CRISPR-Cas9 and Cas12a target site richness reflects genomic diversity in natural populations of Anopheles gambiae and Aedes aegypti mosquitoes.

Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.

LA CROSSE VIRUS VECTOR RESTING BEHAVIORS - FIELD STUDIES WITH PROKOPAK AND RESTING SHELTER COLLECTIONS PROVIDE LOW YIELD.

Resting adult mosquito collections provide opportunities to sample broad physiological conditions (e.g., blood-engorged, gravid, nectar-engorged, and/or parous) that yield important biological information necessary to understand vector and pathogen transmission ecology. In this study, we evaluated Prokopak aspirations of Rhododendron spp. and human-powered pop-up resting shelter collections at 4 residences with historical evidence of proximal La Crosse virus (LACV) transmission from May through September 2022. The goal of this study was to investigate these sampling methods in the context of LACV vector biology-focused principally on Aedes triseriatus (primary LACV vector) and 2 invasive species (Ae. albopictus and Ae. japonicus) that likely serve as secondary LACV vectors. Overall, 304 resting shelters and 80 Prokopak collections yielded a grand total of 33 mosquitoes, of which a third were LACV vectors (Ae. triseriatus [n = 1, 3.0%], Ae. albopictus [n = 4, 12.1%], and Ae. japonicus [n = 6, 18.2%]). Anopheles punctipennis (n = 9, 27.2%) was the most frequently collected species followed by Culex erraticus (n = 7, 21.2%), whereas the least frequently collected species were Ae. triseriatus and Cx. pipiens (n = 1, 3.0%). Despite substantial collection efforts, and concurrent gravid-trap evidence of LACV vectors at the collection sites, Prokopak aspiration of Rhododendron spp. and human-powered pop-up resting shelters did not yield a meaningful number of LACV vectors and thus, as described within, may not be useful adjuncts for the evaluation of LACV ecology and disease risk. Additional approaches to evaluate the resting behavior of these vectors in LACV endemic areas are needed.

Micro-heterogeneity of transmission shapes submicroscopic malaria carriage in coastal Tanzania.

BACKGROUND: Asymptomatic carriage of malaria parasites persists even as malaria transmission declines. Low density infections are often submicroscopic, not detected by rapid diagnostic tests (RDTs) or microscopy, but detectable by PCR. METHODS: To characterize submicroscopic Plasmodium falciparum carriage in an area of declining malaria transmission, asymptomatic persons >5 years of age in rural Bagamoyo District, Tanzania, were screened using RDT, microscopy, and PCR. We investigated the size of the submicroscopic reservoir of infection across villages, determined factors associated with submicroscopic carriage, and assessed the natural history of submicroscopic malaria over four weeks. RESULTS: Among 6,076 participants, P. falciparum prevalence by RDT, microscopy, and PCR was 9%, 9%, and 28%, respectively, with roughly two-thirds of PCR-positive individuals harboring submicroscopic infection. Adult status, female gender, dry season months, screened windows, and bednet use were associated with submicroscopic carriage. Among 15 villages encompassing 80% of participants, the proportion of submicroscopic carriers increased with decreasing village-level malaria prevalence. Over four weeks, 23% (61/266) of submicroscopic carriers became RDT-positive, with half exhibiting symptoms, while half (133/266) were no longer parasitemic at the end of four weeks. Progression to RDT-positive patent malaria occurred more frequently in villages with higher malaria prevalence. CONCLUSIONS: Micro-heterogeneity in transmission observed at the village level appears to impact both the size of the submicroscopic reservoir and the likelihood of submicroscopic carriers developing patent malaria in coastal Tanzania.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining Plasmodium species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI 2.7-18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

Micro-heterogeneity of transmission shapes the submicroscopic malaria reservoir in coastal Tanzania.

