Identification of Salivary Gland Escape Barriers to Western Equine Encephalitis Virus in the Natural Vector, Culex tarsalis

Herein we describe a previously uninvestigated salivary gland escape barrier (SEB) in Culex tarsalis mosquitoes infected with two different strains of Western equine encephalitis virus (WEEV). The WEEV strains were originally isolated either from mosquitoes (IMP181) or a human patient (McMillan). Both IMP181 and McMillan viruses were fully able to infect the salivary glands of Culex tarsalis after intrathoracic injection as determined by expression of mCherry fluorescent protein. IMP181, however, was better adapted to transmission as measured by virus titer in saliva as well as transmission rates in infected mosquitoes. We used chimeric recombinant WEEV strains to show that inclusion of IMP181-derived structural genes partially circumvents the SEB.

Identification of microRNAs in the West Nile virus vector Culex tarsalis

Background: microRNAs (miRNAs) represent a group of small non-coding RNAs that are crucial gene regulators of important biological functions including development and pathogen defense in most living organisms. Presently, there is a lack of availability of information regarding the miRNAs in the mosquito Culex tarsalis, which is one of the most important vectors of West Nile virus (WNV) in the United States. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate the presence of a repertoire of miRNAs in Cx. tarsalis mosquitoes. Results: Using bioinformatic approaches we analyzed small RNA sequencing data from the Cx. tarsalis CT embryonic cell line to discover 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Cx. quinquefasciatus, mi-184 was found to be the most abundant miRNA in Cx tarsalis. We also identified an additional 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both cell line and adult Cx. tarsalis mosquitoes using RT-qPCR and sequencing. Conclusions: Cx. tarsalis is an important vector of many medically important pathogens including WNV and Western Equine encephalitis. Here we report a detailed insight into the miRNA population in Cx. tarsalis mosquitoes. These results will open new avenues of research deciphering the role of miRNAs in different Cx. tarsalis biological events such as development, metabolism, immunity and pathogen infection.

Effect of Incandescent Light on Collection of West Nile Virus Vectors Using CDC Miniature Light Traps in Northern Colorado

To evaluate whether the presence of clear incandescent light was attractive or refractive to host-seeking mosquitoes in northern Colorado, a Bayesian hierarchical model was created to measure differences in trap effectiveness based on presence or absence of phototactic cues. A total of eight CDC miniature light traps (with and without light) were set weekly across four locations in northern Colorado between Weeks 23 and 32 of year 2020. Culex mosquitoes (Diptera: Culicidae) accounted for 81% of all collections in this study with two vectors of West Nile virus being represented. The probability of catching both Culex tarsalis Coquillett and Culex pipiens Linnaeus was reduced when traps were equipped with light, but the difference was not statistically significant for Culex tarsalis. The clear reduction in the number of Culex pipiens caught when these traps were equipped with light indicates negative phototactic behavior and underestimation with current surveillance strategies. Removal of light from these traps may aid our understanding of these species’ distribution within the environment, improve collection efficiency, and help guide implementation of targeted control measures used in public health mosquito control.

Identification of Chemicals Associated Gambusia affinis (Cyprinodontiformes: Poeciliidae), and Their Effect on Oviposition Behavior of Culex tarsalis (Diptera: Culicidae) in the Laboratory

