Prevalence of Permethrin Resistance in Culex Tarsalis Populations in Southern California.

In the western United States, Culex tarsalis is the most important vector of West Nile virus. Insecticides containing permethrin or other pyrethroid compounds are commonly used to control these mosquitoes. Because of the range of environments where Cx. tarsalis are found, this species is under insecticide pressure from both vector control and agricultural spraying. Mosquito populations may evolve resistance through mechanisms such as target site insensitivity, including the frequently identified knockdown resistance (kdr) mutations. Prevalence of permethrin resistance was determined for Cx. tarsalis from 5 southern California field sites representing 2 distinct valley regions (Coachella Valley and Inland Valley), which are geographically separated by the north-south-running Peninsular Mountain Ranges. These two valley regions are >100 km apart and vary considerably in their environmental and habitat characteristics. Permethrin resistance in mosquito populations was determined by the Centers for Disease Control and Prevention (CDC) bottle bioassay, using glass bottles coated with permethrin at 0.19 µg/cm2 of internal surface. Permethrin resistance was evident in Cx. tarsalis populations from the Coachella Valley field sites with all sites showing similar mortality in the bottle bioassay, while Cx. tarsalis from the Inland Valley field sites were largely susceptible to permethrin, with mortality rates that were similar to a susceptible lab strain of Cx. tarsalis.

Characterization of the Blood-Feeding Patterns of Culex quinquefasciatus (Diptera: Culicidae) in San Bernardino County, California.

West Nile virus (WNV) is a zoonotic disease that is endemic in North America and is known to cause a range of symptoms from mild to life threatening in humans. Culex quinquefasciatus is one of the most prominent vectors of WNV in Southern California. The goal of this study was to identify which animal species are most fed upon by these mosquitoes in various habitats in the West Valley area of San Bernardino County, California, and determine the relationship between blood-feeding patterns and WNV activity in the region. Culex quinquefasciatus specimens were collected by West Valley Mosquito and Vector Control District during 2011 from 32 different sites. The bloodmeals of 683 individuals (92.4% of those tested) were identified using the mitochondrial gene cytochrome c oxidase 1 (COI). These bloodmeals comprised 29 vertebrate species across four different habitats. Species richness (ranging from 10 to 17) was not significantly different between habitats when rarified to account for sample size. Across habitats, the highest percentage of avian bloodmeals were taken from house sparrows (18.8-39.1%) and house finches (2.6-31.5%). Bloodmeals were identified from five mammalian species, accounting for 5.1-59.2% of bloodmeals by habitat, including humans (0-4.1%). A seasonal shift towards increased mammalian bloodmeal prevalence, specifically for domestic dog and human bloodmeals, was observed in urban habitats. The WNV activity during 2011 in San Bernardino County occurred mostly in urban and suburban areas as indicated by minimum infection rate (MIR) in Culex quinquefasciatus, notable as all human bloodmeals were identified from these two habitats.

Prevalence of filarial parasites in field-caught mosquitoes in northwestern California.

Mosquitoes were collected in Lake County, CA, in 2014 and tested using standard polymerase chain reaction for filarial parasite DNA. Filarial parasites were detected in 23 out of 1,008 total pools. DNA from Dirofilaria immitis, the parasite causing dog heartworm, was detected in Aedes increpitus (MIR=4.62), Aedes sierrensis (MIR=6.72), Anopheles freeborni (MIR=1.08), and Culex tarsalis (MIR=0.10). Setaria yehi, deer body worm, was detected in Ae. sierrensis (MIR=13.42), Anopheles franciscanus (MIR=0.55), An. freeborni (MIR=2.69), and Culex stigmatosoma (MIR=0.41). The avian parasite Splendidofilaria could not be identified to species but was detected in Cx. tarsalis (MIR=0.20). DNA was also detected for three unidentified filarial parasites in Culex. Filarial-positive pools spanned May-August, with Splendidofilaria earlier in the season and S. yehi later. For D. immitis, MIR tended to be highest in June, when the 130 HDU development threshold was reached. Interestingly, D. immitis was also detected prior to the HDU threshold, and D. immitis was not detected August-September, though HDU remained high enough for development. This suggests that there are other factors influencing dog heartworm transmission in the area.