Background: Asymptomatic malaria may be patent (visible by microscopy) and detectable by rapid malaria diagnostic tests (RDTs), or it may be submicroscopic and only detectable by polymerase chain reaction (PCR). Methods: To characterize the submicroscopic reservoir in an area of declining malaria transmission, asymptomatic persons >5 years of age in Bagamoyo District, Tanzania, were screened using RDT, microscopy, and PCR. We investigated the size of the submicroscopic reservoir across villages, determined factors associated with submicroscopic parasitemia, and assessed the natural history of submicroscopic malaria over four weeks. Results: Among 6,076 participants, Plasmodium falciparum prevalence by RDT, microscopy, and PCR was 9%, 9%, and 28%, respectively, with roughly two-thirds of PCR-positive individuals harboring submicroscopic infection. Adult status, female gender, dry season months, screened windows, and bednet use were associated with submicroscopic carriage. Among 15 villages encompassing 80% of participants, the proportion of submicroscopic carriers increased with decreasing village-level malaria prevalence. Over four weeks, 23% (61/266) of submicroscopic carriers became RDT-positive and were treated, with half exhibiting symptoms. This occurred more frequently in villages with higher malaria prevalence. Conclusions: Micro-heterogeneity in transmission impacts the size of the submicroscopic reservoir and the likelihood of submicroscopic carriers developing patent malaria in coastal Tanzania.

Immune-related transcripts, microbiota and vector competence differ in dengue-2 virus-infected geographically distinct Aedes aegypti populations.

BACKGROUND: Vector competence in Aedes aegypti is influenced by various factors. Crucial new control methods can be developed by recognizing which factors affect virus and mosquito interactions. METHODS: In the present study we used three geographically distinct Ae. aegypti populations and compared their susceptibility to infection by dengue virus serotype 2 (DENV-2). To identify any differences among the three mosquito populations, we evaluated expression levels of immune-related genes and assessed the presence of microbiota that might contribute to the uniqueness in their vector competence. RESULTS: Based on the results from the DENV-2 competence study, we categorized the three geographically distinct Ae. aegypti populations into a refractory population (Vilas do Atlântico), a susceptible population (Vero) and a susceptible but low transmission population (California). The immune-related transcripts were highly expressed in the California population but not in the refractory population. However, the Rel-1 gene was upregulated in the Vilas do Atlântico population following ingestion of a non-infectious blood meal, suggesting the gene's involvement in non-viral responses, such as response to microbiota. Screening of the bacteria, fungi and flaviviruses revealed differences between populations, and any of these could be one of the factors that interfere with the vector competence. CONCLUSIONS: The results reveal potential factors that might impact the virus and mosquito interaction, as well as influence the Ae. aegypti refractory phenotype.

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri species infections in human and mosquito hosts.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining malaria species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/µL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/µL (95% CI( 2.7- 18) for Pow, or 0.1 and 0.8 parasites/µL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/µL (roughly 200 parasites/µL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 10° copies/µL (<1 parasite/µL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to 14 oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovate-infected persons, mixed Poc/Pow infections were detected in 11 (79%). Based on these results, 8/9 P. ovate carriers transmitted both P. ovate species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

The Effects of Light Wavelength and Trapping Habitat on Surveillance of Culicoides Biting Midges (Diptera: Ceratopogonidae) in Alabama.

In the southeastern United States, biting midges transmit agents of hemorrhagic diseases that are enzootic among white-tailed deer (Odocoileus virginianus (Zimmermann), Artiodactyla: Cervidae). Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae), the only confirmed vector of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV) in the United States, is rarely collected in the Southeast, implying that other Culicoides Latreille species act as vectors. Despite multiple surveillance studies, the influence of trapping habitat and light wavelength on Culicoides sampling has yet to be investigated in Alabama. This study sampled Culicoides species at a deer research facility using CO2-baited CDC light traps with three distinct wavelengths. Traps were rotated within three habitats to examine impacts of habitat type and light wavelength on Culicoides abundance and parity status. For most species, midges were more abundant in a pine forest compared to a hardwood-forest riparian zone or a lightly wooded area adjacent to a seasonal pond. The pine forest generally had negative effects on parity status, suggesting that most females in this habitat were foraging for their first bloodmeal. Ultraviolet (UV) black-light (350 nm-360 nm) attracted more midges than incandescent light or UV LED light (385 nm-395 nm), but wavelength had less of an effect on parity than habitat. This study indicates that light wavelength and habitat significantly influence Culicoides sampling outcomes, and that when collecting parous females is desired (e.g., EHDV/BTV surveillance), targeting areas around oviposition sites may be a better strategy than trapping where midges are most abundant.

