Phylogenetic characterization of Orthobunyaviruses isolated from Trinidad shows evidence of natural reassortment.

The genus Orthobunyavirus is a diverse group of viruses in the family Peribunyaviridae, recently classified into 20 serogroups, and 103 virus species. Although most viruses within these serogroups are phylogenetically distinct, the absence of complete genome sequences has left several viruses incompletely characterized. Here we report the complete genome sequences for 11 orthobunyaviruses isolated from Trinidad, French Guiana, Guatemala, and Panama that were serologically classified into six serogroups and 10 species. Phylogenetic analyses of these 11 newly derived sequences indicate that viruses belonging to the Patois, Capim, Guama, and Group C serocomplexes all have a close genetic origin. We show that three of the 11 orthobunyaviruses characterized (belonging to the Group C and Bunyamwera serogroups) have evidence of histories of natural reassortment through the M genome segment. Our data also suggests that two distinct lineages of Group C viruses concurrently circulate in Trinidad and are transmitted by the same mosquito vectors. This study also highlights the importance of complementing serological identification with nucleotide sequencing when characterizing orthobunyaviruses.

An updated checklist of mosquitoes (Diptera: Culicidae) from Chandigarh, India and its surrounding area.

Background & objectives: The affirmation about the prevalence of mosquito species at a particular place and time is very significant, not only to predict the danger of diseases or future outbreaks but also to control the vectors in time. Despite mosquitoes being medically important, the information about its faunal diversity is very scanty as far as Chandigarh in India and its nearby areas are concerned. So, this study was carried out to survey the mosquito fauna from areas in and around Chandigarh in northern India. Methods: Detailed mosquito surveys were carried out to explore the mosquito fauna from various habitats of developed urban areas, gardens, slums and surrounding villages of Chandigarh from June 2017-November 2019 using hand nets and oral aspirators. Results: A total of 34 mosquito species belonging to 8 genera viz; Anopheles, Aedes, Armigeres, Culex, Coquillet-tidia, Mansonia, Mimomyia and Verrallina were recorded, identified and preserved along with detailed collection data, of which eight are new records from Chandigarh. Interpretation & conclusion: The present checklist of mosquito fauna comprising 34 species provides information on the occurrence of mosquito vectors in Chandigarh and its adjoining areas which will be beneficial for the health authorities to adopt appropriate measures in time for the control of these vectors.

AI-Enabled Mosquito Surveillance and Population Mapping Using Dragonfly Robot.

Mosquito-borne diseases can pose serious risks to human health. Therefore, mosquito surveillance and control programs are essential for the wellbeing of the community. Further, human-assisted mosquito surveillance and population mapping methods are time-consuming, labor-intensive, and require skilled manpower. This work presents an AI-enabled mosquito surveillance and population mapping framework using our in-house-developed robot, named 'Dragonfly', which uses the You Only Look Once (YOLO) V4 Deep Neural Network (DNN) algorithm and a two-dimensional (2D) environment map generated by the robot. The Dragonfly robot was designed with a differential drive mechanism and a mosquito trapping module to attract mosquitoes in the environment. The YOLO V4 was trained with three mosquito classes, namely Aedes aegypti, Aedes albopictus, and Culex, to detect and classify the mosquito breeds from the mosquito glue trap. The efficiency of the mosquito surveillance framework was determined in terms of mosquito classification accuracy and detection confidence level on offline and real-time field tests in a garden, drain perimeter area, and covered car parking area. The experimental results show that the trained YOLO V4 DNN model detects and classifies the mosquito classes with an 88% confidence level on offline mosquito test image datasets and scores an average of an 82% confidence level on the real-time field trial. Further, to generate the mosquito population map, the detection results are fused in the robot's 2D map, which will help to understand mosquito population dynamics and species distribution.

Trap Comparison for Surveillance of the Western Tree Hole Mosquito (Diptera: Culicidae).