Abundance and Bloodfeeding Patterns of Mosquitoes (Diptera: Culicidae) in an Oak Woodland on the Eastern Slope of the Northern Coast Range of California.

The abundance and bloodfeeding patterns of mosquitoes was studied from 2008 to 2010 at an 18 ha. oak woodland in Lake County, CA. Host-seeking females were collected weekly from sunset to sunrise by paired dry-ice-baited CDC style traps, whereas resting females were aspirated from paired walk-in red boxes. Sequences of the COI gene amplified from bloodmeals from engorged resting females were used to identify the bloodmeal hosts. Aedes sierrensis (Ludlow) and Aedes increpitus Dyar complex mosquitoes were univoltine, although the timing of emergence and abundance varied temporally and seemed weather dependent. Abundance of both Anopheles franciscanus McCracken and Anopheles freeborni Aitken peaked in mid to late summer. Females of both genera bloodfed primarily on mule deer and black-tailed jackrabbits, and few fed on either dogs or humans that were consistently present within the woodland. In contrast, multivoltine Culex tarsalis Coquillett and Culex stigmatosoma Dyar were abundant throughout summer, especially from July to September. Both Culex species bloodfed on a wide variety of avian hosts, with most bloodmeals originating from California scrub-jay, wild turkey, oak titmouse, and house finch. Culex tarsalis fed on proportionately more mammals as summer progressed, peaking at 33% in September.

Bionomics and Vector Potential of Culex thriambus (Diptera: Culicidae) Mosquitoes in Lake County, California.

California statewide West Nile virus (WNV) minimum infection rates in Culex thriambus Dyar mosquitoes are high; however, few specimens are submitted and tested each year, as their distribution seems limited to larval habitats along riparian systems. To evaluate the role of Cx. thriambus in the amplification, maintenance, and overwintering of WNV in Lake County, CA, the bionomics and vector potential of the species was investigated during 2014 and 2015. Culex thriambus was the most abundant mosquito species, with 1,153 adults and 7,624 immatures collected by vacuum aspiration and dip sampling, respectively, at the primary study site. Detection of WNV in four mosquito pools during September through November coincided with peak seasonality. Females entered and maintained a reproductive diapause during winter under field and seminatural conditions. Diapause was initiated in the majority of Cx. thriambus females by October and was terminated by 30 March. Some parous females (7.1%) and those in host-seeking arrest (7.1%) were collected throughout the winter period. An accrual of 679.51 degree-days (°D) was necessary for diapause termination under seminatural conditions. Culex thriambus females fed on 16 different avian species during spring and summer, and no mammalian feeds were detected. West Nile viral RNA was detected in four of 42 Cx. thriambus pools tested during June through November and infection rates ranged from 3.53-28.15/1,000 tested. In summary, WNV transmission may be increased along riparian corridors throughout California where Cx. thriambus mosquitoes remain relatively abundant.

Allele-specific qRT-PCR demonstrates superior detection of single nucleotide polymorphisms as genetic markers for West Nile virus compared to Luminex® and quantitative sequencing.

To enable in vivo and in vitro competitive fitness comparisons among West Nile viruses (WNV), three reference viruses were marked genetically by site-directed mutagenesis with five synonymous nucleotide substitutions in the envelope gene region of the genome. Phenotypic neutrality of the mutants was assessed experimentally by competitive replication in cell culture and genetic stability of the substituted nucleotides was confirmed by direct sequencing. Luminex(®) technology, quantitative sequencing and quantitative RT-PCR (qRT-PCR) were compared in regard to specificity, sensitivity and accuracy for quantitation of wildtype and genetically marked viruses in mixed samples based on RNA obtained from samples of known viral titers. Although Luminex(®) technology and quantitative sequencing provided semi-quantitative or qualitative measurements, a sequence-specific primer extension approach using a specific reverse primer set in singleplex qRT-PCR demonstrated the best quantitation and specificity in the detection of RNA from wildtype and mutant viruses.

Host selection patterns of Culex tarsalis (Diptera: Culicidae) at wetlands near the Salton Sea, Coachella Valley, California, 1998-2002.

