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.

Cross Resistance in S-Methoprene-Resistant Culex quinquefasciatus (Diptera: Culicidae).

The juvenile hormone analog S-methoprene is the only synthetic biopesticide that is registered with the United States Environmental Protection Agency to control arthropods of economic importance in public health, livestock, pets, urban, and stored products. The high activity, relative target specificity, and benign environmental profile of S-methoprene have been well documented. While the risk of resistance in mosquitoes to S-methoprene is generally low, there is a lack of information regarding cross resistance in S-methoprene-resistant mosquitoes to other pesticides. In this paper, a population of the southern house mosquito Culex quinquefasciatus Say from southern California acquired low levels of resistance to S-methoprene in the field, where the resistance ratios ranged 7.0- to 8.8-fold as compared with a laboratory reference colony. After 30 generations of laboratory selections by S-methoprene when resistance was elevated to 57.4- to 168.3-fold relative to an unselected population, various levels of cross resistance to other commonly used pesticides were revealed in the selected population. Cross resistance to the microbial mosquito larvicide Lysinibacillus sphaericus (Meyer & Neide) (Bacillales: Bacillaceae) was the most profound, amounting to 77.50- to 220.50-fold. The mechanism and potential management tactics toward cross resistance are discussed to preserve the unique value of this synthetic biopesticide.

Susceptibility Profile of Aedes aegypti L. (Diptera: Culicidae) from Montclair, California, to Commonly Used Pesticides, With Note on Resistance to Pyriproxyfen.

The peridomestic anthropophilic Aedes aegypti L. (Diptera: Culicidae) is originated from the wild zoophilic subspecies Aedes aegypti formosus in sub-Saharan Africa, and currently has a broad distribution in human-modified environments of the tropics and subtropics worldwide. In California, breeding populations were initially detected in 2013 in the cities of Fresno, Madera, and San Mateo, and now can be found in 188 cities of 12 counties in the state. Recent genetic studies suggest that this species invaded California on multiple occasions from several regions of the United States and northern Mexico prior to initial detection. As an invasive species and vector for numerous arboviruses, Ae. aegypti is a primary target of surveillance and control in California. In southern California city of Montclair, a population was identified in September 2015, from which a short-term colony was established in an insectary. The susceptibility of this field population to commonly used pesticides with various modes of action, including 15 formulations against larvae and four against adults, was determined, in reference to a susceptible laboratory colony of the same species. No resistance was shown to most pesticides tested. However, tolerance or reduced susceptibility to spinosad, spinetoram, diflubezuron, and fipronil was detected, and modest levels of resistance to pyriproxyfen (resistance ratio = 38.7-fold at IE50 and 81.5-fold at IE90) was observed. Results are discussed based on the field usage and modes of action of the pesticides tested. Strategic selection and application of pesticides against this population of Ae. aegypti in the urban environments should be taken into consideration.

High Resistance to Bacillus sphaericus and Susceptibility to Other Common Pesticides in Culex pipiens (Diptera: Culicidae) from Salt Lake City, UT.

Biorational mosquito larvicides based on microbial organisms and insect growth regulators (IGRs) have played a vital role in integrated mosquito control, particularly since the invasion of West Nile virus to the United States in 1999. Products that are formulated with technical powder of the bacterium, Bacillus sphaericus Neide (recently Lysinibacillus sphaericus Meyer and Neide), are among the ones that have been extensively applied to combat Culex and other mosquito species. Due to the simplicity of the binary toxins, resistance to this pesticide in laboratory and field populations of Culex pipiens L. complex has occurred globally since 1994. A Cx. pipiens population with a high level of resistance to B. sphaericus (VectoLex WDG) was identified in Salt Lake City, UT, in September 2016. The resistance ratios in this population were 20,780.0- and 23,926.9-fold at LC50 and LC90, respectively, when compared with a susceptible population of a laboratory reference colony of the same species. This B. sphaericus-resistant population remained mostly susceptible to other commonly used pesticides to control arthropods of public health and urban significance, including ones based on microbial organisms (Bacillus thuringiensis subsp. israelensis, spinosad, spinetoram, abamectin), IGRs (pyriproxyfen, methoprene, diflubenzuron, novaluron), organophosphate (temephos), neonicotinoid (imidacloprid), phenylpyrazole (fipronil), oxadiazine (indoxacarb), and pyrethroid (permethrin). Results are discussed according to the modes of action of the pesticides tested, and suggestions are made to manage B. sphaericus-resistant mosquito populations.

Impact of Storage and Handling Temperatures On the Activities of Mosquito Larvicides.

