Infection, dissemination, and transmission of lumpy skin disease virus in Aedes aegypti (Linnaeus), Culex tritaeniorhynchus (Giles), and Culex quinquefasciatus (Say) mosquitoes.

Lumpy skin disease virus (LSDV) is a transboundary viral disease in cattle and water buffaloes. Although this Poxvirus is supposedly transmitted by mechanical vectors, only a few studies have investigated the role of local vectors in the transmission of LSDV. This study examined the infection, dissemination, and transmission rates of LSDV in Aedes aegypti, Culex tritaeniorhynchus, and Culex quinquefasciatus following artificial membrane feeding of 102.7, 103.7, 104.7 TCID50/mL LSDV in sheep blood. The results demonstrated that these mosquito species were susceptible to LSDV, with Cx tritaeniorhynchus exhibiting significantly different characteristics from Ae. aegypti and Cx. quinquefasciatus. These three mosquito species were susceptible to LSDV. Ae. aegypti showed it as early as 2 days post-infection (dpi), indicating swift dissemination in this particular species. The extrinsic incubation period (EIP) of LSDV in Cx. tritaeniorhynchus and Cx. quinquefasciatus was 8 and 14 dpi, respectively. Ingestion of different viral titers in blood did not affect the infection, dissemination, or transmission rates of Cx. tritaeniorhynchus and Cx. quinquefasciatus. All rates remained consistently high at 8-14 dpi for Cx. tritaeniorhynchus. In all three species, LSDV remained detectable until 14 dpi. The present findings indicate that, Ae. aegypti, Cx. tritaeniorhynchus, and Cx. quinquefasciatus may act as vectors during the LSDV outbreak; their involvement may extend beyond being solely mechanical vectors.

Impacts of ground ultra-low volume adulticide applications on Culex pipiens and Culex restuans (Diptera: Culicidae) abundance, age structure, and West Nile virus infection in Cook County, Illinois.

Since the introduction of West Nile virus (WNV) to the United States over 20 years ago, thousands of cases of human disease and death have been reported. Yearly seasonal outbreaks continue to persist, and the city and suburbs of Chicago, Illinois, is considered a "hot spot" for WNV activity. To interrupt WNV transmission, ground ultra-low volume (ULV) adulticide applications are regularly used to reduce Culex pipiens L. and Culex restuans Theobold (Diptera: Culicidae) abundance and infection. The real-world effectiveness of adulticide applications has not been comprehensively assessed, and prior studies, including our own investigation, have yielded inconclusive or conflicting results. Therefore, we expanded our prior work and evaluated the effects of 5 sequential weekly truck-mounted ULV adulticide applications in large residential areas in the northern suburbs of Chicago, Illinois, in 2019 and 2020. Each day, Cx. pipiens and Cx. restuans host-seeking and gravid mosquitoes were collected to assess abundance, age structure, and WNV infection rates. Adulticide applications resulted in significant reductions of both host-seeking and gravid abundance on the night of treatment. The reduction in host-seeking mosquitoes was followed by a reduction in gravid mosquitoes trapped 3 and 4 days after adulticide application and an increase in the proportion of nulliparous mosquitoes. WNV infection rates were significantly reduced in treatment sites as compared to untreated sites when infection rates were higher in 2020. This large-scale study provides comprehensive evidence that ground ULV adulticide applications are an effective tool in an integrated mosquito management program for combating WNV vectors and infection risk.

Diversity of Anaplasma and novel Bartonella species in Lipoptena fortisetosa collected from captive Eld's deer in Thailand.