Intrinsic variation in the vertically transmitted core virome of the mosquito Aedes aegypti.

Virome studies among metazoans have revealed the ubiquity of RNA viruses in animals, contributing to a fundamental rethinking of the relationships between organisms and their microbiota. Mosquito viromes, often scrutinized due to their public health relevance, may also provide insight into broadly applicable concepts, such as a "core virome," a set of viruses consistently associated with a host species or population that may fundamentally impact its basic biology. A subset of mosquito-associated viruses (MAVs) could comprise such a core, and MAVs can be categorized as (i) arboviruses, which alternate between mosquito and vertebrate hosts, (ii) insect-specific viruses, which cannot replicate in vertebrate cells, and (iii) viruses with unknown specificity. MAVs have been widely characterized in the disease vector Aedes aegypti, and the occurrence of a core virome in this species has been proposed but remains unclear. Using a wild population previously surveyed for MAVs and a common laboratory strain, we investigated viromes in reproductive tissue via metagenomic RNA sequencing. Virome composition varied across samples, but four groups comprised >97% of virus sequences: a novel partiti-like virus (Partitiviridae), a toti-like virus (Totiviridae), unclassified Riboviria, and four orthomyxo-like viruses (Orthormyxoviridae). Whole or partial genomes for the partiti-like virus, toti-like virus, and one orthomyxo-like virus were assembled and analysed phylogenetically. Multigenerational maintenance of these MAVs was confirmed by RT-PCR, indicating vertical transmission as a mechanism for persistence. This study provides fundamental information regarding MAV ecology and variability in A. aegypti and the potential for vertically maintained core viromes at the population level.

Seasonality and transmissibility of Plasmodium ovale in Bagamoyo District, Tanzania.

BACKGROUND: Plasmodium ovale is a neglected malarial parasite that can form latent hypnozoites in the human liver. Over the last decade, molecular surveillance studies of non-falciparum malaria in Africa have highlighted that P. ovale is circulating below the radar, including areas where Plasmodium falciparum is in decline. To eliminate malaria where P. ovale is endemic, a better understanding of its epidemiology, asymptomatic carriage, and transmission biology is needed. METHODS: We performed a pilot study on P. ovale transmission as part of an ongoing study of human-to-mosquito transmission of P. falciparum from asymptomatic carriers. To characterize the malaria asymptomatic reservoir, cross-sectional qPCR surveys were conducted in Bagamoyo, Tanzania, over three transmission seasons. Positive individuals were enrolled in transmission studies of P. falciparum using direct skin feeding assays (DFAs) with Anopheles gambiae s.s. (IFAKARA strain) mosquitoes. For a subset of participants who screened positive for P. ovale on the day of DFA, we incubated blood-fed mosquitoes for 14 days to assess sporozoite development. RESULTS: Molecular surveillance of asymptomatic individuals revealed a P. ovale prevalence of 11% (300/2718), compared to 29% (780/2718) for P. falciparum. Prevalence for P. ovale was highest at the beginning of the long rainy season (15.5%, 128/826) in contrast to P. falciparum, which peaked later in both the long and short rainy seasons. Considering that these early-season P. ovale infections were low-density mono-infections (127/128), we speculate many were due to hypnozoite-induced relapse. Six of eight P. ovale-infected asymptomatic individuals who underwent DFAs successfully transmitted P. ovale parasites to A. gambiae. CONCLUSIONS: Plasmodium ovale is circulating at 4-15% prevalence among asymptomatic individuals in coastal Tanzania, largely invisible to field diagnostics. A different seasonal peak from co-endemic P. falciparum, the capacity to relapse, and efficient transmission to Anopheles vectors likely contribute to its persistence amid control efforts focused on P. falciparum.