The bloodmeal hosts used by Culex tarsalis Coquillett collected along the Salton Sea in Coachella Valley, CA, during 1998-2002 were identified using sequences of the cytochrome c oxidase I gene identified from Barcode of Life database. Overall, 265 (83.3%) of 318 bloodmeals were identified, of which 76.6% fed on birds, 18.1% on mammals, and 5.3% on reptiles. Forty-seven different hosts were identified, none of which comprised > 12.5% of the total. Although Cx. tarsalis exhibits specific host-seeking flight patterns, bloodmeals seemed to be acquired opportunistically, thereby limiting potential arbovirus transmission efficiency in species-rich environments.

Phenotypic variation among Culex pipiens complex (Diptera: Culicidae) populations from the Sacramento Valley, California: horizontal and vertical transmission of West Nile virus, diapause potential, autogeny, and host selection.

The vector competence and bionomics of Culex pipiens form pipiens L. and Cx. pipiens f. molestus Forskäl were evaluated for populations from the Sacramento Valley. Both f. pipiens and f. molestus females became infected, produced disseminated infections, and were able to transmit West Nile virus. Form molestus females also transmitted West Nile virus vertically to egg rafts and F1 progeny, whereas f. pipiens females only transmitted to egg rafts. Culex pipiens complex from urban Sacramento blood-fed on seven different avian species and two mammalian species. Structure analysis of blood-fed mosquitoes identified K = 4 genetic clusters: f. molestus, f. pipiens, a group of genetically similar hybrids (Cluster X), and admixed individuals. When females were exposed as larvae to midwinter conditions in bioenvironmental chambers, 85% (N = 79) of aboveground Cx. pipiens complex females and 100% (N = 34) of underground f. molestus females did not enter reproductive diapause.

Host-selection patterns of Culex tarsalis (Diptera: Culicidae) determine the spatial heterogeneity of West Nile virus enzootic activity in northern California.

The spatial heterogeneity of West Nile virus (WNV) activity in Sutter County, CA, as measured by mosquito infection rates, was associated with spatial variation in the prevalence of Culex blood feeding on competent passeriform hosts. Overall, 42 vertebrate host species (31 avian, 11 mammal) were identified from 601 blood-fed Culex tarsalis Coquillett and 151 blood-fed Culex pipiens L. complex females using sequences of the cytochrome c oxidase I gene and the Barcode of Life Data Systems database. WNV infection rates were low at sites where the primary vector, Cx. tarsalis, fed frequently on domestic cattle or incompetent galliform birds and high when females fed frequently on American Robins, American Crows, and Yellow-billed Magpies. Opportunistic host selection by Cx. tarsalis in combination with spatial variation in the presence of highly competent corvid hosts appeared to determine the distribution of WNV activity in rural Sutter County, CA.

Evaluating sampling method bias in Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae) bloodmeal identification studies.

Determining the bloodmeal hosts of the Culex vectors of encephalitis viruses such as West Nile virus is essential for understanding the role of these mosquitoes in enzootic and epidemic transmission. Although molecular techniques have increased our knowledge of blood feeding patterns by allowing host identification to the species level, few studies have focused on the role that sampling methods may play in determining these patterns. In the current study, we identified 644 bloodmeals from Culex tarsalis Coquillett and Culex quinquefasciatus Say females collected in CO2 traps (dry ice-baited Center for Disease Control traps), in gravid traps, and aspirated from resting sites. There was no significant difference in the bloodmeal host apportionment in sampling methods such as gravid traps and resting collections that collected fully engorged females. However, CO2 traps that collected partially fed females had a significantly different apportionment of hosts than either gravid or resting collections. Bloodfed females from CO2 traps had either fed on only a small subset of available host species or were biased toward more mammalian and fewer nonpasserine avian feeds than females from other collections. Because both full and partial bloodmeals can contribute to viral transmission, obtaining Culex bloodmeal collections from multiple sampling methods may be important to fully interpret the role of these mosquitoes as maintenance and/or bridge vectors.

Mosquito host selection varies seasonally with host availability and mosquito density.