Larvicides based on Bacillus thuringiensis israelensis, Lysinibacilus sphaericus (formerly Bacillus sphaericus), Saccharopolyspora spinosa and insect growth regulators such as methoprene, and a few others are the most commonly used mosquito control products in the United States and elsewhere because of their relative specificity and high effectiveness against target organisms, safety to nontarget species, and compatibility with the environment. Using standard laboratory bioassay, we have evaluated the loss of larvicidal activities of various formulations after being exposed to different storage and handling temperatures. Transportation of products under outdoor conditions for an extended period of time caused more activity loss compared with other conditions. Results are discussed based on their active ingredients as well as physico-chemical properties of the formulations reported. Recommendations are made for proper inventory control, storage, and handling of larvicides to minimize loss of larvicidal activity.

Resistance to Lysinibacillus sphaericus and Other Commonly Used Pesticides in Culex pipiens (Diptera: Culicidae) from Chico, California.

Bacillus sphaericus Neide, recently renamed as Lysinibacillus sphaericus Meyer and Neide, is a spore-forming bacterium that possesses various levels of larvicidal activity, depending on the strains, against some mosquito species. Products based on most active strains such as 2362, 2297, 1593, C3-41 that bear binary toxins, as well as mosquitocidal toxins at various levels, have been developed to combat mosquito larvae worldwide. Resistance in wild Culex mosquito populations has been reported since 1994 from France, Brazil, India, China, Thailand, and Tunisia. Laboratory studies to evaluate resistance development risk have been conducted by many groups of scientists worldwide. Products based on L. sphaericus strain 2362 were registered in the United States in 1990s, and their use for mosquito control has been increased considerably since invasion of West Nile virus. This report documents the first occurrence of high-level resistance to L. sphaericus in a natural population of Culex pipiens L. in Chico, CA, where resistance ratio was 537.0 at LC50 and 9,048.5 at LC90 when compared with susceptible laboratory colony of the same species. Susceptibility profile to other groups of pesticides with different modes of action was also determined. Various levels of resistance or tolerance were noticed to abamectin, pyriproxyfen, permethrin, and indoxacarb. Resistance management and susceptibility monitoring strategies are discussed and recommended.

Host-Feeding Patterns of Culex stigmatosoma (Diptera: Culicidae) in Southern California.

Knowledge of the blood-feeding patterns exhibited by arthropod vectors is essential for understanding the complex dynamics of vector-borne disease transmission. Some species of mosquitoes belonging to the genus Culex have been implicated as having major roles in the transmission of arboviruses such as West Nile virus, Saint Louis encephalitis virus, and Western equine encephalitis virus. Although the host-feeding patterns for many of these Culex species are well studied, the host-feeding patterns of Culex stigmatosoma Dyar are relatively poorly studied, even though this species is suspected to be an important maintenance vector for West Nile virus and other arboviruses. In the current study, bloodmeals from 976 blood-engorged Cx. stigmatosoma, collected from 30 sites in southern California from 2009-2012, were processed for vertebrate host identification by nucleotide sequencing following polymerase chain reaction to amplify portions of the cytochrome oxidase I and cytochrome b genes of vertebrate animals. Vertebrate DNA was amplified, sequenced, and identified from a total of 647 Cx. stigmatosoma bloodmeals, revealing that 98.6% of bloodmeals were from birds, 1.2% from three mammal species, and a single bloodmeal was from a reptile species. In total, 40 different host species were identified. The greatest number of bloodmeals identified was from domestic chicken (Gallus gallus domesticus L.) (38% of bloodmeals), house sparrow (Passer domesticus L.) (23%), house finch (Haemorhous mexicanus Müller) (17%), American crow (Corvus brachyrhynchos L.) (4%), and mourning dove (Zenaida macroura L.) (3%). However, chicken bloodmeals were identified almost entirely from a single site where mosquito collection devices were placed in the near vicinity of confined domestic chickens. The strongly ornithophilic feeding behavior shown in this study for Cx. stigmatosoma supports the hypothesis that this mosquito species may be an important maintenance (or endemic) vector for arboviruses that circulate among susceptible birds.

Laboratory and field evaluation of spinosad formulation Natular T30 against immature Culex mosquitoes (Diptera: Culicidae).

