A Spatially Resolved and Environmentally Informed Forecast Model of West Nile Virus in Coachella Valley, California.

West Nile virus (WNV) is the most significant arbovirus in the United States in terms of both morbidity and mortality. West Nile exists in a complex transmission cycle between avian hosts and the arthropod vector, Culex spp. mosquitoes. Human spillover events occur when humans are bitten by an infected mosquito and predicting these rates of infection and therefore the risk to humans may be associated with fluctuations in environmental conditions. In this study, we evaluate the hydrological and meteorological drivers associated with mosquito biology and viral development to determine if these associations can be used to forecast seasonal mosquito infection rates with WNV in the Coachella Valley of California. We developed and tested a spatially resolved ensemble forecast model of the WNV mosquito infection rate in the Coachella Valley using 17 years of mosquito surveillance data and North American Land Data Assimilation System-2 environmental data. Our multi-model inference system indicated that the combination of a cooler and dryer winter, followed by a wetter and warmer spring, and a cooler than normal summer was most predictive of the prevalence of West Nile positive mosquitoes in the Coachella Valley. The ability to make accurate early season predictions of West Nile risk has the potential to allow local abatement districts and public health entities to implement early season interventions such as targeted adulticiding and public health messaging before human transmission occurs. Such early and targeted interventions could better mitigate the risk of WNV to humans.

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.

Small RNA sequencing of field Culex mosquitoes identifies patterns of viral infection and the mosquito immune response.

Mosquito-borne disease remains a significant burden on global health. In the United States, the major threat posed by mosquitoes is transmission of arboviruses, including West Nile virus by mosquitoes of the Culex genus. Virus metagenomic analysis of mosquito small RNA using deep sequencing and advanced bioinformatic tools enables the rapid detection of viruses and other infecting organisms, both pathogenic and non-pathogenic to humans, without any precedent knowledge. In this study, we sequenced small RNA samples from over 60 pools of Culex mosquitoes from two major areas of Southern California from 2017 to 2019 to elucidate the virome and immune responses of Culex. Our results demonstrated that small RNAs not only allowed the detection of viruses but also revealed distinct patterns of viral infection based on location, Culex species, and time. We also identified miRNAs that are most likely involved in Culex immune responses to viruses and Wolbachia bacteria, and show the utility of using small RNA to detect antiviral immune pathways including piRNAs against some pathogens. Collectively, these findings show that deep sequencing of small RNA can be used for virus discovery and surveillance. One could also conceive that such work could be accomplished in various locations across the world and over time to better understand patterns of mosquito infection and immune response to many vector-borne diseases in field samples.

Satellite Rearing of Aedes Mosquito Eggs: Synchronized Empirical Test of a Novel Mass Rearing Model.

Mosquito suppression strategies based on "rear and release" of male mosquitoes are attracting renewed interest from governments, municipalities, and private businesses. These include irradiation-based sterile insect technique, Wolbachia-based technologies, and genetic modification. Each of these approaches requires the mass rearing and release of adult male mosquitoes, which typically is accomplished via a rearing facility near the release site. Although some release programs have relied on centralized rearing and shipment of adult males, adult male mosquitoes are relatively fragile, and their fitness can be diminished by temperature fluctuations, humidity, nutritional deficiencies, and other stresses that occur during shipment. Furthermore, expensive, expedited shipment is typically used to maximize the amount of adult lifetime in the field following the release. In contrast, Aedes aegypti and Ae. albopictus eggs can be desiccated and stored for long periods. They are small, and many millions of eggs can be shipped without specialized environmental conditions and using less expensive means. Here we examine a model in which mosquito eggs are centrally produced and then mailed to satellite rearing facilities. As a control, a replicate set of eggs was reared at the factory of origin. At each of the rearing sites, cloud-based software was used to track and compare rearing at the different locations. The results demonstrate similar rearing outcomes (i.e., egg hatch, immature development, and number of adult males) at each of the different sites for both species. We discuss the outcome in relation to downstream applications and potential future studies.