The western tree hole mosquito, Aedes sierrensis (Ludlow), is a common nuisance mosquito and vector of Dirofilaria immitis (Leidy), the etiologic agent of dog heartworm, in western North America. Here, we compare weekly mosquito collections made with Mosquito Magnet (MM) traps, Biogents Sentinel (BGS) traps, and Biogents Bowl (BGS Bowl) traps set in Salt Lake City, UT, from the start of June to mid-August 2017. We found the number of mosquitoes decreased with rainfall and temperature independently of trap type. The highest number of mosquitoes were caught by BGS traps baited with carbon dioxide (CO2) and BG lure, which collected 62% (n = 422) of all mosquitoes, followed by the MM at 31% (n = 213), and both the BGS and BG Bowl with BG lure had 3.5% (n = 24) each. Aedes sierrensis females were caught weekly at similar densities (mean ±â€…SD) in BGS with CO2 and lure (1.17 ±â€…2.93) and the MM (1.17 ±â€…2.66) traps during the study period. Given that BGS with CO2 and lure traps have several operational advantages over MM traps, including a quicker setup, smaller size, and lower cost, we consider BGS with CO2 and lure traps as the best suited surveillance tool to detect and remove Ae. sierrensis in the western United States and similar settings throughout North America.

Using a miniaturized double-net trap (DN-Mini) to assess relationships between indoor-outdoor biting preferences and physiological ages of two malaria vectors, Anopheles arabiensis and Anopheles funestus.

BACKGROUND: Effective malaria surveillance requires detailed assessments of mosquitoes biting indoors, where interventions such as insecticide-treated nets work best, and outdoors, where other interventions may be required. Such assessments often involve volunteers exposing their legs to attract mosquitoes [i.e., human landing catches (HLC)], a procedure with significant safety and ethical concerns. Here, an exposure-free, miniaturized, double-net trap (DN-Mini) is used to assess relationships between indoor-outdoor biting preferences of malaria vectors, Anopheles arabiensis and Anopheles funestus, and their physiological ages (approximated by parity and insemination states). METHODS: The DN-Mini is made of UV-resistant netting on a wooden frame and PVC base. At 100 cm × 60 cm × 180 cm, it fits indoors and outdoors. It has a protective inner chamber where a volunteer sits and collects host-seeking mosquitoes entrapped in an outer chamber. Experiments were conducted in eight Tanzanian villages using DN-Mini to: (a) estimate nightly biting and hourly biting proportions of mosquitoes indoors and outdoors; (b) compare these proportions to previous estimates by HLC in same villages; and, (c) compare distribution of parous (proxy for potentially infectious) and inseminated mosquitoes indoors and outdoors. RESULTS: More than twice as many An. arabiensis were caught outdoors as indoors (p < 0.001), while An. funestus catches were marginally higher indoors than outdoors (p = 0.201). Anopheles arabiensis caught outdoors also had higher parity and insemination proportions than those indoors (p < 0.001), while An. funestus indoors had higher parity and insemination than those outdoors (p = 0.04). Observations of indoor-biting and outdoor-biting proportions, hourly biting patterns and overall species diversities as measured by DN-Mini, matched previous HLC estimates. CONCLUSIONS: Malaria vectors that are behaviourally adapted to bite humans outdoors also have their older, potentially infectious sub-populations concentrated outdoors, while those adapted to bite indoors have their older sub-populations concentrated indoors. Here, potentially infectious An. arabiensis more likely bite outdoors than indoors, while potentially infectious An. funestus more likely bite indoors. These observations validate previous evidence that even outdoor-biting mosquitoes regularly enter houses when young. They also demonstrate efficacy of DN-Mini for measuring indoor-outdoor biting behaviours of mosquitoes, their hourly biting patterns and epidemiologically relevant parameters, e.g., parity and insemination status, without exposure to volunteers. The trap is easy-to-use, easy-to-manufacture and affordable (prototypes cost ~ 100 US$/unit).