Lipoptena insects are important ectoparasites of cervids and may affect humans that are incidentally bitten. The presence of zoonotic pathogen DNA, such as Anaplasma, and Bartonella, raises the importance of Lipoptena insects in veterinary and human medicine. Eld's deer (Rucervus eldii thamin), an endangered wild ruminant in Thailand, are bred and raised in the open zoo. The semi-wild zoo environment suggests ectoparasite infestation and potential risk for mechanical transmission of pathogens to visitors, zoo workers, or other animals. However, epidemiology knowledge of pathogens related to endangered wild ruminants in Thailand is limited. This study aims to determine the prevalence and diversity of Anaplasma and Bartonella in the L. fortisetosa collected from captive Eld's deer in Chon Buri, Thailand. Of the 91 Lipoptena DNA samples obtained, 42 (46.15%) and 25 (27.47%) were positive for Anaplasma and Bartonella by molecular detection, respectively. Further, 42 sequences of Anaplasma (4 nucleotide sequence types) showed 100% identity to those detected in other ruminants and blood-sucking ectoparasites. Twenty-five sequences of Bartonella (8 nucleotide sequence types) showed 97.35-99.11% identity to the novel Bartonella species from sika deer and keds in Japan. Phylogenetic trees revealed Anaplasma sequences were grouped with the clusters of A. bovis and other ruminant-related Anaplasma, while Bartonella sequences were clustered with the novel Bartonella species lineages C, D, and E, which originated from Japan. Interestingly, a new independent lineage of novel Bartonella species was found in obtained specimens. We report the first molecular detection of Anaplasma and Bartonella on L. fortisetosa, which could represent infectious status of captive Eld's deer in the zoo. Wild animals act as reservoirs for many pathogens, thus preventive measures in surrounding areas should be considered to prevent pathogen infection among animals or potential zoonotic infection among humans.

Ultra-low volume (ULV) adulticide treatment impacts age structure of Culex species (Diptera: Culicidae) in a West Nile virus hotspot.

West Nile virus (WNV) invaded the continental United States over 20 years ago and continues to cause yearly seasonal outbreaks of human and veterinary disease. In the suburbs of Chicago, Illinois, ultra-low volume (ULV) truck-mounted adulticide spraying frequently is performed to reduce populations of Culex restuans Theobald and Cx. pipiens L. mosquitoes (Diptera: Culicidae) in an effort to lower the risk of WNV transmission. The effectiveness of this control method has not been rigorously evaluated, and evidence for Culex population reduction after ULV adulticide spraying has been inconclusive. Therefore, we evaluated the results of 5 sequential weekly truck-mounted adulticide applications of Zenivex® E20 (etofenprox) in 2 paired sites located in Cook County, IL, during the summer of 2018. Mosquito population abundance, age structure, and WNV infection prevalence were monitored and compared between paired treatment and nearby control sites. Adulticide treatment did not result in consistent short-term or long-term reductions in target WNV vector Culex abundance. However, there was a significant increase in the proportion of nulliparous females in the treated sites compared to control sites and a decrease in Cx. pipiens WNV infection rates at one of the treated sites. This evidence that ULV adulticide spraying altered the age structure and WNV infection prevalence in a vector population has important implications for WNV transmission risk management. Our findings also underscore the importance of measuring these important indicators in addition to abundance metrics when evaluating the efficacy of control methods.

34-kDa salivary protein enhances duck Tembusu virus infectivity in the salivary glands of Aedes albopictus by modulating the innate immune response.

Duck Tembusu virus (DTMUV) is an important flavivirus that can be transmitted to poultry via Aedes albopictus bites. Furthermore, humans residing in the DTMUV epidemic area display activated antiviral immune responses to local DTMUV isolates during the pathogenic invasion, thereby raising the primary concern that this flavivirus may be transmitted to humans via mosquito bites. Therefore, we identified the gene AALF004421, which is a homolog of the 34-kDa salivary protein (34 kDa) of Ae. albopictus and studied the salivary protein-mediated enhancement of DTMUV infection in Ae. albopictus salivary glands. We observed that double-stranded RNA-mediated silencing of the 34 kDa in mosquito salivary glands demonstrated that the silenced 34 kDa impaired DTMUV infectivity, similar to inhibition through serine protease. This impairment occurred as a consequence of triggering the innate immune response function of a macroglobulin complement-related factor (MCR). 34-kDa in the salivary gland which had similar activity as a serine protease, results in the abrogation of antimicrobial peptides production and strong enhance DTMUV replication and transmission. Although the function of the 34 kDa in Ae. albopictus is currently unknown; in the present study, we showed that it may have a major role in DTMUV infection in mosquito salivary glands through the suppression of the antiviral immune response in the earliest stages of infection. This finding provides the first identification of a prominently expressed 34 kDa protein in Ae. albopictus saliva that could serve as a target for controlling DTMUV replication in mosquito vectors.

A Comparative Analysis of RNAi Trigger Uptake and Distribution in Mosquito Vectors of Disease.