Host selection by vector mosquitoes is a critical component of virus proliferation, particularly for viruses such as West Nile (WNV) that are transmitted enzootically to a variety of avian hosts, and tangentially to dead-end hosts such as humans. Culex tarsalis is a principal vector of WNV in rural areas of western North America. Based on previous work, Cx. tarsalis utilizes a variety of avian and mammalian hosts and tends to feed more frequently on mammals in the late summer than during the rest of the year. To further explore this and other temporal changes in host selection, bloodfed females were collected at a rural farmstead and heron nesting site in Northern California from May 2008 through May 2009, and bloodmeal hosts identified using either a microsphere-based array or by sequencing of the mitochondrial cytochrome c oxidase I (COI) gene. Host composition during summer was dominated by four species of nesting Ardeidae. In addition, the site was populated with various passerine species as well as domestic farm animals and humans. When present, Cx. tarsalis fed predominantly (>80%) upon the ardeids, with Black-crowned Night-Herons, a highly competent WNV host, the most prevalent summer host. As the ardeids fledged and left the area and mosquito abundance increased in late summer, Cx. tarsalis feeding shifted to include more mammals, primarily cattle, and a high diversity of avian species. In the winter, Yellow-billed Magpies and House Sparrows were the predominant hosts, and Yellow-billed Magpies and American Robins were fed upon more frequently than expected given their relative abundance. These data demonstrated that host selection was likely based both on host availability and differences in utilization, that the shift of bloodfeeding to include more mammalian hosts was likely the result of both host availability and increased mosquito abundance, and that WNV-competent hosts were fed upon by Cx. tarsalis throughout the year.

Bloodmeal host congregation and landscape structure impact the estimation of female mosquito (Diptera: Culicidae) abundance using dry ice-baited traps.

Vegetation patterns and the presence of large numbers of nesting herons and egrets significantly altered the number of host-seeking Culex tarsalis Coquillett (Diptera: Culicidae) collected at dry ice-baited traps. The numbers of females collected per trap night at traps along the ecotone of Eucalyptus stands with and without a heron colony were always greater or equal to numbers collected at traps within or under canopy. No Cx. tarsalis were collected within or under Eucaplytus canopy during the peak heron nesting season, even though these birds frequently were infected with West Nile virus and large number of engorged females could be collected at resting boxes. These data indicate a diversion of host-seeking females from traps to nesting birds reducing sampling efficiency.

Blood-feeding patterns of the Culex pipiens complex in Sacramento and Yolo Counties, California.

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.

Effects of warm winter temperature on the abundance and gonotrophic activity of Culex (Diptera: Culicidae) in California.

Culex tarsalis Coquillett, Cx. quinquefasciatus Say, and Cx. pipiens L. were collected during the warm winter of 2009 using dry ice-baited and gravid traps and walk-in red boxes positioned in desert, urban, and agricultural habitats in Riverside, Los Angeles, Kern, and Yolo Counties. Temperatures exceeded the preceding 50 yr averages in all locations for most of January, whereas rainfall was absent or below average. Abundance of Culex species in traps during January ranged from 83 to 671% of the prior 5 yr average in all locations. Few females collected resting were in diapause during January based on follicular measurements. Evidence for early season gonotrophic activity included the detection of freshly bloodfed, gravid, and parous females in resting collections, gravid oviposition site-seeking females in gravid female traps, and nulliparous and parous host-seeking females at dry ice-baited traps. Female Culex seemed to employ multiple overwintering strategies in California, including larval and adult quiescence, adult female diapause, and an intermediate situation with adult females collected with enlarged follicles, but without evident vitellogenesis. West Nile, St. Louis, or western equine encephalitis viruses were not detected in 198 pools of adults or 56 pools of adults reared from field-collected immatures collected during January and February 2009. Our preliminary data may provide insight into how climate change may extend the mosquito season in California.

Impact of aerial spraying of pyrethrin insecticide on Culex pipiens and Culex tarsalis (Diptera: Culicidae) abundance and West Nile virus infection rates in an urban/suburban area of Sacramento County, California.