The challenge of invasive mosquito vectors in the U.K. during 2016-2018: a summary of the surveillance and control of Aedes albopictus.

Mosquito-borne diseases resulting from the expansion of two key vectors, Aedes aegypti and Aedes albopictus (Diptera: Culicidae), continue to challenge whole regions and continents around the globe. In recent years there have been human cases of disease associated with Chikungunya, dengue and Zika viruses. In Europe, the expansion of Ae. albopictus has resulted in local transmission of Chikungunya and dengue viruses. This paper considers the risk that Ae. aegypti and Ae. albopictus represent for the U.K. and details the results of mosquito surveillance activities. Surveillance was conducted at 34 points of entry, 12 sites serving vehicular traffic and two sites of used tyre importers. The most common native mosquito recorded was Culex pipiens s.l. (Diptera: Culicidae). The invasive mosquito Ae. albopictus was detected on three occasions in southern England (September 2016, July 2017 and July 2018) and subsequent control strategies were conducted. These latest surveillance results demonstrate ongoing incursions of Ae. albopictus into the U.K. via ground vehicular traffic, which can be expected to continue and increase as populations in nearby countries expand, particularly in France, which is the main source of ex-continental traffic.

Evaluating perceptions of risk in mosquito experts and identifying undocumented pathways for the introduction of invasive mosquito species into Europe.

In several reported cases of the entry of invasive mosquito species (IMSs) into Europe, the introduction was associated with a specific pathway of introduction or dispersal. The identification of potential pathways for the introduction of IMSs and evaluations of the importance of the different pathways are key to designing proper surveillance strategies to promptly detect and control introductions in non-infested areas. The main goals of the present study were to identify other, previously undocumented, pathways of introduction into Europe, and to identify mosquito experts' perceptions regarding control measures against IMS introductions via different documented pathways. At the European Mosquito Control Association (EMCA) conference in Montenegro in March 2017, a questionnaire was distributed among meeting participants to collect expert data. Results showed that ground transportation (by cars, trucks, etc.), passive natural dispersal and the shipping of used tyres are perceived as the most likely pathways. Introduction via aircraft did not appear to be well known and was not perceived as probable. This study shows that there were no pathways unknown to European experts that could lead to cryptic introductions into the experts' countries. Furthermore, the findings demonstrated that the perceived efficacy of surveillance and control is key to overcoming the constraints experienced and to supporting the implementation of actions against introductions.

Evaluation of enhanced oviposition attractant formulations against Aedes and Culex vector mosquitoes in urban and semi-urban areas.

Surveillance is not only an important tool to assess the population dynamics of vector mosquitoes, but it can also be used to control vector-borne diseases. Mosquito vectors that belong to several genera such as Anopheles, Aedes, and Culex play a crucial role in the transmission of malaria, dengue, chikungunya, Zika, and elephantiasis diseases worldwide. We tested the efficacy of two commercial-grade oviposition attractant formulations that were developed for the container-inhabiting Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus mosquitoes present in urban or semi-urban environments. These attractants can lure gravid females. Field trials were conducted in residential yards during a post-rainy season in September and October. Our data showed considerable efficacy for both attractants. Aedes-attractant collected 1.6-fold more larvae (101.2 ± 10.5 larvae/trap) than the control, and Culex-attractant collected 1.27-fold more larvae (151.2 ± 12.5 larvae/trap) than the control, resulting in 0.8 and 0.7 oviposition attraction indices (OAIs), respectively. Regression analysis indicated that the Aedes-attractant was more stable than the Culex-attractant. Location and time did not alter the efficacy of these attractants. Our experiment suggests that these attractants can be used for the development of species-specific gravid traps to detect, estimate, and control the mosquito population in urban and semi-urban areas.

Spatial-temporal distribution of Anopheles larval habitats in Uganda using GIS/remote sensing technologies.