In mosquitoes, the utilization of RNAi for functional genetics is widespread, usually mediated through introduced double-stranded RNAs (dsRNAs) with sequence identity to a gene of interest. However, RNAi in mosquitoes is often hampered by inconsistencies in target gene knockdown between experimental setups. While the core RNAi pathway is known to function in most mosquito strains, the uptake and biodistribution of dsRNAs across different mosquito species and life stages have yet to be extensively explored as a source of variation in RNAi experiments. To better understand mosquito-RNAi dynamics, the biodistribution of a dsRNA to a heterologous gene, LacZ (iLacZ), was tracked following various routes of exposure in the larval and adult stages of Aedes aegypti, Anopheles gambiae, and Culex pipiens. iLacZ was largely limited to the gut lumen when exposed per os, or to the cuticle when topically applied, but spread through the hemocoel when injected. Uptake of dsRNA was noted in a subset of cells including: hemocytes, pericardial cells of the dorsal vessel, ovarian follicles, and ganglia of the ventral nerve cord. These cell types are all known to undergo phagocytosis, pinocytosis, or both, and as such may actively take up RNAi triggers. In Ae. aegypti, iLacZ was detected for up to one week post exposure by Northern blotting, but uptake and degradation drastically differed across tissues. The results presented here reveal that the uptake of RNAi triggers is distinct and specific to the cell type in vivo.

Extracellular vesicles secreted by Brugia malayi microfilariae modulate the melanization pathway in the mosquito host.

Vector-borne, filarial nematode diseases cause significant disease burdens in humans and domestic animals worldwide. Although there is strong direct evidence of parasite-driven immunomodulation of mammalian host responses, there is less evidence of parasite immunomodulation of the vector host. We have previously reported that all life stages of Brugia malayi, a filarial nematode and causative agent of Lymphatic filariasis, secrete extracellular vesicles (EVs). Here we investigate the immunomodulatory effects of microfilariae-derived EVs on the vector host Aedes aegypti. RNA-seq analysis of an Ae. aegypti cell line treated with B. malayi microfilariae EVs showed differential expression of both mRNAs and miRNAs. AAEL002590, an Ae. aegypti gene encoding a serine protease, was shown to be downregulated when cells were treated with biologically relevant EV concentrations in vitro. Injection of adult female mosquitoes with biologically relevant concentrations of EVs validated these results in vivo, recapitulating the downregulation of AAEL002590 transcript. This gene was predicted to be involved in the mosquito phenoloxidase (PO) cascade leading to the canonical melanization response and correspondingly, both suppression of this gene using RNAi and parasite EV treatment reduced PO activity in vivo. Our data indicate that parasite-derived EVs interfere with critical immune responses in the vector host, including melanization.

Evaluating spatial and temporal patterns of tick exposure in the United States using community science data submitted through a smartphone application.

Research initiatives that engage the public (i.e., community science or citizen science) increasingly provide insights into tick exposures in the United States. However, these data have important caveats, particularly with respect to reported travel history and tick identification. Here, we assessed whether a smartphone application, The Tick App, provides reliable and novel insights into tick exposures across three domains - travel history, broad spatial and temporal patterns of species-specific encounters, and tick identification. During 2019-2021, we received 11,424 tick encounter submissions from across the United States, with nearly all generated in the Midwest and Northeast regions. Encounters were predominantly with human hosts (71%); although one-fourth of ticks were found on animals. Half of the encounters (51%) consisted of self-reported peri­domestic exposures, while 37% consisted of self-reported recreational exposures. Using phone-based location services, we detected differences in travel history outside of the users' county of residence along an urbanicity gradient. Approximately 75% of users from large metropolitan and rural counties had travel out-of-county in the four days prior to tick detection, whereas an estimated 50-60% of users from smaller metropolitan areas did. Furthermore, we generated tick encounter maps for Dermacentor variabilis and Ixodes scapularis that partially accounted for travel history and overall mirrored previously published species distributions. Finally, we evaluated whether a streamlined three-question sequence (on tick size, feeding status, and color) would inform a simple algorithm to optimize image-based tick identification. Visual aides of tick coloration and size engaged and guided users towards species and life stage classification moderately well, with 56% of one-time submitters correctly selecting photos of D. variabilis adults and 76% of frequent-submitters correctly selecting photos of D. variabilis adults. Together, these results indicate the importance of bolstering the use of smartphone applications to engage community scientists and complement other active and passive tick surveillance systems.

