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West Nile virus (WNV) is a mosquito-borne arbovirus that remains a persistent public health challenge in the USA, with seasonal outbreaks that can lead to severe cases. In this study, we detail a real-time prediction system for WNV that incorporates an adapted compartment model to account for the transmission dynamics among birds, mosquitoes and humans, including asymptomatic cases and the influence of weather factors. Using data assimilation techniques, we generate weekly WNV case forecasts for Colorado in 2023, providing valuable insights for public health planning. Comparative analyses underscore the enhanced forecast accuracy achieved by integrating weather variables into our models., Competing Interests: We declare we have no competing interests., (© 2024 The Authors.)

Vesicular stomatitis (VS) is a viral disease that affects horses, cattle, and swine that is transmitted by direct contact and hematophagous insects. In 2023, a multi-state outbreak of vesicular stomatitis New Jersey virus (VSNJV) occurred in California, Nevada, and Texas, infecting horses, cattle, and rhinoceros. To identify possible insect vectors, we conducted insect surveillance at various locations in San Diego County, CA, including at a wildlife park. CO 2 baited traps set from mid-May to mid-August 2023 collected 2357 Culicoides biting midges and 1215 Simulium black flies, which are insect genera implicated in VSNJV transmission. Insects were pooled by species, location, and date, then tested for viral RNA. Nine RNA-positive pools of Culicoides spp. and sixteen RNA-positive pools of Simulium spp were detected. Infectious virus was detected by cytopathic effect in 96% of the RNA-positive pools. This is the first report of VSNJV in wild-caught C. bergi , C. freeborni , C. occidentalis , S. argus , S. hippovorum , and S. tescorum. The vector competency of these species for VSNJV has yet to be determined but warrants examination. Active vector surveillance and testing during disease outbreaks increases our understanding of the ecology and epidemiology of VS and informs vector control efforts.

Japanese encephalitis is a disease caused by the Japanese encephalitis virus (JEV) and is a concern for U.S. military personnel stationed in the Republic of Korea (ROK). The recent literature reports a potential shift from GI to GV as the dominant genotype circulating in east Asia. In the ROK, GV has been reported in a few Culex spp., but not in the main JEV vector, Cx . tritaeniorhynchus . The goal of this surveillance was to shed light on the current knowledge of the epidemiology of JEV in the ROK by analyzing mosquito collection data from three consecutive years, 2021-2023, and molecularly detecting and genotyping JEV in all Culex spp. collected in several military locations across the ROK. In this study, we detected only JEV GI in Cx . tritaeniorhynchus in 2021 samples. In contrast, all 2022 and 2023 positive samples were GV and detected in Cx . bitaeniorhynchus , Cx . orientalis , and Cx . pipiens . Results support a shift in JEV genotype in the ROK and suggest that for GV, Culex spp. other than Cx . tritaeniorhynchus may be playing an important role.

Vesicular stomatitis (VS) is a vector-borne livestock disease caused by the vesicular stomatitis New Jersey virus (VSNJV). This study presents the first application of an SEIR-SEI compartmental model to analyze VSNJV transmission dynamics. Focusing on the 2014-2015 outbreak in the United States, the model integrates vertebrate hosts and insect vector demographics while accounting for heterogeneous competency within the populations and observation bias in documented disease cases. Key epidemiological parameters were estimated using Bayesian inference and Markov chain Monte Carlo (MCMC) methods, including the force of infection, effective reproduction number (Rt), and incubation periods. The model revealed significant underreporting, with only 10-24% of infections documented, 23% of which presented with clinical symptoms. These findings underscore the importance of including competence and imperfect detection in disease models to depict outbreak dynamics and inform effective control strategies accurately. As a baseline model, this SEIR-SEI implementation is intended to serve as a foundation for future refinements and expansions to improve our understanding of VS dynamics. Enhanced surveillance and targeted interventions are recommended to manage future VS outbreaks.

Japanese encephalitis virus (JEV) is associated with encephalitis in humans and reproductive and neurological illness in pigs. JEV has expanded beyond its native distribution in southeast Asia, with identifications in Europe (2010) and Africa (2016), and most recently, its spread into mainland Australia (2021-2022). The introduction of JEV into the United States (US) is a public health risk, and could also impact animal health and the food supply. To efficiently and cost-effectively manage risk, a better understanding of how and where diseases will be introduced, transmitted, and spread is required. To achieve this objective, we updated our group's previous qualitative risk assessment using an established semi-quantitative risk assessment tool (MINTRISK) to compare the overall rate of introduction and risk, including impacts, of JEV in seven US regions. The rate of introduction from the current region of distribution was considered negligible for the Northeast, Midwest, Rocky Mountain, West, Alaska, and Hawaii regions. The South region was the only region with a pathway that had a non-negligible rate of introduction; infected mosquito eggs and larvae introduced via imported used tires (very low; 95% uncertainty interval (UI) = negligible to high). The overall risk estimate for the South was very high (95% UI = very low to very high). Based on this risk assessment, the South region should be prioritized for surveillance activities to ensure the early detection of JEV. The assumptions used in this risk assessment, due to the lack of information about the global movement of mosquitoes, number of feral pigs in the US, the role of non-ardeid wild birds in transmission, and the magnitude of the basic reproduction ratio of JEV in a novel region, need to be fully considered as these impact the estimated probability of establishment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 The Authors.)

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host-pathogen interactions with implications for transmission and evolution.

Culicoides are small biting midges with the capacity to transmit important livestock pathogens around much of the world, and their impacts on animal welfare are likely to expand. Hemorrhagic diseases resulting from Culicoides -vectored viruses, for example, can lead to millions of dollars in economic damages for producers. Chemical insecticides can reduce Culicoides abundance but may not suppress population numbers enough to prevent pathogen transmission. These insecticides can also cause negative effects on non-target organisms and ecosystems. RNA interference (RNAi) is a cellular regulatory mechanism that degrades mRNA and suppresses gene expression. Studies have examined the utility of this mechanism for insect pest control, and with it, have described the hurdles towards producing, optimizing, and applying these RNAi-based products. These methods hold promise for being highly specific and environmentally benign when compared to chemical insecticides and are more transient than engineering transgenic insects. Given the lack of available control options for Culicoides , RNAi-based products could be an option to treat large areas with minimal environmental impact. In this study, we describe the state of current Culicoides control methods, successes and hurdles towards using RNAi for pest control, and the necessary research required to bring an RNAi-based control method to fruition for Culicoides midges.