Effect of Irrigation on the Control of Red Imported Fire Ants (Hymenoptera: Formicidae) by Water-Resistant and Standard Fire Ant Baits.

The red imported fire ant, Solenopsis invicta (Buren), is an invasive pest of agricultural, urban, and natural areas. It is also considered a public health pest due to its painful stings. While it can be efficiently controlled by commercially available fire ant baits formulated with a corn-grit carrier, rain or irrigation is thought to degrade the carrier, compromising bait effectiveness. This study assessed the effect of irrigation on the efficacy of water-resistant and standard fire ant bait formulations, by comparing worker number, brood volume, and queen survivorship after access to water-soaked baits and to irrigated, bait-treated sod. In initial testing, wetted water-resistant and standard baits reduced fire ant colonies less than dry baits, both when baits were given to colonies directly and when colonies were given access to baits broadcasted (i.e., scattered) atop sod. Comparisons of the efficacy of piled versus broadcast applications of water-resistant and standard baits revealed reductions of >88% in adults and brood and no surviving queens for all bait treatments. This result was unexpected because piled baits were hypothesized to be better protected from irrigation than broadcast bait applications. In a field study, irrigated water-resistant and standard baits caused similar and significantly higher reductions in fire ant foraging activity relative to an untreated control. These results indicated that both the water-resistant and standard fire ant bait provided significant fire ant reductions even after irrigation.

St. Louis Encephalitis Virus in the Southwestern United States: A Phylogeographic Case for a Multi-Variant Introduction Event.

Since the reemergence of St. Louis Encephalitis (SLE) Virus (SLEV) in the Southwest United States, identified during the 2015 outbreak in Arizona, SLEV has been seasonally detected within Culex spp. populations throughout the Southwest United States. Previous work revealed the 2015 outbreak was caused by an importation of SLEV genotype III, which had only been detected previously in Argentina. However, little is known about when the importation occurred or the transmission and genetic dynamics since its arrival into the Southwest. In this study, we sought to determine whether the annual detection of SLEV in the Southwest is due to enzootic cycling or new importations. To address this question, we analyzed 174 SLEV genomes (142 sequenced as part of this study) using Bayesian phylogenetic analyses to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Maricopa and Riverside counties form two distinct populations with little evidence of inter-county transmission since the onset of the outbreak. Alternatively, it appears that in 2019, Yuma and Clark counties experienced annual importations of SLEV that originated in Riverside and Maricopa counties. Finally, the earliest representatives of SLEV genotype III in the Southwest form a polytomy that includes both California and Arizona samples. We propose that the initial outbreak most likely resulted from the importation of a population of SLEV genotype III variants, perhaps in multiple birds, possibly multiple species, migrating north in 2013, rather than a single variant introduced by one bird.

No Evidence That Salt Water Ingestion Kills Adult Mosquitoes (Diptera: Culicidae).

Various products and insecticides are available that purport to reduce wild populations of adult mosquitoes. Recently, several manufacturers and general public comments on the internet have promoted devices that claim that ingestion of salt will significantly reduce populations of wild mosquitoes to near zero; there are no known scientific efficacy data that support these claims. We tested the survival of nine mosquito species of pest and public health importance across four adult diets: Water Only, Sugar Water Only (8.00%), Salt Water Only (1.03%), and Sugar + Salt Water. Species included the following: Aedes aegypti (L.), Aedes albopictus (Skuse), Aedes dorsalis (Meigen), Aedes notoscriptus (Skuse), Aedes vigilax (Skuse), Anopheles quadrimaculatus (Say), Culex pipiens (L.), Culex quinquefasciatus (Say), and Culex tarsalis (Coquillett). Male and female mosquitoes were placed in cages and allowed to feed on liquid diets under controlled environmental conditions for 1 wk. For seven of the nine species, adult survival was significantly higher in the presence (Sugar Water, Sugar + Salt Water) versus the absence (Water Only, Salt Only) of sugar, with no indication that salt had any effect on survival. Anopheles quadrimaculatus showed intermediate survival in Sugar + Salt to either Sugar Only or no sugar diets, whereas Aedes dorsalis showed low survival in Salt Only versus other diets. Based on our data and coupled with the fact that mosquitoes have physiological and behavioral adaptations that allow them to avoid or process excess salt (as found in blood meals), we conclude that there is no scientific foundation for salt-based control methods of mosquitoes.