BACKGROUND: Anopheles mosquitoes impose an immense burden on the African population in terms of both human health and comfort. Uganda, in particular, boasts one of the highest malaria transmission rates in the world and its entire population is at risk for infection. Despite the immense burden these mosquitoes pose on the country, very few programmes exist that directly combat the issue at the vector control level and even fewer programmes focus on the vector in its most vulnerable juvenile stages. This study utilizes remote sensing techniques and spatial autocorrelation models to identify and prioritize the most prolific Anopheline larval habitats for control purposes in a rural community in Uganda. METHODS: A community-based mosquito surveillance programme was developed and implemented in Papoli Parish in Eastern Uganda over a 4-month period. Each day, a trained field team sampled the larval habitats of Anopheles mosquitoes within the population-dense areas of the community. Habitats and their productivity were identified and plotted spatially on a daily basis. Daily output was combined and displayed as a weekly habitat time-series. Additional spatial analysis was conducted using the Global and Anselin's Local Moran's I statistic to assess habitat spatial autocorrelation. RESULTS: Spatial models were developed to identify highly significant habitats and dictated the priority of these habitats for larval control purposes. Weekly time-series models identified the locations and productivity of each habitat, while Local Moran's I cluster maps identified statistically significant clusters (Cluster: High) and outliers (High Outlier) that were then interpreted for control priority. Models were stitched together in a temporal format to visually demonstrate the spatial shift of statically significant, high priority habitats over the entire study period. DISCUSSION: The findings show that the spatial outcomes of productive habitats can be made starkly apparent through initial habitat modelling and resulting time-series output. However, mosquito control resources are often limited and it is at this point that the Local Moran's I statistics demonstrates its value. Focusing on habitats identified as Cluster: High and High Outlier outputs allow for the identification of the most influential larval habitats. Utilizing this method for malaria control allows for the optimization of control resources in a real time, community driven, fashion, as well as providing a framework for future control practices.

West Nile virus 'circulation' in Vojvodina, Serbia: Mosquito, bird, horse and human surveillance.

Efforts to detect West Nile virus (WNV) in the Vojvodina province, northern Serbia, commenced with human and mosquito surveillance in 2005, followed by horse (2009) and wild bird (2012) surveillance. The knowledge obtained regarding WNV circulation, combined with the need for timely detection of virus activity and risk assessment resulted in the implementation of a national surveillance programme integrating mosquito, horse and bird surveillance in 2014. From 2013, the system showed highly satisfactory results in terms of area specificity (the capacity to indicate the spatial distribution of the risk for human cases of West Nile neuroinvasive disease - WNND) and sensitivity to detect virus circulation even at the enzootic level. A small number (n = 50) of Culex pipiens (pipiens and molestus biotypes, and their hybrids) females analysed per trap/night, combined with a high number of specimens in the sample, provided variable results in the early detection capacity at different administrative levels (NUTS2 versus NUTS3). The clustering of infected mosquitoes, horses, birds and human cases of WNND in 2014-2015 was highly significant, following the south-west to north-east direction in Vojvodina (NUTS2 administrative level). Human WNND cases grouped closest with infected mosquitoes in 2014, and with wild birds/mosquitoes in 2015. In 2014, sentinel horses showed better spatial correspondence with human WNND cases than sentinel chickens. Strong correlations were observed between the vector index values and the incidence of human WNND cases recorded at the NUTS2 and NUTS3 levels. From 2010, West Nile virus was detected in mosquitoes sampled at 43 different trap stations across Vojvodina. At 14 stations (32.56%), WNV was detected in two different (consecutive or alternate) years, at 2 stations in 3 different years, and in 1 station during 5 different years. Based on these results, integrated surveillance will be progressively improved to allow evidence-based adoption of preventive public health and mosquito control measures.

Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.

BACKGROUND: Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives. METHODS: Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration. RESULTS: The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India. CONCLUSIONS: A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments.