Mosquitoes pose a significant risk to millions of people worldwide since they can transmit pathogens. Current methods to control mosquito populations include the use of synthetic pesticides. Nanotechnology may be a solution to develop new mosquito control. However, one barrier to expanding the impact of nanomaterials is the ability to mass-produce the particles. Here, we report a novel hybrid particle synthesis combining micro- and nanoparticles using the coprecipitation technique with the potential for mass production. These particles may have applications as a mosquito larvacide. The particles reported here were designed using a microparticle zein polymer as the core and a nanoparticle silver as the active ingredient. The hybrid NPs reported here targeted a late-stage mosquito larvae and that resulted in a high larval mortality concentration (1.0 ppm, LC 90 ) and suppression of pupal emergence at 0.1 ppm. This research demonstrates the efficacy of a plant-based core with a metal-based AI coating (AgNPs) against larval mosquitoes., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Dengue fever remains a significant public health concern in many tropical and subtropical countries, and there is still a need for a system that can effectively combine global risk assessment with timely incidence forecasting. This research describes an integrated application called PICTUREE-Aedes, which can collect and analyze dengue-related data, display simulation results, and forecast outbreak incidence. PICTUREE-Aedes automatically updates global temperature and precipitation data and contains historical records of dengue incidence (1960-2012) and Aedes mosquito occurrences (1960-2014) in its database. The application utilizes a mosquito population model to estimate mosquito abundance, dengue reproduction number, and dengue risk. To predict future dengue outbreak incidence, PICTUREE-Aedes applies various forecasting techniques, including the ensemble Kalman filter, recurrent neural network, particle filter, and super ensemble forecast, which are all based on user-entered case data. The PICTUREE-Aedes' risk estimation identifies favorable conditions for potential dengue outbreaks, and its forecasting accuracy is validated by available outbreak data from Cambodia.

The most prevalent insect sampling and surveillance problem is powering insect traps in the field. Most modern light traps use 6-V power supplies such as the Centers for Disease Control and Prevention (CDC) suction trap. Buck converter modules efficiently reduce 12-V direct current power to 6-V, which permits the use of higher voltage batteries with lower voltage traps, resulting in longer operational duration and reduced labor requirements associated with replacing and recharging batteries in the field. We evaluated several battery configurations of 6- and 12-V lead-acid batteries in various sizes (10-20 ampere-hours) and addressed, in the circuit design, common problems that occur when using the buck converter (such as crossing polarity and excessive battery depletion). The efficacy of each configuration was assessed by measuring the voltage and suction while powering a 6-V CDC light trap. The buck converter permitted the use of cheaper and more commonly available 12-V batteries to run the CDC light traps and resulted in longer effective operation time as measured by air speed., (Copyright © 2023 by The American Mosquito Control Association, Inc.)

Background: West Nile virus (WNV) is the leading cause of mosquito-borne illness in the continental USA. WNV occurrence has high spatiotemporal variation, and current approaches to targeted control of the virus are limited, making forecasting a public health priority. However, little research has been done to compare strengths and weaknesses of WNV disease forecasting approaches on the national scale. We used forecasts submitted to the 2020 WNV Forecasting Challenge, an open challenge organized by the Centers for Disease Control and Prevention, to assess the status of WNV neuroinvasive disease (WNND) prediction and identify avenues for improvement., Methods: We performed a multi-model comparative assessment of probabilistic forecasts submitted by 15 teams for annual WNND cases in US counties for 2020 and assessed forecast accuracy, calibration, and discriminatory power. In the evaluation, we included forecasts produced by comparison models of varying complexity as benchmarks of forecast performance. We also used regression analysis to identify modeling approaches and contextual factors that were associated with forecast skill., Results: Simple models based on historical WNND cases generally scored better than more complex models and combined higher discriminatory power with better calibration of uncertainty. Forecast skill improved across updated forecast submissions submitted during the 2020 season. Among models using additional data, inclusion of climate or human demographic data was associated with higher skill, while inclusion of mosquito or land use data was associated with lower skill. We also identified population size, extreme minimum winter temperature, and interannual variation in WNND cases as county-level characteristics associated with variation in forecast skill., Conclusions: Historical WNND cases were strong predictors of future cases with minimal increase in skill achieved by models that included other factors. Although opportunities might exist to specifically improve predictions for areas with large populations and low or high winter temperatures, areas with high case-count variability are intrinsically more difficult to predict. Also, the prediction of outbreaks, which are outliers relative to typical case numbers, remains difficult. Further improvements to prediction could be obtained with improved calibration of forecast uncertainty and access to real-time data streams (e.g. current weather and preliminary human cases)., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Proper vector surveillance relies on the ability to identify species of interest accurately and efficiently, though this can be difficult in groups containing cryptic species. Culicoides Latreille is a genus of small biting flies responsible for the transmission of numerous pathogens to a multitude of vertebrates. Regarding pathogen transmission, the C. variipennis species complex is of particular interest in North America. Of the six species within this group, only C. sonorensis Wirth & Jones is a proven vector of bluetongue virus and epizootic hemorrhagic disease virus. Unfortunately, subtle morphological differences, cryptic species, and mitonuclear discordance make species identification in the C. variipennis complex challenging. Recently, single-nucleotide polymorphism (SNP) analysis enabled discrimination between the species of this group; however, this demanding approach is not practical for vector surveillance., Methods: The aim of the current study was to develop a reliable and affordable way of distinguishing between the species within the C. variipennis complex, especially C. sonorensis. Twenty-five putative microsatellite markers were identified using the C. sonorensis genome and tested for amplification within five species of the C. variipennis complex. Machine learning was then used to determine which markers best explain the genetic differentiation between species. This led to the development of a subset of four and seven markers, which were also tested for species differentiation., Results: A total of 21 microsatellite markers were successfully amplified in the species tested. Clustering analyses of all of these markers recovered the same species-level identification as the previous SNP data. Additionally, the subset of seven markers was equally capable of accurately distinguishing between the members of the C. variipennis complex as the 21 microsatellite markers. Finally, one microsatellite marker (C508) was found to be species-specific, only amplifying in the vector species C. sonorensis among the samples tested., Conclusions: These microsatellites provide an affordable way to distinguish between the sibling species of the C. variipennis complex and could lead to a better understanding of the species dynamics within this group. Additionally, after further testing, marker C508 may allow for the identification of C. sonorensis with a single-tube assay, potentially providing a powerful new tool for vector surveillance in North America., (© 2022. The Author(s).)

