Primary Vector Species Research Articles

Genetic diversity of soybean dwarf virus in two regions of mainland Australia

Soybean dwarf virus (SbDV; family Tombusviridae, genus Luteovirus, species Luteovirus glycinis) is an RNA plant virus that is transmitted solely by aphids in a persistent, circulative and non-propagative manner. SbDV causes significant losses in cultivated Fabaceae, especially in subterranean clover (Trifolium subterraneum) pastures of mainland Australia. SbDV isolates are classified into four phenotypically distinguishable strains: YP, YS, DP, and DS. Y and D strains differ primarily in their host range, and P and S strains in their primary vector species. Genetically, Y and D strains separate into two clades in every genomic region except for the N-terminal region of the readthrough domain (N-RTD), in which P and S strains separate. SbDV diversity in Australia has yet to be investigated, so in this study, 41 isolates were collected from six different host species across two production regions of Australia: the south coast of Western Australia (‘south-west’) and northern New South Wales/southern Queensland (‘north-east’). A near-complete genome sequence of each isolate was obtained, and together with all 50 whole-genome sequences available in the GenBank database, underwent phylogenetic analysis of the whole genome nt and the N-RTD aa sequences. At the whole-genome level, the isolates separated into D and Y clades. At the N-RTD level, most of the isolates separated into P and S clades. All south-west isolates and 11 of the 31 north-east isolates were in the Y clade, and the remaining 20 north-east isolates were in the D clade. Except for one isolate that fell outside the P and S clades, all south-west and north-east isolates were in the P clade, suggesting that they are transmitted by Acyrthosiphon pisum and Myzus persicae. Available biological data largely supported the phenotypic inferences made from the phylogenetic analysis, suggesting that genetic data can provide critical epidemiological insights, provided that sufficient biological data have been collected.

Molecular xenomonitoring as an indicator of microfilaraemia prevalence for lymphatic filariasis in Samoa in 2019

BackgroundLymphatic filariasis (LF) is a globally significant, vector-borne, neglected tropical disease that can result in severe morbidity and disability. As the World Health Organization (WHO) Global Programme to Eliminate Lymphatic Filariasis makes progress towards LF elimination, there is greater need to develop sensitive strategies for post-intervention surveillance. Molecular xenomonitoring (MX), the detection of pathogen DNA in vectors, may provide a sensitive complement to traditional human-based surveillance techniques, including detection of circulating filarial antigen and microfilaraemia (Mf). This study aims to explore the relationship between human Mf prevalence and the prevalence of polymerase chain reaction (PCR)-positive mosquitoes using MX.MethodsThis study compared Mf and MX results from a 2019 community-based survey conducted in 35 primary sampling units (PSUs) in Samoa. This study also investigated concordance between presence and absence of PCR-positive mosquitoes and Mf-positive participants at the PSU level, and calculated sensitivity and negative predictive values for each indicator using presence of any Mf-positive infection in humans or PCR-positive mosquitoes as a reference. Correlation between prevalence of filarial DNA in mosquitoes and Mf in humans was estimated at the PSU and household/trap level using mixed-effect Bayesian multilevel regression analysis.ResultsMf-positive individuals were identified in less than half of PSUs in which PCR-positive mosquito pools were present (13 of 28 PSUs). Prevalence of PCR-positive mosquitoes (each species separately) was positively correlated with Mf prevalence in humans at the PSU level. Analysed at the species level, only Aedes polynesiensis demonstrated strong evidence of positive correlation (r) with human Mf prevalence at both PSU (r: 0.5, 95% CrI 0.1–0.8) and trap/household levels (r: 0.6, 95% CrI 0.2–0.9).ConclusionsFindings from this study demonstrate that MX can be a sensitive surveillance method for identifying residual infection in low Mf prevalence settings. MX identified more locations with signals of transmission than Mf-testing. Strong correlation between estimated PCR-positive mosquitoes in the primary vector species and Mf in humans at small spatial scales demonstrates the utility of MX as an indicator for LF prevalence in Samoa and similar settings. Further investigation is needed to develop MX guidelines to strengthen the ability of MX to inform operational decisions.Graphical abstract

Potential distribution of malaria vectors in Central Vietnam: A MaxEnt modeling approach.

In Central Vietnam, Anopheles dirus and Anopheles minimus are the primary malaria vector species. These Anopheles spp.' distribution and prevalence are determined by environmental, climatic, and socioeconomic conditions. This study aimed to predict the potential distribution of these two Anopheles spp. in this region. This study was conducted in 15 Central Vietnamese provinces. From 2014 to 2018, we utilized An. dirus and An. minimus presence records. Proxy data from the Google Earth Engine platform for the study area, encompassing environmental, climatic, and socioeconomic factors. MaxEnt software predicted the potential environmental, climatic, and socioeconomic suitability of these two Anopheles spp. in Central Vietnam. The test area under the curve values for An. dirus and An. minimus MaxEnt models averaged 0.801 and 0.806, respectively, showing excellent performance. Minimum air temperature had the greatest impact on the distribution of both species. A negative correlation between precipitation and normalized difference water index influences the occurrence of An. dirus. In the temperature range of 13-19.5°C, An. minimus is most likely to be present, with nighttime light detrimentally influencing its distribution. The Central Highlands region is inhabited by both species, with some presence in North-Central and South-Central Coastal areas. The importance of temperature in determining the presence of both species is emphasized by our findings, with subtle differences in the temperature-related factors shaping their distributions. The results highlight the need for focused malaria vector control and surveillance initiatives in the study area.