Assessment of Truck-Mounted Area-Wide S-methoprene Applications to Manage West Nile Virus Vector Species in the Suburbs of Chicago, IL, USA.

West Nile virus remains the leading cause of arboviral neuroinvasive disease in the United States, despite extensive efforts to control the mosquito vectors involved in transmission. In this study, we evaluated the effectiveness of Altosid SR-20 (active ingredient, S-methoprene 20%) larvicide applications using truck-mounted ultra-low volume (ULV) dispersal equipment to target Culex pipiens Linnaeus (Diptera: Culicidae) and Cx. restuans (Theobald)larvae. A combination of emergence bioassays, open-field measurements of deposited S-methoprene and spray distribution using gas chromatography-mass spectrometry, and assessments of adult Culex spp. populations in response to applications were conducted over the summer of 2020 within the North Shore Mosquito Abatement District (IL, USA). Open-field applications revealed that dispersed Altosid SR-20 using ULV equipment was effective (75% emergence inhibition in susceptible lab strain Cx. pipiens larvae) up to 53 m. In suburban neighborhood applications, we found that S-methoprene deposition and larval emergence inhibition (EI) in front yards did not differ significantly from backyards. An overall EI of 46% and 28% were observed for laboratory strain Cx. pipiens and wild Cx. restuans larvae respectively, and both had an EI significantly higher than the untreated control group. The EI of exposed wild Cx. pipiens larvae did not differ from the untreated controls, suggesting an increased tolerance to S-methoprene. No difference in abundance of gravid or host-seeking adult Culex spp. post-application was detected between treated and untreated sites. These results document the ability of area-wide application to distribute S-methoprene, but this strategy will need further modifications and evaluation for Culex spp. management.

RNA Nanovaccine Protects against White Spot Syndrome Virus in Shrimp.

In the last 15 years, crustacean fisheries have experienced billions of dollars in economic losses, primarily due to viral diseases caused by such pathogens as white spot syndrome virus (WSSV) in the Pacific white shrimp Litopenaeus vannamei and Asian tiger shrimp Penaeus monodon. To date, no effective measures are available to prevent or control disease outbreaks in these animals, despite their economic importance. Recently, double-stranded RNA-based vaccines have been shown to provide specific and robust protection against WSSV infection in cultured shrimp. However, the limited stability of double-stranded RNA is the most significant hurdle for the field application of these vaccines with respect to delivery within an aquatic system. Polyanhydride nanoparticles have been successfully used for the encapsulation and release of vaccine antigens. We have developed a double-stranded RNA-based nanovaccine for use in shrimp disease control with emphasis on the Pacific white shrimp L. vannamei. Nanoparticles based on copolymers of sebacic acid, 1,6-bis(p-carboxyphenoxy)hexane, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane exhibited excellent safety profiles, as measured by shrimp survival and histological evaluation. Furthermore, the nanoparticles localized to tissue target replication sites for WSSV and persisted through 28 days postadministration. Finally, the nanovaccine provided ~80% protection in a lethal WSSV challenge model. This study demonstrates the exciting potential of a safe, effective, and field-applicable RNA nanovaccine that can be rationally designed against infectious diseases affecting aquaculture.

Variation in Susceptibility to Permethrin in Culex pipiens and Culex restuans Populations in the Great Lakes Region of the United States.

Two Culex pipiens form Pipiens colony strains and a field population of Cx. restuans from Michigan were susceptible and a Cx. pipiens form Molestus colony strain was comparatively less susceptible to a dose of 43 µg/ml of permethrin in Centers for Disease Control and Prevention (CDC) bottle bioassays. Using this diagnostic dose and these populations as controls, adult female Cx. pipiens and Cx. restuans were reared from egg rafts from 28 sites in Illinois, Ohio, Michigan, Iowa, Indiana, Wisconsin, and Minnesota. Tested mosquitoes showed high mortality in populations from 12 sites, less mortality (90-96%) at 9 sites, and less than 90% mortality from 7 sites during 30-min exposures. However, all tested populations showed 97-100% mortality at 60 min, indicating low phenotypic penetrance of resistance factors. These results indicate variation in susceptibility to permethrin in populations of West Nile virus vectors in the Great Lakes region of the United States, with evidence of modest resistance at 7 of 28 (25%) of the sampled populations.