The inability to distinguish between species can be a serious problem in groups responsible for pathogen transmission. Culicoides biting midges transmit many pathogenic agents infecting wildlife and livestock. In North America, the C. variipennis species complex contains three currently recognized species, only one of which is a known vector, but limited species-specific characters have hindered vector surveillance. Here, genomic data were used to investigate population structure and genetic differentiation within this species complex. Single nucleotide polymorphism data were generated for 206 individuals originating from 17 locations throughout the United States and Canada. Clustering analyses suggest the occurrence of two additional cryptic species within this complex. All five species were significantly differentiated in both sympatry and allopatry. Evidence of hybridization was detected in three different species pairings indicating incomplete reproductive isolation. Additionally, COI sequences were used to identify the hybrid parentage of these individuals, which illuminated discordance between the divergence of the mitochondrial and nuclear datasets., (© 2022. The Author(s).)

Epizootic hemorrhagic disease (EHD) is an insect-transmitted viral disease of wild and domestic ruminants. It was first described following a 1955 epizootic in North American white-tailed deer ( Odocoileus virginianus ), a species which is highly susceptible to the causative agent of EHD, epizootic hemorrhagic disease virus (EHDV). EHDV has been detected globally across tropical and temperate regions, largely corresponding to the presence of Culicoides spp. biting midges which transmit the virus between ruminant hosts. It regularly causes high morbidity and mortality in wild and captive deer populations in endemic areas during epizootics. Although cattle historically have been less susceptible to EHDV, reports of clinical disease in cattle have increased in the past two decades. There is a pressing need to identify new methods to prevent and mitigate outbreaks and reduce the considerable impacts of EHDV on livestock and wildlife. This review discusses recent research advancements towards the control of EHDV, including the development of new investigative tools and progress in basic and applied research focused on virus detection, disease mitigation, and vector control. The potential impacts and implications of these advancements on EHD management are also discussed.

Mosquito larvae encounter diverse assemblages of bacteria (i.e., "microbiota") and fungi in the aquatic environments that they develop in. However, while a number of studies have addressed the diversity and function of microbiota in mosquito life history, relatively little is known about mosquito-fungus interactions outside several key fungal entomopathogens. In this study, we used high-throughput sequencing of internal transcribed spacer 2 (ITS2) metabarcode markers to provide the first simultaneous characterization of the fungal communities in field-collected Aedes albopictus larvae and their associated aquatic environments. Our results reveal unprecedented variation in fungal communities among adjacent but discrete larval breeding habitats. Our results also reveal a distinct fungal community assembly in the mosquito gut versus other tissues, with gut-associated fungal communities being most similar to those present in the environment where larvae feed. Altogether, our results identify the environment as the dominant factor shaping the fungal community associated with mosquito larvae, with no evidence of environmental filtering by the gut. These results also identify mosquito feeding behavior and fungal mode of nutrition as potential drivers of tissue-specific fungal community assembly after environmental acquisition. IMPORTANCE The Asian tiger mosquito, Aedes albopictus, is the dominant mosquito species in the United States and an important vector of arboviruses of major public health concern. One aspect of mosquito control to curb mosquito-borne diseases has been the use of biological control agents such as fungal entomopathogens. Recent studies also demonstrate the impact of mosquito-associated microbial communities on various mosquito traits, including vector competence. However, while much research attention has been dedicated to understanding the diversity and function of mosquito-associated bacterial communities, relatively little is known about mosquito-associated fungal communities. A better understanding of the factors that drive fungal community diversity and assembly in mosquitoes will be essential for future efforts to target mosquito-associated bacteria and fungi for mosquito and mosquito-borne disease control.

Mosquito-borne West Nile virus (WNV) is the causative agent of West Nile disease in humans, horses, and some bird species. Since the initial introduction of WNV to the United States (US), approximately 30,000 horses have been impacted by West Nile neurologic disease and hundreds of additional horses are infected each year. Research describing the drivers of West Nile disease in horses is greatly needed to better anticipate the spatial and temporal extent of disease risk, improve disease surveillance, and alleviate future economic impacts to the equine industry and private horse owners. To help meet this need, we integrated techniques from spatiotemporal epidemiology, eco-phylogenetics, and distributional ecology to assess West Nile disease risk in horses throughout the contiguous US. Our integrated approach considered horse abundance and virus exposure, vector and host distributions, and a variety of extrinsic climatic, socio-economic, and environmental risk factors. Birds are WNV reservoir hosts, and therefore we quantified avian host community dynamics across the continental US to show intra-annual variability in host phylogenetic structure and demonstrate host phylodiversity as a mechanism for virus amplification in time and virus dilution in space. We identified drought as a potential amplifier of virus transmission and demonstrated the importance of accounting for spatial non-stationarity when quantifying interaction between disease risk and meteorological influences such as temperature and precipitation. Our results delineated the timing and location of several areas at high risk of West Nile disease and can be used to prioritize vaccination programs and optimize virus surveillance and monitoring.

Vesicular stomatitis (VS) is a vector-borne livestock disease caused by vesicular stomatitis New Jersey virus (VSNJV) or vesicular stomatitis Indiana virus (VSIV). The disease circulates endemically in northern South America, Central America, and Mexico and only occasionally causes outbreaks in the United States. Over the past 20 years, VSNJV outbreaks in the southwestern and Rocky Mountain regions occurred with incursion years followed by virus overwintering and subsequent expansion outbreak years. Regulatory response by animal health officials is deployed to prevent spread from lesioned animals. The 2019 VS incursion was the largest in 40 years, lasting from June to December 2019 with 1144 VS-affected premises in 111 counties in eight states (Colorado, Kansas, Nebraska, New Mexico, Oklahoma, Texas, Utah, and Wyoming) and was VSIV serotype, last isolated in 1998. A subsequent expansion occurred from April to October 2020 with 326 VS-affected premises in 70 counties in eight states (Arizona, Arkansas, Kansas, Missouri, Nebraska, New Mexico, Oklahoma, and Texas). The primary serotype in 2020 was VSIV, but a separate incursion of VSNJV occurred in south Texas. Summary characteristics of the outbreaks are presented along with VSV-vector sampling results and phylogenetic analysis of VSIV isolates providing evidence of virus overwintering.