The western mosquitofish, Gambusia affinis (Baird & Girard), has been used worldwide for the control of larval mosquitoes for more than 100 yr. We found that the western encephalitis mosquito, Culex tarsalis Coquillett (Diptera: Culicidae), can detect the presence of G. affinis in oviposition sites based on associated chemicals, leading to a decrease in the number of egg rafts laid. Three volatile chemical compounds were identified in the headspace above the water where G. affinis had been held for 24 h. Oviposition bioassays conducted using standards of the volatile compounds identified (dimethyl disulfide [DMDS], dimethyl trisulfide [DMTS], and S-methyl methanethiosulphonate) found that females reduced oviposition only when low concentrations of DMTS were present, but this response was not consistent across all trials and concentrations tested. DMDS, DMTS, and S-methyl methanethiosulphonate are known bacterial metabolic waste products and may be the source of the compounds. Two nonvolatile compounds of interest were found to be present in the Gambusia-exudate water. After tasting Cx. tarsalis were deterred from ovipositing onto Gambusia-treated water from which the bacteria had been removed by filtration, indicating that the kairomone may consist of nonvolatile compound(s). One of the nonvolatile compounds isolated from the Gambusia-treated water has a benzene ring structure similar to that of cholesterol but the structure of the two nonvolatile deterrents remains to be fully characterized. Our research shows that three volatile compounds and two nonvolatile compounds are present in water associated with G. affinis (Poeciliidae: Gambusia) and affect the oviposition behavior of Cx. tarsalis in laboratory bioassays.

Laboratory demonstration of the vertical transmission of Rift Valley fever virus by Culex tarsalis mosquitoes

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus with proven ability to emerge into naïve geographic areas. Limited field evidence suggests that RVFV is transmitted vertically from parent mosquito to offspring, but until now this mechanism has not been confirmed in the laboratory. Furthermore, this transmission mechanism has allowed for the prediction of RVFV epizootics based on rainfall patterns collected from satellite information. However, in spite of the relevance to the initiation of epizootic events, laboratory confirmation of vertical transmission has remained an elusive research aim for thirty-five years. Herein we present preliminary evidence of the vertical transmission of RVFV by Culex tarsalis mosquitoes after oral exposure to RVFV. Progeny from three successive gonotrophic cycles were reared to adults, with infectious RVFV confirmed in each developmental stage. Virus was detected in ovarian tissues of parental mosquitoes 7 days after imbibing an infectious bloodmeal. Infection was confirmed in progeny as early as the first gonotrophic cycle, with infection rates ranging from 2.0–10.0%. Virus titers among progeny were low, which may indicate a host mechanism suppressing replication.

Susceptibility of Midge and Mosquito Vectors to SARS-CoV-2

SARS-CoV-2 is a recently emerged, highly contagious virus and the cause of the current COVID-19 pandemic. It is a zoonotic virus, although its animal origin is not clear yet. Person-to-person transmission occurs by inhalation of infected droplets and aerosols, or by direct contact with contaminated fomites. Arthropods transmit numerous viral, parasitic, and bacterial diseases; however, the potential role of arthropods in SARS-CoV-2 transmission is not fully understood. Thus far, a few studies have demonstrated that SARS-CoV-2 replication is not supported in cells from certain insect species nor in certain species of mosquitoes after intrathoracic inoculation. In this study, we expanded the work of SARS-CoV-2 susceptibility to biting insects after ingesting a SARS-CoV-2-infected bloodmeal. Species tested included Culicoides sonorensis (Wirth & Jones) (Diptera: Ceratopogonidae) biting midges, as well as Culex tarsalis (Coquillett) and Culex quinquefasciatus (Say) mosquitoes (Diptera: Culicidae), all known biological vectors for numerous RNA viruses. Arthropods were allowed to feed on SARS-CoV-2-spiked blood and at a time point postinfection analyzed for the presence of viral RNA and infectious virus. Additionally, cell lines derived from C. sonorensis (W8a), Aedes aegypti (Linnaeus) (Diptera: Culicidae) (C6/36), Cx. quinquefasciatus (HSU), and Cx. tarsalis (CxTrR2) were tested for SARS-CoV-2 susceptibility. Our results indicate that none of the biting insects, nor the insect cell lines evaluated support SARS-CoV-2 replication, suggesting that these species are unable to be biological vectors of SARS-CoV-2.

