggested that the most common chicken flock size of 10 birds could be reduced to six to seven without substantial reductions in timeliness or sensitivity. We conclude that a surveillance system that uses the testing of dead birds reported by the public complemented by strategically timed mosquito and chicken sampling as agency resources allow would detect viral activity efficiently in terms of effort and costs, so long as susceptible bird species that experience a high mortality rate from infection with WNV, such as corvids, are present in the area.

Ultra-low-volume (ULV) aerosol applications of insecticides are used to manage high densities of adult mosquitoes. We used two-dimensional probabilistic risk assessment methodologies to evaluate three pyrethroid insecticides (phenothrin, resmethrin, and permethrin), pyrethrins, and two organophosphate insecticides (malathion and naled), applied by truck-mounted ULV sprayer. Piperonyl butoxide, a synergist commonly used in pyrethroid and pyrethrins formulations, was also assessed. The objective of our study was to evaluate probabilistically if a deterministic human-health risk assessment of mosquito insecticides was sufficiently conservative to protect human-health. Toddlers and infants were the highest risk groups while adult males were the lowest risk group assessed in this study. Total acute exposure ranged from 0.00003 to 0.0003 mg/kg day -1 for the chemicals and subgroups assessed examining inhalation, dermal, oral, and hand-to-mouth exposure. We used the risk quotient (RQ) method for our risk assessment, which is calculated by dividing the total potential exposure for each subgroup and chemical by its ingestion toxic endpoint value (RfD). Mean RQs ranged from 0.000004 to 0.034 for all subgroups and chemicals, with none exceeding the RQ level of concern. Naled had the highest RQs of any chemical assessed while PBO had the lowest. Sensitivity analysis demonstrated that the exposure from inhalation and deposition contributed the largest variance to the model output. Results support the findings of previous studies that the risks from adult mosquito management are most likely negligible, and that the human-health deterministic risk assessment is most likely sufficiently conservative.

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Mosquitoes in the Culex pipiens complex are competent vectors of West Nile virus (WNV; family Flaviviridae, genus Flavivirus) in the laboratory, and field-collected mosquitoes have tested positive for the virus in California and elsewhere. A better understanding of Cx. pipiens complex blood-feeding patterns will help define the threat that these mosquitoes pose to human health and their role in WNV amplification in northern California. We collected blood-engorged Cx. pipiens complex mosquitoes from resting sites near and away from human habitation in Sacramento and Yolo Counties. Cytochrome c oxidase 1 gene sequences were used to identify the vertebrate species from which blood meals were taken. Of 330 engorged mosquitoes collected at 28 sites from June through August 2007 and May through August 2008, >99% fed on an avian host. Three mosquitoes contained bovine blood and none had fed on a human. American Robins (Turdus migratorius) were bitten most often, and the proportion of American Robin blood meals increased significantly over the summer. Other important avian hosts included House Finches (Carpodacus mexicanus), Barn Swallows (Hirundo rustica), Western Meadowlarks (Sturnella neglecta), and Mourning Doves (Zenaida macroura). In rural areas, Barn Swallows, Brewer's Blackbirds (Euphagus cyanocephalus), and House Sparrows (Passer domesticus) were frequent hosts. In settings near human habitation, Mourning Doves and Western Meadowlarks were common hosts. Our data indicate that in north central California mosquitoes in the Cx. pipiens complex may be more important as epiornitic than epidemic vectors of WNV.