Spinosad consisting of spinosyn A and D is derived from a naturally occurring, soil-dwelling bacterium, Saccharopolyspora spinosa. Spinosyns are neurotoxins that activate postsynaptic nicotinic acetylcholine and gamma-aminobutyric acid receptors and cause rapid excitation of the insect nervous system and ultimately exhaustion and death of the targets. During the past 30 yr, numerous spinosad-based formulations have been developed and applied to control various arthropod pests of agricultural importance. Natular T-30 is a new slow-release formulation containing 8.33% spinosad for use in mosquito larval control programs. High-level larvicidal activity, as indicated by low LC50 and LC90 levels, was demonstrated against Culex quinquefasciatus Say in the laboratory. Larvicidal efficacy was evaluated in semifield microcosms, field mesocosms, and underground storm drains. Fair performance against larval populations of Culex spp. and other mosquito species was achieved, although low efficacy during the initial few days posttreatment was encountered. This slow-release formulation will play an important role in controlling mosquitoes in persistent breeding sources.

Laboratory selection of resistance to spinosad in Culex quinquefasciatus (Diptera: Culicidae).

A southern house mosquito Culex quinquefasciatus Say colony was established from surviving late instars and pupae from a semifield evaluation on Natular XRG (a granular formulation containing 2.5% spinosad). The initial lethal levels ofNatular XRG against this colony were determined in the laboratory for the first-generation progeny (designated as F1). Selection pressure was applied at LC70-90 levels to 10,000-15,000 late third- and early fourth-instar larvae of each generation with Natular XRG. Susceptibility changes in response to selection were determined every other generation, where a gradual and steady decline in susceptibility occurred from generation F1 to F35, followed by significant decline from generations F37 to F45 For reference purposes, susceptibility of freshly collected wild populations as well as a laboratory colony of the same species was also determined concurrently, which fluctuated within a slightly wider range for the wild populations and a tighter range for the laboratory colony. By comparing with wild populations and laboratory reference colony, tolerance to spinosad was observed from generations up to F9 in the selected population. Resistance levels increased gradually from generation F11 to F35, and elevated significantly from generations F37 to F45, when resistance ratios reached 1,415.3- to 2,229.9-fold at LC50 and 9,613.1- to 17,062.6-fold at LC90. Possible mechanisms of resistance development to spinosad were discussed.

Cross resistances in spinosad-resistant Culex quinquefasciatus (Diptera: Culicidae).

A Culex quinquefasciatus Say colony was selected for 45 generations at LC70-90 levels using Natular XRG, a granular formulation of 2.5% spinosad for induction of spinosad resistance. Resistance to spinosad was noticed in early generations (F1-F9). Resistance levels increased gradually from generations F11-F35, and elevated significantly from generation F37 through F47, when resistance ratios reached 2,845-2,907-fold at LC50 and 11,948-22,928-fold at LC90 The spinosad-resistant Cx. quinquefasciatus colony was found not to be cross-resistant to Bacillus thuringiensis israelensis (Bti), a combination of Bti and Bacillus sphaericus, methoprene, pyriproxyfen, diflubenzuron, novaluron, temephos, or imidacloprid. However, it showed various levels of cross-resistance to B. sphaericus, spinetoram, abamectin, and fipronil. Conversely, a laboratory colony of Cx. quinquefasciatus that is highly resistant to B. sphaericus did not show cross-resistance to spinosad and spinetoram. Field-collected and laboratory-selected Cx. quinquefasciatus that showed low to moderate resistance to methoprene did not show cross-resistance to spinosad and spinetoram. Mechanisms of cross-resistance among several biorational pesticides were discussed according to their modes of actions.

Laboratory and Field Evaluations of Mosquiron® 0.12CRD, a New Formulation of Novaluron, Against Culex Mosquitoes.

Laboratory and field evaluations were conducted to assess the activity and efficacy of Mosquiron® 0.12CRD, a new formulation containing 0.12% novaluron, against immature Culex mosquitoes. In laboratory bioassays, this formulation was highly active against Culex quinquefasciatus as indicated by low inhibition of emergence (IE) values (IE50 and IE90). When Mosquiron 0.12CRD was applied at 1 briquet per underground stormwater vault, counts of late instars showed a significant reduction on day 28 posttreatment. When the late instars and pupae collected from Mosquiron-treated water were brought back to the laboratory for posttreatment observation, emergence inhibition was >90% on day 7. When the late instars from a laboratory colony of Cx. quinquefasciatus were exposed to the Mosquiron-treated water, 99% and 95% emergence inhibition was noted on day 7 and day 14, respectively. When Mosquiron 0.12CRD was applied at 11 briquets per vault, significant reductions of larval populations were encountered on days 7 and 35 posttreatment for early instars, and on days 14, 21, and 35 posttreatment for late instars. Laboratory observation of late instars and pupae sampled from the treated vault water showed nearly complete emergence inhibition from day 7 to day 28 posttreatment. A similar trend was observed in laboratory-reared late instars of Cx. quinquefasciatus when exposed to the treated water. Preliminary evaluations indicated that Mosquiron 0.12CRD is a useful new tool to control Culex mosquitoes breeding in persistent sources.