Geographic Partitioning of Dengue Virus Transmission Risk in Florida.

Dengue viruses (DENVs) cause the greatest public health burden globally among the arthropod-borne viruses. DENV transmission risk has also expanded from tropical to subtropical regions due to the increasing range of its principal mosquito vector, Aedes aegypti. Focal outbreaks of dengue fever (dengue) in the state of Florida (FL) in the USA have increased since 2009. However, little is known about the competence of Ae. aegypti populations across different regions of FL to transmit DENVs. To understand the effects of DENV genotype and serotype variations on vector susceptibility and transmission potential in FL, we orally infected a colony of Ae. aegypti (Orlando/ORL) with low passage or laboratory DENV-1 through -4. Low passage DENVs were more infectious to and had higher transmission potential by ORL mosquitoes. We used these same DENVs to examine natural Ae. aegypti populations to determine whether spatial distributions correlated with differential vector competence. Vector competence across all DENV serotypes was greater for mosquitoes from areas with the highest dengue incidence in south FL compared to north FL. Vector competence for low passage DENVs was significantly higher, revealing that transmission risk is influenced by virus/vector combinations. These data support a targeted mosquito-plus-pathogen screening approach to more accurately estimate DENV transmission risk.

Efficacy of extended release formulations of Natular™ (spinosad) against larvae and adults of Anopheles mosquitoes in western Kenya.

BACKGROUND: Larval source management is recommended as a supplementary vector control measure for the prevention of malaria. Among the concerns related to larviciding is the feasibility of implementation in tropical areas with large numbers of habitats and the need for frequent application. Formulated products of spinosad that are designed to be effective for several weeks may mitigate some of these concerns. METHODS: In a semi-field study, three formulations of spinosad (emulsifiable concentrate, extended release granules and tablet formulations) were tested in naturalistic habitats in comparison to an untreated control. Cohorts of third instar Anopheles gambiae (Diptera: Culicidae) were introduced into the habitats in screened cages every week up to four weeks after application and monitored for survivorship over three days. A small-scale field trial was then conducted in two villages. Two of the spinosad formulations were applied in one village over the course of 18 months. Immature mosquito populations were monitored with standard dippers in sentinel sites and adult populations were monitored by pyrethrum spray catches. RESULTS: In the semi-field study, the efficacy of the emulsifiable concentrate of spinosad waned 1 week after treatment. Mortality in habitats treated with the extended release granular formulation of spinosad was initially high but declined gradually over 4 weeks while mortality in habitats treated with the dispersable tablet formulation was low immediately after treatment but rose to 100% through four weeks. In the field study, immature and adult Anopheles mosquito populations were significantly lower in the intervention village compared to the control village during the larviciding period. Numbers of collected mosquitoes were lower in the intervention village compared to the control village during the post-intervention period but the difference was not statistically significant. CONCLUSIONS: The extended release granular formulation and the dispersible tablet formulations of spinosad are effective against larval Anopheles mosquitoes for up to four weeks and may be an effective tool as part of larval source management programmes for reducing adult mosquito density and malaria transmission.

Biosafety at Home: How to Translate Biomedical Laboratory Safety Precautions for Everyday Use in the Context of COVID-19.

Population adoption of social distancing measures during the COVID-19 pandemic is at times deficient, increasing the risk of SARS-CoV-2 transmission. Healthcare workers and those living in areas of intense transmission may benefit from implementing biosafety measures in their daily lives. A mixed-methods approach, combining components of single negotiation text and the Delphi method, was used to create a COVID-19 biosafety-at-home protocol. A consensus building coordinator liaised with 12 experts to develop the protocol over 11 iterations. Experts had more than 200 years of combined experience in epidemiology, virology, infectious disease prevention, and public health. A flyer, created from the final protocol, was professionally designed and initially distributed via social media and institutional websites/emails in Ecuador beginning on May 2, 2020. Since then, it has been distributed in other countries, reaching ∼7,000 people. Translating research laboratory biosafety measures for the home/street environment might be challenging. The biosafety-at-home flyer addresses this challenge in a user-friendly format.