Bluetongue virus (BTV) is a segmented RNA virus transmitted by Culicoides midges. Climatic factors, animal movement, vector species, and viral mutation and reassortment may all play a role in the occurrence of BTV outbreaks among susceptible ruminants. We used two enzootic strains of BTV (BTV-2 and BTV-10) to explore the potential for Culicoides sonorensis , a key North American vector, to be infected with these viruses, and identify the impact of temperature variations on virogenesis during infection. While BTV-10 replicated readily in C. sonorensis following an infectious blood meal, BTV-2 was less likely to result in productive infection at biologically relevant exposure levels. Moreover, when C. sonorensis were co-exposed to both viruses, we did not detect reassortment between the two viruses, despite previous in vitro findings indicating that BTV-2 and BTV-10 are able to reassort successfully. These results highlight that numerous factors, including vector species and exposure dose, may impact the in vivo replication of varying BTV strains, and underscore the complexities of BTV ecology in North America.

West Nile virus (WNV) is the most common arthropod-borne virus (arbovirus) in the United States (US) and is the leading cause of viral encephalitis in the country. The virus has affected tens of thousands of US persons total since its 1999 North America introduction, with thousands of new infections reported annually. Approximately 1% of humans infected with WNV acquire neuroinvasive West Nile Disease (WND) with severe encephalitis and risk of death. Research describing WNV ecology is needed to improve public health surveillance, monitoring, and risk assessment. We applied Bayesian joint-spatiotemporal modeling to assess the association of vector surveillance data, host species richness, and a variety of other environmental and socioeconomic disease risk factors with neuroinvasive WND throughout the conterminous US. Our research revealed that an aging human population was the strongest disease indicator, but climatic and vector-host biotic interactions were also significant in determining risk of neuroinvasive WND. Our analysis also identified a geographic region of disproportionately high neuroinvasive WND disease risk that parallels the Continental Divide, and extends southward from the US-Canada border in the states of Montana, North Dakota, and Wisconsin to the US-Mexico border in western Texas. Our results aid in unraveling complex WNV ecology and can be applied to prioritize disease surveillance locations and risk assessment.

Vector-borne disease risk assessment is crucial to optimize surveillance, preventative measures (vector control), and resource allocation (medical supplies). High arthropod abundance and host interaction strongly correlate to vector-borne pathogen transmission. Increasing host density and movement increases the possibility of local and long-distance pathogen transmission. Therefore, we developed a risk-assessment framework using climate (average temperature and rainfall) and host demographic (host density and movement) data, particularly suitable for regions with unreported or underreported incidence data. This framework consisted of a spatiotemporal network-based approach coupled with a compartmental disease model and nonhomogeneous Gillespie algorithm. The correlation of climate data with vector abundance and host-vector interactions is expressed as vectorial capacity-a parameter that governs the spreading of infection from an infected host to a susceptible one via vectors. As an example, the framework is applied for dengue in Bangladesh. Vectorial capacity is inferred for each week throughout a year using average monthly temperature and rainfall data. Long-distance pathogen transmission is expressed with human movement data in the spatiotemporal network. We have identified the spatiotemporal suitability of dengue spreading in Bangladesh as well as the significant-incidence window and peak-incidence period. Analysis of yearly dengue data variation suggests the possibility of a significant outbreak with a new serotype introduction. The outcome of the framework comprised spatiotemporal suitability maps and probabilistic risk maps for spatial infection spreading. This framework is capable of vector-borne disease risk assessment without historical incidence data and can be a useful tool for preparedness with accurate human movement data.

Biting midges of the genus Culicoides transmit disease-causing agents resulting in a significant economic impact on livestock industries in many parts of the world. Localized control efforts, such as removal of larval habitat or pesticide application, can be logistically difficult, expensive and ineffective if not instituted and maintained properly. With these limitations, a population-level approach to the management of Culicoides midges should be investigated as a means to replace or supplement existing control strategies. Next-generation control methods such as Wolbachia- and genetic-based population suppression and replacement are being investigated in several vector species. Here we assess the feasibility and applicability of these approaches for use against biting midges. We also discuss the technical and logistical hurdles needing to be addressed for each method to be successful, as well as emphasize the importance of addressing community engagement and involving stakeholders in the investigation and development of these approaches.

Mosquito-borne viruses will continue to emerge and generate a significant public health burden around the globe. Here, we provide a longitudinal perspective on how the emergence of mosquito-borne viruses in the Americas has triggered reactionary funding by sponsored agencies, stimulating a number of publications, innovative development of traps, and augmented capacity. We discuss the return on investment (ROI) from the oscillation in federal funding that influences demand for surveillance and control traps and leads to innovation and research productivity., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Japanese encephalitis (JE) is a zoonotic, emerging disease transmitted by mosquito vectors infected with the Japanese encephalitis virus (JEV). Its potential for emergence into susceptible regions is high, including in the United States (US), and is a reason of economic concern among the agricultural community, and to public health due to high morbidity and mortality rates in humans. While exploring the complexities of interactions involved with viral transmission, we proposed a new outlook on the role of vectors, hosts and the environment under changing conditions. For instance, the role of feral pigs may have been underappreciated in our previous work, given research keeps pointing to the importance of susceptible populations of wild swine in naïve regions as key elements for the introduction of emergent vector-borne diseases. High risk of JEV introduction has been associated with the transportation of infected mosquitoes via aircraft. Nonetheless, no JEV outbreaks have been reported in the US to date and results from a qualitative risk assessment considered the risk of establishment to be negligible under the current conditions (environmental, vector, pathogen, and host). In this work, we discuss virus-vector-host interactions and ecological factors important for virus transmission and spread, review research on the risk of JEV introduction to the US considering the implications of risk dismissal as it relates to past experiences with similar arboviruses, and reflect on future directions, challenges, and implications of a JEV incursion., (Copyright © 2020 Oliveira, Cohnstaedt, Noronha, Mitzel, McVey and Cernicchiaro.)