Resistance development in Culex quinquefasciatus to spinosad: a preliminary report.

A Culex quinquefasciatus colony was established from surviving late instars and pupae from a semifield microcosm test with Natular XRG (a granular formulation containing 2.5% spinosad) applied at 14.2 kg/ha. The initial lethal concentrations for 50% and 90% of the population (LC50 and LC90) against Natular XRG were determined in the laboratory for the 1st generation progeny (F1). Selection was applied at LC70-90 levels to 10,000-15,000 of late 3rd and early 4th instars each generation. Susceptibility changes were determined every other generation and referenced to a susceptible laboratory colony as well as freshly collected wild populations of the same species concurrently. Tolerance to spinosad (resistance ratio < 5.0 fold) was observed up to F8 generation in the selected population. Thereafter, resistance ratios increased significantly from F10 to F16 generations, being 7.26- to 20.52-fold at the LC50 and 7.48- to 20.08-fold at the LC90 level.

Rapid screening for sentinel chicken seroconversions against arboviruses.

A novel test procedure can screen seroconversions in sentinel chickens with the commercially available VecTest designed to detect viral antigens, such as West Nile virus. The test requires minimum laboratory equipment and skills, and provides qualitative results in about 45 min, which are immediately available to vector control agencies for making decisions to manage mosquito populations in order to interrupt arbovirus transmission.

Vector-host interactions governing epidemiology of West Nile virus in Southern California.

Southern California remains an important focus of West Nile virus (WNV) activity, with persistently elevated incidence after invasion by the virus in 2003 and subsequent amplification to epidemic levels in 2004. Eco-epidemiological studies of vectors-hosts-pathogen interactions are of paramount importance for better understanding of the transmission dynamics of WNV and other emerging mosquito-borne arboviruses. We investigated vector-host interactions and host-feeding patterns of 531 blood-engorged mosquitoes in four competent mosquito vectors by using a polymerase chain reaction (PCR) method targeting mitochondrial DNA to identify vertebrate hosts of blood-fed mosquitoes. Diagnostic testing by cell culture, real-time reverse transcriptase-PCR, and immunoassays were used to examine WNV infection in blood-fed mosquitoes, mosquito pools, dead birds, and mammals. Prevalence of WNV antibodies among wild birds was estimated by using a blocking enzyme-linked immunosorbent assay. Analyses of engorged Culex quinquefasciatus revealed that this mosquito species acquired 88.4% of the blood meals from avian and 11.6% from mammalian hosts, including humans. Similarly, Culex tarsalis fed 82% on birds and 18% on mammals. Culex erythrothorax fed on both birds (59%) and mammals (41%). In contrast, Culex stigmatosoma acquired all blood meals from avian hosts. House finches and a few other mostly passeriform birds served as the main hosts for the blood-seeking mosquitoes. Evidence of WNV infection was detected in mosquito pools, wild birds, dead birds, and mammals, including human fatalities during the study period. Our results emphasize the important role of house finches and several other passeriform birds in the maintenance and amplification of WNV in southern California, with Cx. quinquefasciatus acting as both the principal enzootic and "bridge vector" responsible for the spillover of WNV to humans. Other mosquito species, such as Cx. tarsalis and Cx. stigmatosoma, are important but less widely distributed, and also contribute to spatial and temporal transmission of WNV in southern California.

Genetic relationships among three squirrel monkey types: Implications for taxonomy, biomedical research, and captive breeding.

Fourteen electrophoretically variable and 12 monomorphic erythrocytic and serum proteins were used to determine the genetic relationships among Bolivian squirrel monkeys (Saimiri boliviensis boliviensis), Peruvian squirrel monkeys (Saimiri boliviensis peruviensis), and Guyanese squirrel monkeys (Saimiri sciureus sciureus). The results supported the classification scheme of Hershkovitz (AMERICAN JOURNAL OF PRIMATOLOGY 7:155-210, 1984), which is used above. A profile of marker phenotypes can unambiguously discriminate between the two species examined, and can discriminate most S. b. boliviensis from most S. b. peruviensis. All three groups can be distinguished unambiguously by the location of the centromeres (acrocentric or submetacentric) on chromosomes 15 and 16; however, the biochemical genetic markers provide a powerful means of detecting admixture that might not be detected cytogenetically in instances where hybridization occurred earlier than the preceding generation. The use of this panel of biochemical genetic markers, combined with karyotypic analysis, can ensure a high degree of certainty that animals selected for experimental protocols are uniform with respect to unique physiological characteristics of each species and subspecies. They also can ensure that animals selected to be members of breeding colonies are of a single species/subspecies type and reproductively compatible.