Under-the-Radar Dengue Virus Infections in Natural Populations of Aedes aegypti Mosquitoes.

The incidence of locally acquired dengue infections increased during the last decade in the United States, compelling a sustained research effort concerning the dengue mosquito vector, Aedes aegypti, and its microbiome, which has been shown to influence virus transmission success. We examined the "metavirome" of four populations of Aedes aegypti mosquitoes collected in 2016 to 2017 in Manatee County, FL. Unexpectedly, we discovered that dengue virus serotype 4 (DENV4) was circulating in these mosquito populations, representing the first documented case of such a phenomenon in the absence of a local DENV4 human case in this county over a 2-year period. We confirmed that all of the mosquito populations carried the same DENV4 strain, assembled its full genome, validated infection orthogonally by reverse transcriptase PCR, traced the virus origin, estimated the time period of its introduction to the Caribbean region, and explored the viral genetic signatures and mosquito-specific virome associations that potentially mediated DENV4 persistence in mosquitoes. We discuss the significance of prolonged maintenance of the DENV4 infections in A. aegypti that occurred in the absence of a DENV4 human index case in Manatee County with respect to the inability of current surveillance paradigms to detect mosquito vector infections prior to a potential local outbreak.IMPORTANCE Since 1999, dengue outbreaks in the continental United States involving local transmission have occurred only episodically and only in Florida and Texas. In Florida, these episodes appear to be coincident with increased introductions of dengue virus into the region through human travel and migration from countries where the disease is endemic. To date, the U.S. public health response to dengue outbreaks has been largely reactive, and implementation of comprehensive arbovirus surveillance in advance of predictable transmission seasons, which would enable proactive preventative efforts, remains unsupported. The significance of our finding is that it is the first documented report of DENV4 transmission to and maintenance within a local mosquito vector population in the continental United States in the absence of a human case during two consecutive years. Our data suggest that molecular surveillance of mosquito populations in high-risk, high-tourism areas of the United States may enable proactive, targeted vector control before potential arbovirus outbreaks.

Seasonal Changes of Host Use by Culiseta melanura (Diptera: Culicidae) in Central Florida.

The mosquito Culiseta melanura (Coquillett) is the primary enzootic vector of eastern equine encephalitis virus (EEEV), a zoonotic Alphavirus endemic to eastern North America. In its northern range, Cs. melanura is considered a strict avian biter, transmitting EEEV among susceptible birds in a cycle of enzootic amplification. In its southern range, however, Cs. melanura is more general in host use, feeding heavily upon birds but also reptiles and mammals. The goal of this study was to better understand how host use of Cs. melanura changes throughout the year in Florida, where year-round EEEV transmission is observed. Mosquitoes were sampled in 2018 from nine sites across three central Florida counties. In total, 213 Cs. melanura bloodmeals were identified by PCR consisting of 39 species of birds, reptiles, and mammals. Avian bloodmeals were prominent throughout the year (range = 30-85%), and songbirds were a large portion of identified bloodmeals (37.1%). Reptiles surpassed birds only in spring (April-June), and brown anole (Anolis sagrei Duméril and Bibron, 1837 [Reptilia: Dactyloidae]) was the most commonly detected single host species (22.1% overall). Mammalian bloodmeals were mainly observed in summer, with humans being the most fed on mammal (12.7% overall). This study reveals that in southern foci of EEEV transmission, Cs. melanura host use varies throughout the year with reptiles providing the majority of blood meals in spring (51.3%), and birds are fed on more than other host groups during all other seasons (50.6-70.1%). In addition, feeding on mammals increases during summer months, which may implicate Cs. melanura in epizootic transmission in Florida.

Biting Rates and Onchocerca Infectivity Status of Black Flies from the Simulium damnosum Complex (Diptera: Simuliidae) in Osun State, Nigeria.