Mosquito Surveillance for 15 Years Reveals High Genetic Diversity Among West Nile Viruses in Israel.

West Nile Virus (WNV) is endemic in Israel and has been the cause of several outbreaks in recent years. In 2000, a countrywide mosquito survey was established to monitor WNV activity and characterize viral genotypes in Israel. We analyzed data from 7135 pools containing 277 186 mosquitoes collected over the past 15 years and, here, report partial sequences of WNV genomes obtained from 102 of the 336 positive mosquito pools. Phylogenetic analysis demonstrated that cluster 4 and the Mediterranean and Eastern European subtypes of cluster 2 within WNV lineage 1 circulated in Israel, as did WNV lineage 2, highlighting a high genetic diversity of WNV genotypes in our region. As a major crossroads for bird migration between Africa and Eurasia and with a long history of human infection, Israel serves as a resource hub for WNV in Africa and Eurasia and provides valuable information on WNV circulation in these regions.

Emergence of Aedes koreicus (Diptera: Culicidae) in an urban area, Hungary, 2016.

In June 2016, three adult females of Aedes koreicus mosquitoes were trapped in the urban area of Pécs, Southwest Hungary. The introduction of this invasive mosquito species in this region, along with the recent detection in Germany, may indicate the capability of the species to spread across Europe. Along with Aedes albopictus and Aedes japonicus mosquitoes, this is the third invasive mosquito species occurred in Hungary.

First molecular identification of Dirofilaria spp. (Onchocercidae) in mosquitoes from Serbia.

Dirofilariosis is a common and widespread veterinary health issue in several European countries with notable zoonotic potential. The causative agents are Dirofilaria immitis and Dirofilaria repens nematoda species which are transmitted by different mosquito vectors. Similar to other mosquito-borne infections, the knowledge about mosquito species involved in disease transmission is crucial for the complex understanding of local transmission cycles. Since there is no available data on mosquito species, potentially involved in disease transmission from Serbia, 6369 female mosquito individuals were retrospectively tested for Dirofilaria nematodes, collected from 13 localities in Vojvodina province, Serbia, in 2013. Altogether, 8.33 % of tested pools showed positivity, composed of five mosquito species, mainly, Culex pipiens and Aedes vexans. D. immitis and D. repens were both detected from multiple localities, during the whole period of mosquito breeding season, which provides the first data on local transmission characteristics regarding mosquitoes from the Balkans.

Field Comparison of Cyclopentanone Versus Carbon Dioxide as an Attractant for Adult Mosquitoes in Southeast Queensland, Australia.

Cyclopentanone is a saturated monoketone typically used as an intermediate in the manufacture of pharmaceuticals, biologicals, insecticides, and rubber chemicals. Recently, it has been demonstrated that cyclopentanone activates the cpA CO2 receptor neuron on the maxillary palp of mosquitoes, suggesting that it may be a viable alternative to CO2 as an attractant for mosquitoes. Furthermore, semifield experiments showed that traps baited with cyclopentanone attract Culex quinquefasciatus Say at a similar rate to those baited with CO2. We evaluated the field efficacy of cyclopentanone as an alternative to CO2 in Centers for Disease Control (CDC) light traps and counterflow geometry (CFG) traps commonly used to collect mosquitoes in surveillance programs. Three pairwise trials and four Latin square trials were conducted across three peri-urban sites, comprising two saltwater sites and one freshwater site, in southeast Queensland, Australia. In all trials, CO2-baited traps outperformed traps baited with cyclopentanone. Carbon dioxide-baited CDC traps collected significantly more total mosquitoes, Aedes vigilax (Skuse), Culex sitiens Weidemann, and Culex annulirostris Skuse, than those baited with ≥99% cyclopentanone in pairwise trials. Similarly, in almost all Latin square trials, CO2-baited CDC and CFG traps collected significantly greater numbers of total mosquitoes, Ae. vigilax, Cx. annulirostris, Culex orbostiensis Dobrotworsky, and Cx. sitiens when compared with CFG traps baited with 20% cyclopentanone. Our trials indicate that cyclopentanone is not effective as a mosquito attractant in the field and cannot be used as a simple substitute for CO2 in commonly used mosquito surveillance traps.