As an emerging infectious disease, the 2019 coronavirus disease (COVID-19) has developed into a global pandemic. During the initial spreading of the virus in China, we demonstrated the ensemble Kalman filter performed well as a short-term predictor of the daily cases reported in Wuhan City. Second, we used an individual-level network-based model to reconstruct the epidemic dynamics in Hubei Province and examine the effectiveness of non-pharmaceutical interventions on the epidemic spreading with various scenarios. Our simulation results show that without continued control measures, the epidemic in Hubei Province could have become persistent. Only by continuing to decrease the infection rate through 1) protective measures and 2) social distancing can the actual epidemic trajectory that happened in Hubei Province be reconstructed in simulation. Finally, we simulate the COVID-19 transmission with non-Markovian processes and show how these models produce different epidemic trajectories, compared to those obtained with Markov processes. Since recent studies show that COVID-19 epidemiological parameters do not follow exponential distributions leading to Markov processes, future works need to focus on non-Markovian models to better capture the COVID-19 spreading trajectories. In addition, shortening the infectious period via early case identification and isolation can slow the epidemic spreading significantly., Competing Interests: None., (© 2020 The Authors.)

Background: Biting midges of the genus Culicoides vector multiple veterinary pathogens and are difficult to control. Endosymbionts particularly Wolbachia pipientis may offer an alternative to control populations of Culicoides and/or impact disease transmission in the form of population suppression or replacement strategies., Methods: Culicoides sonorensis cell lines were transfected with a Wolbachia infection using a modified shell vial technique. Infections were confirmed using PCR and cell localization using fluorescent in situ hybridization (FISH). The stability of Wolbachia infections and density was determined by qPCR. qPCR was also used to examine immune genes in the IMD, Toll and JACK/STAT pathways to determine if Wolbachia were associated with an immune response in infected cells., Results: Here we have transfected two Culicoides sonorensis cell lines (W3 and W8) with a Wolbachia infection (walbB) from donor Aedes albopictus Aa23 cells. PCR and FISH showed the presence of Wolbachia infections in both C. sonorensis cell lines. Infection densities were higher in the W8 cell lines when compared to W3. In stably infected cells, genes in the immune Toll, IMD and JAK/STAT pathways were upregulated, along with Attacin and an Attacin-like anti-microbial peptides., Conclusions: The successful introduction of Wolbachia infections in C. sonorensis cell lines and the upregulation of immune genes, suggest the utility of using Wolbachia for a population replacement and/or population suppression approach to limit the transmission of C. sonorensis vectored diseases. Results support the further investigation of Wolbachia induced pathogen inhibitory effects in Wolbachia-infected C. sonorensis cell lines and the introduction of Wolbachia into C. sonorensis adults via embryonic microinjection to examine for reproductive phenotypes and host fitness effects of a novel Wolbachia infection.

The recent outbreaks of the insect-vectored Zika virus have demonstrated its potential to be sexually transmitted, which complicates modeling and our understanding of disease dynamics. Autochthonous outbreaks in the US mainland may be a consequence of both modes of transmission, which affect the outbreak size, duration, and virus persistence. We propose a novel individual-based interconnected network model that incorporates both insect-vectored and sexual transmission of this pathogen. This model interconnects a homogeneous mosquito vector population with a heterogeneous human host contact network. The model incorporates the seasonal variation of mosquito abundance and characterizes host dynamics based on age group and gender in order to produce realistic projections. We use a sexual contact network which is generated on the basis of real world sexual behavior data. Our findings suggest that for a high relative transmissibility of asymptomatic hosts, Zika virus shows a high probability of sustaining in the human population for up to 3 months without the presence of mosquito vectors. Zika outbreaks are strongly affected by the large proportion of asymptomatic individuals and their relative transmissibility. The outbreak size is also affected by the time of the year when the pathogen is introduced. Although sexual transmission has a relatively low contribution in determining the epidemic size, it plays a role in sustaining the epidemic and creating potential endemic scenarios.

West Nile virus (WNV)-a mosquito-borne arbovirus-entered the USA through New York City in 1999 and spread to the contiguous USA within three years while transitioning from epidemic outbreaks to endemic transmission. The virus is transmitted by vector competent mosquitoes and maintained in the avian populations. WNV spatial distribution is mainly determined by the movement of residential and migratory avian populations. We developed an individual-level heterogeneous network framework across the USA with the goal of understanding the long-range spatial distribution of WNV. To this end, we proposed three distance dispersal kernels model: 1) exponential-short-range dispersal, 2) power-law-long-range dispersal in all directions, and 3) power-law biased by flyway direction -long-range dispersal only along established migratory routes. To select the appropriate dispersal kernel we used the human case data and adopted a model selection framework based on approximate Bayesian computation with sequential Monte Carlo sampling (ABC-SMC). From estimated parameters, we find that the power-law biased by flyway direction kernel is the best kernel to fit WNV human case data, supporting the hypothesis of long-range WNV transmission is mainly along the migratory bird flyways. Through extensive simulation from 2014 to 2016, we proposed and tested hypothetical mitigation strategies and found that mosquito population reduction in the infected states and neighboring states is potentially cost-effective., Competing Interests: The authors have declared that no competing interests exist.

This work describes the use of entomological studies combined with in silico models (computer simulations derived from numerical models) to assess the efficacy of a novel device for controlled release of spatial repellents. Controlled Release Devices (CRDs) were tested with different concentrations of metofluthrin and tested against An. quadrimaculatus mosquitoes using arm-in cage, semi-field, and outdoor studies. Arm-in-cage trials showed an approximate mean values for mosquito knockdown of 40% and mosquito bite reduction of 80% for the optimal metofluthrin formulation for a 15-minute trial. Semi-field outdoor studies showed a mean mortality of a 50% for 24 hour trial and 75% for a 48 hour trial for optimal concentrations. Outdoors studies showed an approximate mean mortality rate of 50% for a 24 hour trial for optimal concentrations. Numerical simulations based on Computational Fluid Dynamics (CFD) were performed in order to obtain spatial concentration profiles for 24 hour and 48 hour periods. Experimental results were correlated with simulation results in order to obtain a functional model that linked mosquito mortality with the estimated spatial concentration for a given period of time. Such correlation provides a powerful insight in predicting the effectiveness of the CRDs as a vector-control tool. While CRDs represent an alternative to current spatial repellent delivery methods, such as coils, candles, electric repellents, and passive emanators based on impregnated strips, the presented method can be applied to any spatial vector control treatment by correlating entomological endpoints, i.e. mortality, with in-silico simulations to predict overall efficacy. The presented work therefore presents a new methodology for improving design, development and deployment of vector-control tools to reduce transmission of vector-borne diseases, including malaria and dengue., Competing Interests: NME and PG have been associated with GearJump Technologies, LLC during performance of this research work. MP and NME work for the US Army. The other authors have declared that no competing interests exist.