The Simulium damnosum Theobald complex transmits Onchocerca volvulus Leuckart (Spirurida: Onchocercidae), the causative agent of onchocerciasis. Recent evidence suggests that control efforts have strongly suppressed parasite populations, but vector surveillance is needed in parts of Africa where the disease remains endemic. Here, studies on biting rates and infectivity status of suspected vector species were conducted in three onchocerciasis-endemic areas, namely Iwo, Ede, and Obokun, in Osun State, Nigeria. A total of 3,035 black flies were collected between October 2014 and September 2016, and examined for parity and parasites using standard methods. A separate collection of 2,000 black flies was pool-screened for infectivity using polymerase chain reaction (PCR) amplification of the O-150 marker. Results showed that parous flies were significantly less common than nulliparous flies with overall parous rates of 8.02% in Iwo and 35.38% in Ede at the end of the study period. Obokun had a parous rate of 22.22% obtained in the first year only. None of the dissected parous flies were infected with O. volvulus and PCR assays showed no amplification of O-150 O. volvulus-specific repeats in head and body pools. However, annual biting rates exceeded the World Health Organization threshold of 1,000 bites/person/yr. Thus it appears that, with such high rates of biting, even low levels of vector infection can sustain onchocerciasis in African communities.

Aedes Vector Surveillance in the Southeastern United States Reveals Growing Threat of Aedes japonicus japonicus (Diptera: Culicidae) and Aedes albopictus.

Mosquito-borne diseases are on the rise globally, and have the potential to thrive along the Gulf Coast of the United States, where subtropical conditions may facilitate the introduction or movement of mosquito vectors. Despite surveillance efforts, Aedes aegypti (L.) had not been detected in the Gulf state of Alabama for nearly three decades. The detection of Ae. aegypti in Alabama may suggest remnant or reemergent populations of this vector. We conducted adult sampling between May and August of 2018 to capture mosquitoes during a time frame when all species should be active. This was to ensure no species were missed due to overwintering and to identify the distributions of Aedes mosquitoes of medical importance. No Ae. aegypti were detected in Alabama over the period of this study. We detected Aedes albopictus (Skuse) in 65 counties and the recently invasive Aedes japonicus japonicus (Theobald) in 30 counties across the state. These results indicate that while Ae. aegypti was recently reported from parts of Alabama, the state is not experiencing a major resurgence of the species, whereas Ae. albopictus remains ubiquitous. Further, results indicate that a third wave of Aedes invasion may be occurring, that of Ae. japonicus japonicus. All three of these species are medically important vectors and may pose threats to the public health of the Gulf Coast of the United States.

Field data implicating Culicoides stellifer and Culicoides venustus (Diptera: Ceratopogonidae) as vectors of epizootic hemorrhagic disease virus.

BACKGROUND: Epizootic hemorrhagic disease virus (EHDV) is an Orbivirus of veterinary importance which is transmitted by biting midges of the genus Culicoides (Diptera: Ceratopogonidae) to ruminants. Culicoides sonorensis Wirth & Jones, the only confirmed vector of EHDV in the USA, is rare in the southeastern states where transmission persists, suggesting that other Culicoides species transmit EHDV in this region. The present study aimed to determine which Culicoides species transmitted EHDV in Florida and Alabama, two states in the southeastern USA. Viral RNA was detected in field-collected midges using molecular methods. These data are presented alongside data on Culicoides blood meal analysis, white-tailed deer (Odocoileus virginianus) aspiration, and seasonality to demonstrate an interaction between potential vector species and EHDV hosts. RESULTS: Out of 661 pools tested, 20 pools were positive for EHDV viral RNA, including six pools from Culicoides stellifer (Coquillett) and 14 pools from Culicoides venustus Hoffman. The overall infection rate was 0.06% for C. stellifer and 2.18% for C. venustus. No positive pools were identified for a further 17 species. Serotypes identified in Culicoides included EHDV-2, EHDV-6, and coinfections of EHDV-2 and EHDV-6 and were identified in similar proportions to serotypes in deer at 3 of 4 deer farms. Viral detections conducted in Alabama also identified one positive pool of C. venustus. Blood meal analysis revealed that both Culicoides species fed on white-tailed deer (verified through aspiration), fallow deer, and elk, species for which EHDV viremia has been documented. Seasonality data indicated that both species were present throughout the period in which viral transmission occurred to EHDV hosts in 2016 in addition to the 2017 epizootic. CONCLUSIONS: Our finding of EHDV positive pools of field-collected C. stellifer and C. venustus and an interaction between these species and EHDV hosts satisfy two of the four criteria for vector incrimination as set by the World Health Organization. Determining the vectors of EHDV is an important step towards developing sound strategies for the control of vector Culicoides and management of EHDV in the southeastern USA.