A checklist of the 67 mosquito species of Ontario, Canada.

We provide an updated checklist of 67 endemic mosquito species known from Ontario, Canada. Nine endemic species are added to the checklist found in Darsie and Ward (2005) : Aedes cantator, Ae. churchillensis, Ae. nigripes, Ae. pullatus, Anopheles perplexens, An. crucians, An. smaragdinus, Culex erraticus, and Cx. salinarius. Only 4 specimens of Ae. albopictus have been recorded in Ontario since 2001 despite concerted efforts to find this species; therefore, it is considered an "accidental" species and is excluded from the checklist.

Characterizing the seasonal abundance and reproductive activity of overwintering Anopheles (Diptera: Culicidae) mosquitoes.

In temperate regions of the United States, female Anopheles mosquitoes respond to low temperatures and short photoperiods by entering an overwintering dormancy or diapause. Diapause in Anopheles results in reduced frequency of blood-feeding and reproductive arrest, indicating a period when pathogen transmission by these mosquitoes is unlikely. However, it is unclear precisely how late into the fall and how early in the spring these mosquitoes are biting, reproducing, and potentially transmitting pathogens. This is further complicated by the lack of clear markers of diapause in Anopheles (e.g., changes in egg follicle length). Our goal was to characterize the seasonal reproductive activity of female Anopheles in central Ohio, United States and evaluate egg follicle length as an indicator of Anopheles diapause. We used traditional mosquito traps and aspirators to collect Anopheles from urban woodlots and culverts, respectively, from late September 2021 through mid-May 2022 in central Ohio. By measuring their egg follicle length, reproductive status, and blood-feeding status, we found that egg follicle length is not a reliable indicator of Anopheles diapause. We also found that a small proportion of An. punctipennis (Say), An. perplexens (Ludlow), and An. quadrimaculatus (Say) continued to bite and reproduce into early November 2021 and that females of these species terminated reproductive dormancy and began biting by mid-March 2022. This period of reproductive activity extends beyond current mosquito surveillance and control in Ohio. Our findings suggest that within temperate regions of North America, Anopheles have the capacity to transmit pathogens throughout the spring, summer, and fall.

Multiplex PCR method for MinION sequencing of Bagaza virus isolated from wild caught mosquitoes in South Africa.

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions. BAGV was detected in a mosquito pool during surveillance of mosquitoes collected in central South Africa between November 2019 and March 2023. Homogenized mosquito pools were screened for flaviviral RNA using conventional RT-PCR and virus isolation was attempted on positive samples. BAGV was detected and subsequently isolated using cell culture. A multiplex tiling PCR method for targeted enrichment using a PCR based or amplicon sequencing approach of the complete genome of BAGV was developed and optimized. Primers were designed using alignment of complete genome sequence data retrieved from GenBank to identify suitable primer sites that would generate overlapping fragments spanning the complete genome. Six forward primers and eight reverse primers were identified that target the complete genome and amplified nine overlapping fragments, that ranged in length from 1954 to 2039 with an overlap ranging from 71 to 711 base pairs. The design strategy included multiple forward and reverse primer pairs for the 5' and 3' ends. Phylogenetic analysis with other isolates was performed and BAGV isolate VBD 74/23/3 was shown to share high similarity with previous BAGV isolates from all regions, with genetic distance ranging from 0.026 to 0.083. VBD 74/23/3 was most closely related to previous isolates from southern Africa, ZRU96/16/2 isolated from a post-mortem sample from a pheasant in 2016 and MP-314-NA-2018 isolated from mosquitoes in northwestern Namibia with genetic distance 0.0085 and 0.016 respectively. Currently there is limited complete genome sequence data available for many of the arboviruses circulating in Africa. The multiplex tiling method provided a simple and cost-effective method for obtaining complete genome sequence. This method can be readily applied to other viruses using sequence data from publicly available databases and would have important application facilitating genomic surveillance of arboviruses in low resource countries.