The diversity of nonculicid biting flies was surveyed in Sunset Zoo, Manhattan, KS, by carbon dioxide-baited traps. A total of 8,399 nonculicid biting-fly females representing 32 species and 5 families were collected. Twenty-one biting midge (Ceratopogonidae: Culicoides ) and 7 black fly (Simuliidae) species were collected, including new state records of 3 Culicoides and 1 simuliid. The species richness of Culicoides and Simuliidae within the zoo represents 72.4% and 41.2%, respectively, of the fauna known to occur in Kansas. Trap type significantly influenced ( P < 0.05) collections of the 5 species analyzed, and trapping period affected 3 species. The diversity and abundance of nonculicid biting flies in the zoo as related to animal health and wellness is discussed.

Background: The emergence of mosquitoes that can avoid indoor-deployed interventions, such as treated bed nets and indoor residual spraying, threatens the mainstay of malaria control in Zambia. Furthermore, the requirement for high coverage of these tools poses operational challenges. Spatial repellents are being assessed to supplement these vector control tools, but limitations exist in the residual effect of the repellent and the need for external power or heat for diffusion of the volatiles., Methods: A semi-field evaluation of a novel controlled release spatial repellent device (CRD) was conducted in Macha, Zambia. These devices emanate metofluthrin with no need for external power. Devices were deployed in huts within the semi-field system (SFS). Female Anopheles gambiae sensu stricto released within the SFS were trapped overnight by light traps and collected by aspiration the next morning inside and outside of huts to determine the extent of mosquito repellency and the impact on host-seeking and survival. Experiments studied the impact of number of devices as well as the presence of hut occupants. The study was complemented with numerical methods based on computational fluid dynamics to simulate spatial distribution of metofluthrin., Results: Presence of CRDs was associated with significant reductions in indoor counts of mosquitoes, regardless of whether huts were occupied or not. Repellency ranged from 15 to 60% compared to huts with no devices. Reducing the number of devices from 16 to 4 had little impact on repellency. When huts were occupied, indoor mosquito host-seeking was higher in the presence of CRDs, whilst survival was significantly reduced., Conclusions: This study demonstrated that deployment of as few as four CRDs within a hut was associated with reduced indoor mosquito densities. As would be expected, presence of occupants within huts, resulted in greater indoor catches (both with and without devices). The increased indoor mosquito host-seeking and mortality in huts when devices were present may be explained by the excito-repellency activity of metofluthrin. These semi-field experiments provide preliminary data on the utility of CRD spatial repellents to reduce indoor densities of An. gambiae mosquitoes. Studies will further investigate the impact of CRDs on mosquito behaviour as well as epidemiological protective efficacy.

Honey bees (Apis mellifera) are effective foragers for floral and extra-floral sources of sugars and as a result may easily be exposed to toxicants in the environment, such as pesticides. Toxic sugar baits (TSB) or insecticide-laced baits are designed for insect vector management but may be a danger to foraging honey bees and their hives. During a mosquito movement study at a zoological institution, nonflowering foliage surrounding the zoo was marked with sugar solution dyed with over the counter food-coloring. Mosquitoes and other insects foraged on the marked sugar and were collected within the zoo. Additionally, four of six honey bee hives within the zoo had workers that also foraged the dyed sugar and consequently colored approximately 57 kg of honey within the hives. This observation identifies a possibility route of toxicant accumulation within the hives from TSB and possible exposure may have whole hive health implications not previously described on an individual bee level.

The objective of this work was to summarize and quantify Japanese encephalitis virus (JEV) infection, dissemination, and transmission rates in mosquitoes, using a meta-analysis approach. Data were obtained from experimental studies, gathered by means of a systematic review of the literature. Random-effects subgroup meta-analysis models by mosquito species were fitted to estimate pooled estimates and to calculate the variance between studies for three outcomes of interest: JEV infection, dissemination, and transmission rates in mosquitoes. To identify sources of heterogeneity among studies and to assess the association between different predictors (mosquito species, virus administration route, incubation period, and diagnostic method) with the outcome JEV infection rate in vectors, we fitted univariable meta-regression models. Mosquito species and administration route represented the main sources of heterogeneity associated with JEV infection rate in vectors. This study provided summary effect size estimates to be used as reference for other investigators when assessing transmission efficiency of vectors and explored sources of variability for JEV infection rates in vectors. Because transmission efficiency, as part of vector competence assessment, is an important parameter when studying the relative contribution of vectors to JEV transmission, our findings contribute to further our knowledge, potentially moving us toward more informed and targeted actions to prevent and control JEV in both affected and susceptible regions worldwide.