Infection, Dissemination, and Transmission Potential of North American Culex quinquefasciatus, Culex tarsalis, and Culicoides sonorensis for Oropouche Virus

Oropouche virus (OROV), a vector-borne Orthobunyavirus circulating in South and Central America, causes a febrile illness with high rates of morbidity but with no documented fatalities. Oropouche virus is transmitted by numerous vectors, including multiple genera of mosquitoes and Culicoides biting midges in South America. This study investigated the vector competence of three North American vectors, Culex tarsalis, Culex quinquefasciatus, and Culicoides sonorensis, for OROV. Cohorts of each species were fed an infectious blood meal containing 6.5 log10 PFU/mL OROV and incubated for 10 or 14 days. Culex tarsalis demonstrated infection (3.13%) but not dissemination or transmission potential at 10 days post infection (DPI). At 10 and 14 DPI, Cx. quinquefasciatus demonstrated 9.71% and 19.3% infection, 2.91% and 1.23% dissemination, and 0.97% and 0.82% transmission potential, respectively. Culicoides sonorensis demonstrated 86.63% infection, 83.14% dissemination, and 19.77% transmission potential at 14 DPI. Based on these data, Cx. tarsalis is unlikely to be a competent vector for OROV. Culex quinquefasciatus demonstrated infection, dissemination, and transmission potential, although at relatively low rates. Culicoides sonorensis demonstrated high infection and dissemination but may have a salivary gland barrier to the virus. These data have implications for the spread of OROV in the event of a North American introduction.

UPDATED COUNTY MOSQUITO SPECIES RECORDS FOR NORTHWEST FLORIDA

This report updates the mosquito species composition for Santa Rosa, Okaloosa, Walton, Holmes, Washington, Jackson, Calhoun, Liberty, Gadsden, Leon, Wakulla, Jefferson, Madison, and Taylor Counties, through collections made in a centralized surveillance program operated from 2002-2020 in northwest Florida. 91 county species records were documented. The most notable discoveries included finding Mansonia titillans (Walker) in eleven of the fourteen surveyed counties, Psorophora horrida (Dyar and Knab) in nine, Anopheles perplexens Ludlow in eight and Culex erraticus (Dyar and Knab) and Uranotaenia lowii Theobald in seven. Psorophora mathesoni Belkin and Heinemann and Aedes japonicus japonicus (Theobald) were found in six new counties. Culex pilosus (Dyar and Knab) was found solely in Calhoun and Liberty Cos., while Culex peccator Dyar and Knab and Culex tarsalis Coquillett were recovered in Calhoun Co. and Santa Rosa Co., respectively. Mansonia titillans, Cx. erraticus, Cx. tarsalis and Ae. j. japonicus are known arbovirus vectors, thus increasing the disease risk in this region.

Whole-genome assembly of Culex tarsalis

The mosquito, Culex tarsalis, is a key vector in the western United States due to its role in transmission of zoonotic arboviruses that affect human health. Extensive research has been conducted on Cx. tarsalis ecology, feeding behavior, vector competence, autogeny, diapause, genetics, and insecticide resistance. Population genetic analyses in the western U.S. have identified at least three genetic clusters that are geographically distinct. However, in-depth genetic studies have been hindered by the lack of a reference genome. In this study, we present the first whole-genome assembly of this mosquito species (CtarK1) based on PacBio HiFi reads from high-molecular-weight DNA extracted from a single male. The CtarK1 assembly is 790 Mb with an N50 of 58 kb, which is 27% larger than Culex quinquefasciatus (578 Mb). This difference appears to be mostly composed of transposable elements. To annotate CtarK1, we used a previously assembled Cx. tarsalis transcriptome and approximately 17,456 protein genes from Cx. quinquefasciatus (N = 17,456). Genome completeness was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) tool, which identified 84.8% of the 2799 Dipteran BUSCO genes. Using a Bayesian phylogeny based on mitochondrial genomes, we place Cx. tarsalis in the context of other mosquito species and estimate the divergence between Cx. tarsalis and Cx. quinquefasciatus to be between 15.8 and 22.2 million years ago (MYA). Important next steps from this work include characterizing the genetic basis of diapause and sex determination in Culex mosquitoes.