Snow-Covered Tires Generate Microhabitats That Enhance Overwintering Survival of Aedes albopictus (Diptera: Culicidae) in the Midwest, USA.

The Asian tiger mosquito, Aedes albopictus (Skuse), is a public health threat because it can potentially transmit multiple pathogenic arboviruses, exhibits aggressive diurnal biting, and is highly invasive. As Ae. albopictus moved northward into the United States, the limits of expansion were predicted as locations with a mean January temperature warmer than -2.5°C. We postulated that the range of Ae. albopictus could exceed these temperature limits if eggs in diapause overwinter in tires that provide an insulating effect from extreme temperatures. Fifteen tires with Ae. albopictus and Aedes triseriatus (Say) eggs, a native cold hardy species, were placed outside at five locations along a latitudinal gradient in Wisconsin and Illinois during the winter of 2018-2019; notably, in January 2019, a regional arctic air event brought the lowest temperatures recorded in over 20 yr. External and internal tire temperatures were recorded at 3 hr intervals, and egg survival was recorded after six months. Aedes albopictus eggs survived only from tires at northernmost locations. The mean internal January temperature of tires that supported survival was -1.8°C, while externally the mean temperature was -5.3°C, indicating that tires provided an average of +3.5°C of insulation. Tires that supported egg survival also had over 100 mm of snow cover during January. In the absence of snow cover, tires across the study area provided an average +0.79°C [95% CI 0.34-1.11] insulation. This work provides strong argument for the inclusion of microhabitats in models of dispersal and establishment of Ae. albopictus and other vector species.

Outbreak Investigation: Jamestown Canyon Virus Surveillance in Field-Collected Mosquitoes (Diptera: Culicidae) From Wisconsin, USA, 2018-2019.

During the summers of 2017-2019, 60 human cases of Jamestown Canyon virus-associated disease were reported in the State of Wisconsin, U.S.A; by comparison, there were 28 cases in the 5 years prior. Jamestown Canyon virus (JCV, Peribunyaviridae: Orthobunyavirus) is a zoonotic, mosquito-borne virus that is endemic throughout North America. The proposed transmission cycle for JCV involves horizontal transmission by a variety of mammal-feeding mosquito species and deer hosts, and transseasonal maintenance by vertical transmission in Aedes mosquito species. Although some of the earliest work on JCV transmission and disease was done in Wisconsin (WI), little is known about the spectrum of mosquitoes that are currently involved in transmission and maintenance of JCV, which is key to inform the approach to control and prevent JCV transmission, and to understand why case numbers have increased dramatically in recent years. Therefore, we undertook an intensive surveillance effort in Sawyer and Washburn counties, WI between April and August of 2018 and 2019, in an area with a concentration of JCV human cases. Larval and adult stages of mosquitoes were surveyed using larval dippers and emergence traps, light traps, resting boxes, a Shannon-style trap, and backpack aspirator. In total, 14,949 mosquitoes were collected in 2018, and 28,056 in 2019; these specimens represent 26 species in 7 genera. Suspect vector species were tested for JCV by polymerase chain reaction (PCR); of 23 species that were tested, only Aedes provocans yielded JCV positive results. In 2018, a single pool of Ae. provocans tested positive. In 2019, with more focused early season surveillance, we detected JCV in 4 pools of adult mosquitoes, and one pool that consisted of lab-raised adults that were collected as larvae. Material from all of these PCR-positive samples also yielded infectious virus in cell culture. Overall, these data provide new insight into the seasonality and habitat preferences for 26 mosquito species in Northern WI, which will be useful to inform future surveillance efforts for JCV. The results underscore the importance of Ae. provocans as a vector species involved in transseasonal maintenance of JCV in this region.

Knowledge, attitudes, and behaviors regarding tick-borne disease prevention in Lyme disease-endemic areas of the Upper Midwest, United States.