Homologs of Human Dengue-Resistance Genes, FKBP1B and ATCAY, Confer Antiviral Resistance in Aedes aegypti Mosquitoes.

Dengue virus (DENV) is transmitted by mosquitoes and is a major public health concern. The study of innate mosquito defense mechanisms against DENV have revealed crucial roles for the Toll, Imd, JAK-STAT, and RNAi pathways in mediating DENV in the mosquito. Often overlooked in such studies is the role of intrinsic cellular defense mechanisms that we hypothesize to work in concert with the classical immune pathways to affect organismal defense. Our understanding of the molecular interaction of DENV with mosquito host cells is limited, and we propose to expand upon the recent results from a genome-scale, small interfering RNA (siRNA)-based study that identified mammalian host proteins associated with resistance to dengue/West Nile virus (DENV/WNV) infection. The study identified 22 human DENV/WNV resistance genes (DVR), and we hypothesized that a subset would be functionally conserved in Aedes aegypti mosquitoes, imparting cellular defense against flaviviruses in this species. We identified 12 homologs of 22 human DVR genes in the Ae. aegypti genome. To evaluate their possible role in cellular resistance/antiviral defense against DENV, we used siRNA silencing targeted against each of the 12 homologs in an Ae. aegypti cell line (Aag2) infected with DENV2 and identified that silencing of the two candidates, AeFKBP1 and AeATCAY, homologs of human FKBP1B and ATCAY, were associated with a viral increase. We then used dsRNA to silence each of the two genes in adult mosquitoes to validate the observed antiviral functions in vivo. Depletion of AeFKBP1 or AeATCAY increased viral dissemination through the mosquito at 14 days post-infection. Our results demonstrated that AeFKBP1 and AeATCAY mediate resistance to DENV akin to what has been described for their homologs in humans. AeFKBP1 and AeATCAY provide a rare opportunity to elucidate a DENV-resistance mechanism that may be evolutionarily conserved between humans and Ae. aegypti.

Detection of Aedes (Stegomyia) aegypti (Diptera: Culicidae) Populations in Southern Alabama Following a 26-yr Absence and Public Perceptions of the Threat of Zika Virus.

With the establishment of Zika virus in the Americas, an accurate understanding of the geographic range of its primary vector, Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae), is vital to assessing transmission risk. In an article published in June 2016, Hahn and colleagues compiled county-level records in the United States for the presence of Ae. aegypti and Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) reported between January 1995 and March 2016. Despite ecological suitability for both mosquito species along the Gulf Coast, Ae. aegypti was not reported in Alabama during the time interval, a result consistent with research suggesting that interactions between these two species often result in displacement of Ae. aegypti. Herein, we report the detection of Ae. aegypti populations in Mobile, Alabama, after a 26-yr absence and present findings on human perceptions of Zika virus relevant to transmission. It is unclear whether the specimens (69 out of 1074) represent a recent re-introduction or belong to a previously undetected remnant population. Sequencing of mtDNA from identified Ae. aegypti matched closest to a specimen collected in Kerala, India. A survey of residents in the surveillance area suggests high encounter rates with mosquitoes in and around homes. Despite high self-reported knowledge about Zika virus, the survey revealed gaps in knowledge regarding its transmission cycle and relative degrees of vulnerability to serious illness among segments of the human population. These findings highlight the importance of continued surveillance, vector control, and public-health education in Gulf Coast states, as well as the potential threat of Ae. Aegypti-transmitted pathogens in southern Alabama.