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.

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.

Identification of Volatiles From Plants Infested With Honeydew-Producing Insects, and Attraction of House Flies (Diptera: Muscidae) to These Volatiles.

House flies (Musca domestica L.) are mechanical vectors of food-borne pathogens including Salmonella spp., Escherichia coli O157:H7, and Shigella spp., resulting in increased risk of diarrheal disease in areas where flies are abundant. Movement of house flies into food crops may be increased by the presence of honeydew-producing insects feeding on these crops. Using gas chromatography-electroantennogram detection (GC-EAD) and gas chromatography-mass spectrometry (GC-MS), volatile odors that elicited house fly antennal response were identified from naval orange (Osbeck) (Sapindales: Rutaceae) and Marsh grapefruit (Macfad.) (Sapindales: Rutaceae) leaves infested with whitefly (Hemiptera: Aleyrodidae) and from whole faba (L.) (Fabales: Fabaceae) bean plants infested with aphids (Hemiptera: Aphididae). Volatiles identified included benzaldehyde, butyl hexanoate, ß-caryophyllene, Δ3-carene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (Z)-3-hexenyl acetate, myrcene, limonene, linalool, and naphthalene. This was followed by semifield bioassays of volatile blends and individual volatiles to determine house fly attraction to these volatiles. Although fly capture rates in the semifield setting were low, benzaldehyde and (Z)-3-hexenyl acetate were consistently attractive to house flies as individual compounds and as components of volatile blends.

House Fly (Musca domestica L.) Attraction to Insect Honeydew.

House flies are of major concern as vectors of food-borne pathogens to food crops. House flies are common pests on cattle feedlots and dairies, where they develop in and feed on animal waste. By contacting animal waste, house flies can acquire human pathogenic bacteria such as Escherichia coli and Salmonella spp., in addition to other bacteria, viruses, or parasites that may infect humans and animals. The subsequent dispersal of house flies from animal facilities to nearby agricultural fields containing food crops may lead to pre-harvest food contamination with these pathogens. We hypothesized that odors from honeydew, the sugary excreta produced by sucking insects feeding on crops, or molds and fungi growing on honeydew, may attract house flies, thereby increasing the risk of food crop contamination. House fly attraction to honeydew-contaminated plant material was evaluated using a laboratory bioassay. House flies were attracted to the following plant-pest-honeydew combinations: citrus mealybug on squash fruit, pea aphid on faba bean plants, whitefly on navel orange and grapefruit leaves, and combined citrus mealybug and cottony cushion scale on mandarin orange leaves. House flies were not attracted to field-collected samples of lerp psyllids on eucalyptus plants or aphids on crepe myrtle leaves. Fungi associated with field-collected honeydews were isolated and identified for further study as possible emitters of volatiles attractive to house flies. Two fungal species, Aureobasidium pullulans and Cladosporium cladosporioides, were repeatedly isolated from field-collected honeydew samples. Both fungal species were grown in potato dextrose enrichment broth and house fly attraction to volatiles from these fungal cultures was evaluated. House flies were attracted to odors from A. pullulans cultures but not to those of C. cladosporioides. Identification of specific honeydew odors that are attractive to house flies could be valuable for the development of improved house fly baits for management of this pest species.

Indirect effects of temperature on stink bug fitness, via maintenance of gut-associated symbionts.

Impacts of climate change on organisms are already apparent, with effects ranging from the individual to ecosystem scales. For organisms engaged in mutualisms, climate may affect population performance directly or indirectly through mediated effects on their mutualists. We tested this hypothesis for two stink bugs, Acrosternum hilare and Murgantia histrionica, and their gut-associated symbionts. We reared these species at two constant temperatures, 25 and 30 degrees C, and monitored population demographic parameters and the presence of gut-associated symbionts with diagnostic PCR primer sets. Both stink bugs lost their respective gut symbionts within two generations at 30 degrees C. In addition, the insect survivorship and reproductive rates of both A. hilare and M. histrionica at 30 degrees C were lower than at 25 degrees C. Other demographic parameters also indicated a decrease in overall insect fitness at the high temperature. Collectively our data showed that the decrease in host fitness was coupled with, and potentially mediated by, symbiont loss at 30 degrees C. This work illustrates the need to better understand the biology of animal-symbiont associations and the consequences of local climate for the dynamics of these interactions.