Lyme disease and other tick-borne diseases are a major public health threat in the Upper Midwestern United States, including Michigan, Minnesota, and Wisconsin. To prevent tick bites and tick-borne diseases, public health officials commonly recommend personal protective measures and property management techniques. Adoption of tick-borne disease prevention behaviors and practices by individuals are, however, highly variable. We aimed to characterize current tick-borne disease knowledge, attitudes, and prevention behaviors (KAB) practiced by the public in these states, as well as their willingness to use specific tick control methods. We conducted a population-based survey in summer 2019 in 48 high-risk counties (those having a five-year average (2013-2017) Lyme disease incidence of ≥ 10 cases per 100,000 persons per year), in Michigan, Minnesota, and Wisconsin. A total of 2713 surveys were analyzed; survey weights were used to account for household selection probability and post-stratified to match county-level joint age and sex population distributions in population-level inference. An estimated 98% of the population had heard of Lyme disease, with most perceiving it as very or extremely serious (91%); however, only an estimated 25% perceived tick-borne diseases as very or extremely common in their community. Among those who spent time in places with ticks from April through October, an estimated 68% check themselves thoroughly for ticks most of the time or always and 43% use bug repellent on skin or clothing most of the time or always. An estimated 13% of the population had ever treated their property with a pesticide to kill ticks, and 3% had ever used devices that apply pesticide to rodents to kill ticks on their property. Willingness to practice tick bite prevention behaviors, however, was estimated to be much higher; with 82% being willing to perform tick checks at least once a day, and more than 60% willing to use bug repellent, tick control products on pets, or to bathe within two hours of being outdoors. We found that residents would likely be willing to support a county-wide tick control program to reduce the risk of tick-borne disease in their community (81%) or to apply tick control products to their property to reduce the risk of tick-borne disease in their household (79%). Tick checks were more likely to be practiced among participants who perceived tick-borne diseases to be highly prevalent in their community, if they or a household member had been previously diagnosed with a tick-borne disease?, or if they perceived tick exposure to be likely around their home, cabin, or vacation home. In addition, property-based tick control methods were associated with perceived risk of encountering ticks around the home, cabin, or vacation home. Participants who had seen information from state health departments were also more likely to practice preventive measures. The most common reported barriers to using any of these methods were forgetfulness, safety concerns, and lack of awareness. Our survey findings shed light on how residents from these Upper Midwest states may adopt tick control and tick bite prevention measures and how public health outreach may be most effective for this population.

Biology and Transmission Dynamics of Aedes flavivirus.

Aedes albopictus (Skuse) and Aedes aegypti (Linnaeus) (Diptera: Culicidae) mosquitoes transmit pathogenic arthropod-borne viruses, including dengue, chikungunya, and Zika viruses, with significant global health consequences. Both Ae. albopictus and Ae. aegypti also are susceptible to Aedes flavivirus (AEFV), an insect-specific flavivirus (ISF) first isolated in Japan from Ae. albopictus and Ae. flavopictus. ISFs infect only insect hosts and evidence suggests that they are maintained by vertical transmission. In some cases, ISFs interfere with pathogenic flavivirus infection, and may have potential use in disease control. We explored the host range of AEFV in 4 genera of mosquitoes after intrathoracic injection and observed greater than 95% prevalence in the species of Aedes and Toxorhynchites tested. Anopheles and Culex species were less permissive to infection. Vertical transmission studies revealed 100% transovarial transmission and a filial infection rate of 100% for AEFV in a persistently-infected colony of Ae. albopictus. Horizontal transmission potential was assessed for adult and larval mosquitoes following per os exposures and in venereal transmission experiments. No mosquitoes tested positive for AEFV infection after blood feeding, and infection with AEFV after sucrose feeding was rare. Similarly, 2% of adult mosquitoes tested positive for AEFV after feeding on infected cells in culture as larvae. Venereal transmission of AEFV was most frequently observed from infected males to uninfected females as compared with transmission from infected females to uninfected males. These results reveal new information on the infection potential of AEFV in mosquitoes and expand our understanding of both vertical and horizontal transmission of ISFs.

Effects of Aedes aegypti salivary protein on duck Tembusu virus replication and transmission in salivary glands.

Duck Tembusu virus (DTMUV) infection is an arthropod-borne viral disease that affects many poultry species, including ducks, chickens, and geese. Aedes aegypti mosquito is an important vector of DTMUV. This study sought to determine whether any individual Ae. aegypti salivary protein modulated DTMUV replication in the mosquito salivary gland. Ae. aegypti salivary gland protein of 34 kDa (AaSG34) was found to be expressed explicitly in mosquito salivary glands and was upregulated following DTMUV infection. Thus, AaSG34 was silenced in mosquitoes via RNA interference using double strand RNA (dsRNA), and the mosquitoes were then infected with DTMUV to elucidate their effects on DTMUV replication and transmission. Transcripts of the DTMUV genome in salivary glands and virus titer in saliva were significantly diminished when AaSG34 was silenced, indicating that its presence enhances DTMUV replication in the salivary glands and DTMUV dissemination to saliva. Furthermore, the expression of antimicrobial peptides (AMPs) was upregulated upon AaSG34 silenced. Our results demonstrate that AaSG34 may play a vital role in the suppression of antiviral immune responses to enhance DTMUV replication and transmission. We thus provide new information on the effect of the AaSG34 salivary protein on DTMUV replication in Ae. aegypti as the mechanism of blocking virus transmission to the host.