Background: Japanese encephalitis (JE) is a zoonosis in Southeast Asia vectored by mosquitoes infected with the Japanese encephalitis virus (JEV). Japanese encephalitis is considered an emerging exotic infectious disease with potential for introduction in currently JEV-free countries. Pigs and ardeid birds are reservoir hosts and play a major role on the transmission dynamics of the disease. The objective of the study was to quantitatively summarize the proportion of JEV infection in vectors and vertebrate hosts from data pertaining to observational studies obtained in a systematic review of the literature on vector and host competence for JEV, using meta-analyses., Methods: Data gathered in this study pertained to three outcomes: proportion of JEV infection in vectors, proportion of JEV infection in vertebrate hosts, and minimum infection rate (MIR) in vectors. Random-effects subgroup meta-analysis models were fitted by species (mosquito or vertebrate host species) to estimate pooled summary measures, as well as to compute the variance between studies. Meta-regression models were fitted to assess the association between different predictors and the outcomes of interest and to identify sources of heterogeneity among studies. Predictors included in all models were mosquito/vertebrate host species, diagnostic methods, mosquito capture methods, season, country/region, age category, and number of mosquitos per pool., Results: Mosquito species, diagnostic method, country, and capture method represented important sources of heterogeneity associated with the proportion of JEV infection; host species and region were considered sources of heterogeneity associated with the proportion of JEV infection in hosts; and diagnostic and mosquito capture methods were deemed important contributors of heterogeneity for the MIR outcome., Conclusions: Our findings provide reference pooled summary estimates of vector competence for JEV for some mosquito species, as well as of sources of variability for these outcomes. Moreover, this work provides useful guidelines when interpreting vector and host infection proportions or prevalence from observational studies, and contributes to further our understanding of vector and vertebrate host competence for JEV, elucidating information on the relative importance of vectors and hosts on JEV introduction and transmission.

During the last 20 years, the epidemiology of Japanese encephalitis virus (JEV) has changed significantly in its endemic regions due to the gradual displacement of the previously dominant genotype III (GIII) with clade b of GI (GI-b). Whilst there is only limited genetic difference distinguishing the two GI clades (GI-a and GI-b), GI-b has shown a significantly wider and more rapid dispersal pattern in several regions in Asia than the GI-a clade, which remains restricted in its geographic distribution since its emergence. Although previously published molecular epidemiological evidence has shown distinct phylodynamic patterns, characterization of the two GI clades has only been limited to in vitro studies. In this study, Culex quinquefasciatus, a known competent JEV mosquito vector species, was orally challenged with three JEV strains each representing GI-a, GI-b, and GIII, respectively. Infection and dissemination were determined based on the detection of infectious viruses in homogenized mosquitoes. Detection of JEV RNA in mosquito saliva at 14 days post infection indicated that Cx. quinquefasciatus can be a competent vector species for both GI and GIII strains. Significantly higher infection rates in mosquitoes exposed to the GI-b and GIII strains than the GI-a strain suggest infectivity in arthropod vectors may lead to the selective advantage of previously and currently dominant genotypes. It could thus play a role in enzootic transmission cycles for the maintenance of JEV if this virus were ever to be introduced into North America., Competing Interests: The authors have declared that no competing interests exist.

Insecticidal sugar baits for mosquitoes and house ies have proven e cacy to reduce insect populations and consequently, disease transmission rates. The new insecticidal sugar trap (IST) is designed speci cally for controlling biting midge disease vector populations around livestock and near larval habitats. The trap operates by combining light-emitting diode (LED) technology with insecticidal sugar baits. The positive photo attraction of Culicoides elicited by the LEDs, draws the insects to the insecticidal sugar bait, which can be made from various commercial insecticide formulations (pyrethroids, neonicotinoids, etc.) or naturally derived formulations (boric acid, garlic oil, etc.) lethal to Culicoides. Insecticidal sugar trap advantages include: customizable LED lights, they can be used with several di erent oral insecticides that have di erent modes of action to help combat the evolution of pesticide resistance, screening on the trap reduces non-target insect feeding (for example bees and butter ies), targets males and females of the species because both must feed on sugar, and low energy LEDs and a solar panel reduce trap maintenance to re lling sugar baits, rather than replacing batteries. This article discusses key components of an IST, which increase the traps e ectiveness for biting midge control.

Rift Valley fever (RVF) is a zoonotic disease endemic in sub-Saharan Africa with periodic outbreaks in human and animal populations. Mosquitoes are the primary disease vectors; however, Rift Valley fever virus (RVFV) can also spread by direct contact with infected tissues. The transmission cycle is complex, involving humans, livestock, and multiple species of mosquitoes. The epidemiology of RVFV in endemic areas is strongly affected by climatic conditions and environmental variables. In this research, we adapt and use a network-based modeling framework to simulate the transmission of RVFV among hypothetical cattle operations in Kansas, US. Our model considers geo-located livestock populations at the individual level while incorporating the role of mosquito populations and the environment at a coarse resolution. Extensive simulations show the flexibility of our modeling framework when applied to specific scenarios to quantitatively evaluate the efficacy of mosquito control and livestock movement regulations in reducing the extent and intensity of RVF outbreaks in the United States., Competing Interests: The authors have declared that no competing interests exist.

Crowdsourcing is obtaining needed services, ideas, or content by soliciting contributions from a large group of people. This new method of acquiring data works well for single reports, but fails when long-term data collection is needed, mainly due to reporting fatigue or failure of repeated sampling by individuals. To establish a crowdsourced collections network researchers must recruit, reward, and retain contributors to the project. These 3 components of crowdsourcing are discussed using the United States Department of Agriculture social networks, the Cervid Disease Network, and the North American Mosquito Project. The North American Mosquito Project is a large network of professional mosquito control districts and public health agencies, which collects mosquito specimens for genetic studies. The Cervid Disease Network is a crowd-sourced disease monitoring system, which uses voluntary sentinel farms or wildlife programs throughout the United States of America to report the onset and severity of diseases in local areas for pathogen surveillance studies.

Background: Animal hosts may vary in their attraction and acceptability as components of the host location process for assessing preference, and biting rates of vectors and risk of exposure to pathogens. However, these parameters remain poorly understood for mosquito vectors of the Rift Valley fever (RVF), an arboviral disease, and for a community of mosquitoes., Methods: Using three known livestock amplifiers of RVF virus including sheep, goat and cattle as bait in enclosure traps, we investigated the host-feeding patterns for a community of mosquitoes in Naivasha, an endemic area of Rift Valley fever (RVF), in a longitudinal study for six months (June-November 2015). We estimated the incidence rate ratios (IRR) where mosquitoes chose cow over the other livestock hosts by comparing their attraction (total number collected) and engorgement rate (proportion freshly blood-fed) on these hosts., Results: Overall, significant differences were observed in host preference parameters for attraction (F2,15 = 4.1314, P = 0.037) and engorgement (F2,15 = 6.24, P = 0.01) with cow consistently attracting about 3-fold as many mosquitoes as those engorged on sheep (attraction: IRR = 2.9, 95 % CI 1.24-7.96; engorgement: IRR = 3.2, 95 % CI = 1.38-7.38) or goat (attraction: IRR = 2.7, 95 % CI 1.18-7.16; engorgement: IRR = 3.28, 95 % CI 1.47-7.53). However, there was no difference between the attraction elicited by sheep and goat (IRR = 1.08; 95 % CI 0.35-3.33 or engorgement rate (IRR = 0.96, 95 % CI 0.36-2.57)., Conclusion: Despite the overall attractive pattern to feed preferentially on cows, the engorgement rate was clearly independent of the number attracted for certain mosquito species, notably among the flood water Aedes spp., largely incriminated previously as primary vectors of RVF. Our findings suggest that insecticide treated cattle (ITC) can be exploited in enclosure traps as contact bait in the monitoring and control of disease-causing mosquitoes in RVF endemic areas.