In response to an epidemic amplification of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), the Sacramento and Yolo Mosquito and Vector Control District (SYMVCD) sprayed ultralow-volume (ULV) formulations of pyrethrin insecticide (Evergreen EC 60-6: 6% pyrethrin insecticide, 60% piperonylbutoxide; MGK, Minneapolis, MN, applied as 0.003 kg/ha [0.0025 lb/acre] ) over 218 km2 in north Sacramento and 243.5 km2 in south Sacramento on three consecutive evenings in August 2005. We evaluated the impact of this intervention in north Sacramento on the abundance and WNV infection rates of Culex pipiens L. and Culex tarsalis Coquillett. Mortality rates of caged Cx. tarsalis sentinels ranged from 0% under dense canopy to 100% in open fields. A comparison of weekly geometric mean mosquito abundance in CO2-baited traps in sprayed and unsprayed areas before and after treatment indicated a 75.0 and 48.7% reduction in the abundance of Cx. pipiens and Cx. tarsalis, respectively. This reduction was statistically significant for Cx. pipiens, the primary vector of WNV, with highest abundance in this urban area, but not for Cx. tarsalis, which is more associated with rural areas. The infection rates of WNV in Cx. pipiens and Cx. tarsalis collected from the spray zone were 8.2 and 4.3 per 1,000 female mosquitoes in the 2 wk before and the 2 wk after applications of insecticide, respectively. In comparison, WNV infection rates in Cx. pipiens and Cx. tarsalis collected at same time interval in the unsprayed zone were 2.0 and 8.7 per 1,000, respectively. Based on the reduction in vector abundance and its effects on number of infective bites received by human population, we concluded that the aerial application ofpyrethrin insecticide reduced the transmission intensity of WNV and decreased the risk of human infection.

Effects of vegetation control on mosquitoes in seasonal freshwater wetlands.

Wetland restorations benefit wildlife, but wetland vegetation may promote mosquitoes. We studied the effects of removing joint grass (Paspalum distichum L.) on the abundance and distribution of immature mosquitoes in seasonal freshwater wetlands in California. In 4 >4-ha plots and one 29.8-ha field, joint grass was removed via herbicide and disking. Four untreated plots and one 4.1-ha field were controls. Fields were sampled every 7-14 days in early autumn by taking 75 dips along levees, plus a 25-dip circular transect 6 m from field edges. Untreated plots had 7 times more larvae and 20 times more pupae than did vegetation-removal plots. Similar densities of mosquitoes were found along edges and within fields.

Longitudinal survey of knockdown resistance to pyrethroid (kdr) in Mali, West Africa, and evidence of its emergence in the Bamako form of Anopheles gambiae s.s.

Studies aimed at monitoring the spread of knockdown resistance to pyrethroids (kdr) in time and space are particularly useful for detecting barriers to gene flow among the chromosomal and molecular forms of Anopheles gambiae. We used a recently developed polymerase chain reaction assay to estimate changes in kdr frequency that occurred in several mixed-form populations from Mali, West Africa, in the past decade. We found that the kdr allele significantly increased in frequency in most populations but was still absent from the M molecular form. Importantly, within the S molecular form, kdr was detected for the first time in the Bamako chromosomal form. These results provide important insights on the patterns of spread and emergence of pyrethroid knockdown resistance in West Africa.

Reduced recombination rate and genetic differentiation between the M and S forms of Anopheles gambiae s.s.

Genetic differentiation between the largely sympatric molecular forms M and S of Anopheles gambiae appears mostly limited to division 6 and part of division 5 of the X chromosome. This region is adjacent to the centromere and includes the rDNA that was used to define these forms. This localized differentiation between populations that experience gene flow strongly suggests that this region contains genes responsible for reproductive isolation. Regions adjacent to centromeres are known to experience less recombination in several species and it has recently been suggested that low recombination rates can facilitate the accumulation and maintenance of isolation genes in partially isolated populations. Therefore, we measured the recombination rate in division 5D/6 directly and estimate that it is at least 16-fold reduced across this region compared to the remainder of the X chromosome. Additionally, sequence data from four loci from field-collected mosquitoes from several West African countries show very strong differentiation between the molecular forms in division 5D/6, whereas none was observed in two loci elsewhere on the X chromosome. Furthermore, genetic variation was substantially lower in division 5D/6 compared to the two reference loci, and the inferred genealogies of the division 5D/6 genes show patterns consistent with selective sweeps. This suggests that the reduced recombination rate has increased the effect of selection on this region and that our data are consistent with the hypothesis that reduced recombination rates can play a role in the accumulation of isolation genes in the face of gene flow.