Characterizing oogenesis and programmed cell death in the eastern tree hole mosquito Aedes (Protomacleaya) triseriatus.

Oogenesis in flies manifests as a carefully orchestrated cascade of developmental gates and growth events, punctuated by programmed cell death (PCD) and follicular resorption events. In anautogenous mosquitoes, a blood meal stimulates growth of primary follicles, but the timing of developmental stages is species-specific, and few species have been characterized. Here, we characterize the first gonotrophic cycle of oogenesis in Aedes triseriatus (Diptera: Culicidae), the principal vector of La Crosse Virus (LACV), a major cause of pediatric encephalitis in North America. We note significant differences in the timing and appearance of developmental stages from previous studies of other mosquito species, particularly Aedes aegypti. We also describe the appearance and timing of PCD events including atresia, nurse cell death, and follicular epithelium death and show that the majority of follicular epithelium cells do not undergo apoptosis during oogenesis but persist in the ovariole at least until the second gonotrophic cycle. This thorough characterization of oogenesis and PCD in Ae. triseriatus, through which LACV must persist in order to achieve filial infection, also serves as a baseline to study host-pathogen interactions during transovarial transmission.

Identification of public submitted tick images: A neural network approach.

Ticks and tick-borne diseases represent a growing public health threat in North America and Europe. The number of ticks, their geographical distribution, and the incidence of tick-borne diseases, like Lyme disease, are all on the rise. Accurate, real-time tick-image identification through a smartphone app or similar platform could help mitigate this threat by informing users of the risks associated with encountered ticks and by providing researchers and public health agencies with additional data on tick activity and geographic range. Here we outline the requirements for such a system, present a model that meets those requirements, and discuss remaining challenges and frontiers in automated tick identification. We compiled a user-generated dataset of more than 12,000 images of the three most common tick species found on humans in the U.S.: Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. We used image augmentation to further increase the size of our dataset to more than 90,000 images. Here we report the development and validation of a convolutional neural network which we call "TickIDNet," that scores an 87.8% identification accuracy across all three species, outperforming the accuracy of identifications done by a member of the general public or healthcare professionals. However, the model fails to match the performance of experts with formal entomological training. We find that image quality, particularly the size of the tick in the image (measured in pixels), plays a significant role in the network's ability to correctly identify an image: images where the tick is small are less likely to be correctly identified because of the small object detection problem in deep learning. TickIDNet's performance can be increased by using confidence thresholds to introduce an "unsure" class and building image submission pipelines that encourage better quality photos. Our findings suggest that deep learning represents a promising frontier for tick identification that should be further explored and deployed as part of the toolkit for addressing the public health consequences of tick-borne diseases.

Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism.

BACKGROUND: Dogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with mild to moderate disease showed significantly higher transmission to the vector than dogs with subclinical or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. CONCLUSIONS/SIGNIFICANCE: Together, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies.

The Taxonomic History of Ochlerotatus Lynch Arribálzaga, 1891 (Diptera: Culicidae).

A review of all taxonomic actions within the subgenus Ochlerotatus Lynch Arribálzaga, 1891 (Diptera: Culicidae) sensu Reinert et al. (2008) is provided. In particular, the complex historical taxonomic treatment of the type species of this group is dissected and explained in detail. Additionally, current challenges with the definition of the subgenus and its constituents are discussed, as are the requisite steps for a successful revision of the taxon. Going forward, we conclude that a taxonomic revision of the species should include a neotype designation for Ochlerotatus scapularis (Rondani, 1848) from topotypical material. Additionally, we provide a review of the characters and taxa that need to be re-evaluated and well-described in order to stabilize the taxonomy of the subgenus. This effort represents a key step towards a stable nomenclature of the Tribe Aedini.