A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes.

BACKGROUND & OBJECTIVES: Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations. METHODS: Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes. RESULTS: In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x104 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100. In full combination assays, CHIKV was detected to 1.32x104, DENV1-4 to 3.79x101 and ZIKV to 1.07x100. INTERPRETATION & CONCLUSION: This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.

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.

Establishment of the Invasive Aedes aegypti (Diptera: Culicidae) in the West Valley Area of San Bernardino County, CA.

The yellow fever mosquito, Aedes aegypti (Linnaeus, 1762), is the most aggressive invasive mosquito species with worldwide distribution. In addition to being a notorious nuisance species, it can pose significant public health concern because of its ability to transmit various viral pathogens. The first adult capture in the West Valley area of San Bernardino County, CA, occurred in September 2015 in Montclair. A strategic surveillance plan was implemented accordingly by the West Valley Mosquito and Vector Control District to document the infestation. The Biogent Sentinel (BG-2) trap augmented with BG-Lure and carbon dioxide (CO2) was deployed as a routine surveillance tool during 2017-2019. Extensive trapping revealed an expanding infestation, when positive trap nights (TN) increased from 14.2% in 2017 to 23.9% in 2018 and 55.6% in 2019. The average counts/TN increased from 0.65 in 2017 and 0.90 in 2018 to 3.83 in 2019. The cities of Montclair, Chino, and Ontario had much higher infestation than other cities in the district with the highest positive TN of 46.0% in Montclair, and highest average trap count of 3.23/TN in Chino. It was interesting to note that males coincided with females with more profound trend during warmer months of July to October when ratios of males ranged 28.4-35.0%. The BG-2 trap significantly outperformed the CO2 trap and gravid trap. The establishment of this invasive species in semiarid inland Southern California was further confirmed by concurrent larval collections.

Deployment and Fact Analysis of the In2Care® Mosquito Trap, A Novel Tool for Controlling Invasive Aedes Species.

During April-October 2019, the West Valley Mosquito and Vector Control District (Ontario, CA) deployed large numbers of In2Care® mosquito traps in a preliminary study to evaluate the trap's potential effectiveness at controlling invasive Aedes aegypti (L.) and Ae. albopictus (Skuse) in 6 cities of San Bernardino County, CA. The trap was used to attract ovipositing females, expose them to the juvenile hormone mimic pyriproxyfen and the entomopathogenic fungus Beauveria bassiana, and autodisseminate pyriproxyfen to other water sources prior to their death from fungal infection. The trap attracted Ae. aegypti and Culex quinquefasciatus, with the latter species predominating at much higher larval densities in the trap reservoirs. Field-collected larvae and pupae from the trap reservoirs showed complete adult emergence inhibition. Furthermore, the trap reservoirs retained high levels of residual larvicidal, pupicidal, and emergence inhibition activity after they were retrieved from the field, as indicated by laboratory bioassays against laboratory colony of Cx. quinquefasciatus. Results of this study support more detailed quantitative local evaluations on trap efficacy to measure the impact of the In2Care mosquito trap on wild invasive Aedes and Culex populations in future mosquito control efforts.

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.

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 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.