Sunshine versus gold: The effect of population age on genetic structure of an invasive mosquito.

The genetic diversity and structure of invasive species are affected by the time since invasion, but it is not well understood how. We compare likely the oldest populations of Aedes aegypti in continental North America with some of the newest to illuminate the range of genetic diversity and structure that can be found within the invasive range of this important disease vector. Aedes aegypti populations in Florida have probably persisted since the 1600-1700s, while populations in southern California derive from new invasions that occurred in the last 10 years. For this comparison, we genotyped 1,193 individuals from 28 sites at 12 highly variable microsatellites and a subset of these individuals at 23,961 single nucleotide polymorphisms (SNPs). This is the largest sample analyzed for genetic structure for either region, and it doubles the number of southern California populations previously analyzed. As predicted, the older populations (Florida) showed fewer indicators of recent founder effect and bottlenecks; in particular, these populations have dramatically higher genetic diversity and lower genetic structure. Geographic distance and driving distance were not good predictors of genetic distance in either region, especially southern California. Additionally, southern California had higher levels of genetic differentiation than any comparably sized documented region throughout the worldwide distribution of the species. Although population age and demographic history are likely driving these differences, differences in climate and transportation practices could also play a role.

Evaluation of Two Entomopathogenic Fungi for Control of Culex quinquefasciatus (Diptera: Culicidae) in Underground Storm Drains in the Coachella Valley, California, United States.

Commercially available formulations of two entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae), were assessed for control of Culex quinquefasciatus Say (Diptera: Culicidae) in underground storm drain systems (USDS) in the Coachella Valley of southern California. Each of three treatments, the two fungi or a water control, was applied to 1 m2 of vertical wall at eight USDS sites in spring and autumn of 2015. Fungal infectivity and lethality were assessed at 1 d and 1, 2, and 4 wk post-application. Overnight bioassays using adult lab-reared female mosquitoes were carried out on the treated USDS wall areas and then mosquitoes were held in the laboratory for up to 21 d to allow fungal infections to be expressed. Postmortem fungal sporulation was assessed up to 2 wk at 100% humidity. Mosquito-fungal interactions also were assessed in bioassays of the three treatments on filter paper exposed to USDS conditions during autumn. Metarhizium anisopliae killed mosquitoes faster than B. bassiana; nevertheless, both freshly applied formulations caused greater than 80% mortality. Fungal persistence declined significantly after 1 wk under USDS conditions, but some infectivity persisted for more than 4 wk. Beauveria bassiana was more effective against Cx. qinquefasciatus in the spring, while M. anisopliae was more effective in the cooler conditions during autumn. USDS environmental conditions (e.g., temperature, relative humidity, standing water) influenced fungal-related mortality and infection of Cx. quinquefasciatus. The utility of these fungal formulations for mosquito abatement in the Coachella Valley and implications for fungal control agents in USDS environments are discussed.

Ultra-Low Volume Application of Spinosad (Natular 2EC) as a Residual in a Hot-Arid Environment Against Aedes aegypti.

The invasive Aedes aegypti is an important disease vector increasing in frequency in hot-arid regions of the USA such as the Southwest. Within hot-arid surroundings this mosquito may be confined to peridomestic locations that tend to be cooler and humid, such as in lush, irrigated ornamental vegetation surrounding homes. However, to reach these habitat refugia, ultra-low volume (ULV) applications of insecticides targeting this mosquito must retain efficacy after being sprayed from the air or street where hot-arid conditions are prevalent. We investigated the efficacy of a biologically based larvicide, spinosad (Natular 2EC), applied as a ULV in a hot-arid environment targeting Aedes aegypti. We found that this pesticide is able to penetrate this environment and has the potential to act as a residual.