Dirofilaria sp. and Blood Meal Analysis in Mosquitoes Collected in Vojvodina and Macva, and the First Report of Setaria tundra (Issaitshikoff & Rajewskaya, 1928) in Serbia.

Dirofilaria immitis and D. repens are the two most widespread and important species of mosquito-borne nematodes, posing a significant threat to veterinary health and particularly affecting canines and felines. While D. immitis causes cardiopulmonary dirofilariasis, D. repens causes subcutaneous infections in dogs and other carnivores. Despite the extensive knowledge on these parasites, little is known about their natural vectors in Serbia. The parasite Setaria tundra, known to infect deer, has not yet been detected in Serbia but has been documented in neighboring countries. Thus, the aim of this study was to (i) further map out Dirofilaria sp. hotspots in the Vojvodina Province and detect S. tundra for the first time, (ii) detect positive mosquito species that can provide insights into how the nematodes spread in Serbia, and (iii) analyze the blood-fed female mosquitoes of species found to be infected, in order to identify the potential source of parasite infection. A total of 2902 female mosquitoes were collected across 73 locations during 2021 and 2022. Molecular biology methods, based on conventional PCR, were used to analyze non-blood-fed (2521 specimens) and blood-fed (381 specimens) female mosquitos, in order to detect filarial nematode presence and identify blood-meal sources, respectively. When the parasite genome was detected, the amplicon (cox1 gene, 650 bp fragment) was sent for Sanger sequencing, further confirming the presence of nematodes and species assignation. D. immitis was detected in three Culex pipiens mosquitoes collected in Zrenjanin (August 2021) and Glogonj and Svetozar Miletic (both in July 2021). Additionally, Setaria tundra was detected in Aedes vexans collected in Idos (mid-August 2021) and Aedes caspius, which was collected in Mali Idos (end of July 2021). This work identifies two new locations where D. immitis occurs in Vojvodina, and is the first report of S. tundra in Serbian territory. Blood-meal analysis provided insights into the preferences of mosquitoes that were positive for Dirofilaria sp. and S. tundra.

Abundance, distribution, and dynamics of Anopheles species (Diptera: Culicidae) in Iowa, United States.

Malaria was once endemic in the United States prior to its elimination in 1951. However, due to consistent introductions of travel-associated malaria cases and the presence of several native Anopheles species (Diptera: Culicidae) that are competent vectors of malaria, the potential for local (autochthonous) malaria transmission remains a persistent threat in the United States. While several intermittent cases of local malaria transmission have occurred in the United States in the decades since elimination, the emergence of autochthonous transmission in 4 states in 2023 demonstrates the continued risk for future outbreaks. Moreover, these recent examples also highlight significant gaps in current mosquito surveillance efforts that have predominantly focused on threats of arboviral disease, such that our understanding of Anopheles distributions relies only on historical records and offers limited insight into the ecological factors that influence their abundance. Herein, we summarize mosquito surveillance data collected over the last 20 years (2004-2023) across 59 Iowa counties to provide essential information into the spatial distribution, temporal abundance, and trap preferences of Anopheles species in the state. Further analyses of the 2 most abundant species, Anopheles punctipennis Say and Anopheles quadrimaculatus Say, reveal the additional influence of precipitation and forested habitats in defining An. punctipennis abundance. Together, we believe these results provide an increased understanding of previously neglected Anopheles species that have the potential for autochthonous malaria transmission in Iowa and that can be extended to other regions of the United States to enhance preparedness for future malaria outbreaks.