Rift Valley fever is a vector-borne zoonotic disease which causes high morbidity and mortality in livestock. In the event Rift Valley fever virus is introduced to the United States or other non-endemic areas, understanding the potential patterns of spread and the areas at risk based on disease vectors and hosts will be vital for developing mitigation strategies. Presented here is a general network-based mathematical model of Rift Valley fever. Given a lack of empirical data on disease vector species and their vector competence, this discrete time epidemic model uses stochastic parameters following several PERT distributions to model the dynamic interactions between hosts and likely North American mosquito vectors in dispersed geographic areas. Spatial effects and climate factors are also addressed in the model. The model is applied to a large directed asymmetric network of 3,621 nodes based on actual farms to examine a hypothetical introduction to some counties of Texas, an important ranching area in the United States of America. The nodes of the networks represent livestock farms, livestock markets, and feedlots, and the links represent cattle movements and mosquito diffusion between different nodes. Cattle and mosquito (Aedes and Culex) populations are treated with different contact networks to assess virus propagation. Rift Valley fever virus spread is assessed under various initial infection conditions (infected mosquito eggs, adults or cattle). A surprising trend is fewer initial infectious organisms result in a longer delay before a larger and more prolonged outbreak. The delay is likely caused by a lack of herd immunity while the infection expands geographically before becoming an epidemic involving many dispersed farms and animals almost simultaneously. Cattle movement between farms is a large driver of virus expansion, thus quarantines can be efficient mitigation strategy to prevent further geographic spread.

Background: Mosquitoes' response to artificial lights including color has been exploited in trap designs for improved sampling of mosquito vectors. Earlier studies suggest that mosquitoes are attracted to specific wavelengths of light and thus the need to refine techniques to increase mosquito captures following the development of super-bright light-emitting diodes (LEDs) which emit narrow wavelengths of light or very specific colors. Therefore, we investigated if LEDs can be effective substitutes for incandescent lamps used in CDC light traps for mosquito surveillance, and if so, determine the best color for attraction of important Rift Valley Fever (RFV) vectors., Methods: The efficiency of selected colored LED CDC light traps (red, green, blue, violet, combination of blue-green-red (BGR)) to sample RVF vectors was evaluated relative to incandescent light (as control) in a CDC light trap in two RVF hotspots (Marigat and Ijara districts) in Kenya. In field experiments, traps were baited with dry ice and captures evaluated for Aedes tricholabis, Ae. mcintoshi, Ae. ochraceus, Mansonia uniformis, Mn. africana and Culex pipiens, following Latin square design with days as replicates. Daily mosquito counts per treatment were analyzed using a generalized linear model with Negative Binomial error structure and log link using R. The incidence rate ratios (IRR) that mosquito species chose other treatments instead of the control, were estimated., Results: Seasonal preference of Ae.mcintoshi and Ae. ochraceus at Ijara was evident with a bias towards BGR and blue traps respectively in one trapping period but this pattern waned during another period at same site with significantly low numbers recorded in all colored traps except blue relative to the control. Overall results showed that higher captures of all species were recorded in control traps compared to the other LED traps (IRR < 1) although only significantly different from red and violet., Conclusion: Based on our trapping design and color, none of the LEDs outcompeted the standard incandescent light. The data however provides preliminary evidence that a preference might exist for some of these mosquito species based on observed differential attraction to these light colors requiring future studies to compare reflected versus transmitted light and the incorporation of colored light of varying intensities.

A common method of adult mosquito control consists of residual application on surfaces and aerial spraying often using pyrethroids. However, not all insects that contact insecticides are killed. Sublethal exposure to neurotoxic compounds can negatively affect sensory organs and reduce efficiency of host location. Flight tracks of host-seeking female Culex quinquefasciatus, Anopheles albimanus, and Aedes aegypti in a wind tunnel were video-recorded to compare activation of host-seeking and patterns of flight orientation to host odors. During host-seeking flights, all three mosquito species differed significantly in flight duration, velocity, turn angle, and angular velocity. Mosquitoes were then exposed to sublethal levels (LD(25) ) of pyrethroid insecticides to evaluate the effects of the neurotoxicants 24 hours post-exposure. Significant reductions in time of activation to flight and flight direction were observed in mosquitoes exposed to deltamethrin and permethrin. Additionally, pesticide-treated Cx. quinquefasciatus mosquitoes flew significantly slower, spent more time in flight, and turned more frequently than untreated controls., (© 2011 The Society for Vector Ecology.)

Within the sand fly genus Lutzomyia, the Verrucarum species group contains several of the principal vectors of American cutaneous leishmaniasis and human bartonellosis in the Andean region of South America. The group encompasses 40 species for which the taxonomic status, phylogenetic relationships, and role of each species in disease transmission remain unresolved. Mitochondrial cytochrome c oxidase I (COI) phylogenetic analysis of a 667-bp fragment supported the morphological classification of the Verrucarum group into series. Genetic sequences from seven species were grouped in well-supported monophyletic lineages. Four species, however, clustered in two paraphyletic lineages that indicate conspecificity--the Lutzomyia longiflocosa-Lutzomyia sauroida pair and the Lutzomyia quasitownsendi-Lutzomyia torvida pair. COI sequences were also evaluated as a taxonomic tool based on interspecific genetic variability within the Verrucarum group and the intraspecific variability of one of its members, Lutzomyia verrucarum, across its known distribution.