The impact of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr long-lasting insecticidal nets on density of primary malaria vectors Anopheles gambiae s.s. and Anopheles coluzzii in Benin: a secondary analysis of a cluster randomised controlled trial

BackgroundLong-lasting insecticidal nets (LLINs) may have different impacts on distinct mosquito vector species. We assessed the efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs on the density of Anopheles gambiae s.s. and An. coluzzii compared to pyrethroid-only nets in a three-arm cluster randomised control trial in Benin.MethodsIndoor and outdoor collections of adult mosquitoes took place in 60 clusters using human landing catches at baseline and every 3 months for 2 years. After morphological identification, around 15% of randomly selected samples of An. gambiae s.l. were dissected to determine parity, species (using PCR).ResultsOverall, a total of 46,613 mosquito specimens were collected at baseline and 259,250 in the eight quarterly collections post-net distribution. Post-net distribution, approximately 70% of the specimens of An. gambiae s.l. speciated were An. coluzzii, while the rest were mostly composed of An. gambiae s.s. with a small proportion (< 1%) of hybrids (An. gambiae/coluzzii). There was no evidence of a significant reduction in vector density indoors in either primary vector species [An. coluzzii: DR (density ratio) = 0.62 (95% CI 0.21–1.77), p = 0.3683 for the pyrethroid-pyriproxyfen LLIN and DR = 0.56 (95% CI 0.19–1.62), p = 0.2866 for the pyrethroid-chlorfenapyr LLIN, An. gambiae s.s.: DR = 0.52 (95% CI 0.18–1.46), p = 0.2192 for the pyrethroid-pyriproxyfen LLIN and DR = 0.53 (95% CI 0.19–1.46), p = 0.2222 for the pyrethroid-chlorfenapyr]. The same trend was observed outdoors. Parity rates of An. gambiae s.l. were also similar across study arms.ConclusionsCompared with pyrethroid-only LLINs, pyrethroid-chlorfenapyr LLINs and pyrethroid-pyriproxyfen LLINs performed similarly against the two primary mosquito species An. gambiae s.s. and An. coluzzii in Benin.Graphical

Microsporidia MB in the primary malaria vector Anopheles gambiae sensu stricto is avirulent and undergoes maternal and horizontal transmission

BackgroundThe demonstration that the recently discovered Anopheles symbiont Microsporidia MB blocks malaria transmission in Anophelesarabiensis and undergoes vertical and horizontal transmission suggests that it is a promising candidate for the development of a symbiont-based malaria transmission-blocking strategy. The infection prevalence and characteristics of Microsporidia MB in Anopheles gambiae sensu stricto (s.s.), another primary vector species of malaria in Kenya, were investigated.MethodsField-collected females were confirmed to be Microsporidia MB-positive after oviposition. Egg counts of Microsporidia MB-infected and non-infected individuals were used to infer the effects of Microsporidia MB on fecundity. The time to pupation, adult sex ratio and survival were used to determine if Microsporidia MB infection has similar characteristics in the host mosquitoes An. gambiae and An. arabiensis. The intensity of Microsporidia MB infection in tissues of the midgut and gonads, and in carcasses, was determined by quantitative polymerase chain reaction. To investigate horizontal transmission, virgin males and females that were either Microsporidia MB-infected or non-infected were placed in standard cages for 48 h and allowed to mate; transmission was confirmed by quantitative polymerase chain reaction targeting Microsporidia MB genes.ResultsMicrosporidia MB was found to naturally occur at a low prevalence in An. gambiae s.s. collected in western Kenya. Microsporidia MB shortened the development time from larva to pupa, but other fitness parameters such as fecundity, sex ratio, and adult survival did not differ between Microsporidia MB-infected and non-infected hosts. Microsporidia MB intensities were high in the male gonadal tissues. Transmission experiments indicated that Microsporidia MB undergoes both maternal and horizontal transmission in An. gambiae s.s.ConclusionsThe findings that Microsporidia MB naturally infects, undergoes maternal and horizontal transmission, and is avirulent in An. gambiae s.s. indicate that many of the characteristics of its infection in An. arabiensis hold true for the former. The results of the present study indicate that Microsporidia MB could be developed as a tool for the transmission-blocking of malaria across different Anopheles species.Graphical

Feeding rates of malaria vectors from a prototype attractive sugar bait station in Western Province, Zambia: results of an entomological validation study

BackgroundAttractive targeted sugar bait (ATSB) stations are a promising new approach to malaria vector control that could compliment current tools by exploiting the natural sugar feeding behaviors of mosquitoes. Recent proof of concept work with a prototype ATSB® Sarabi Bait Station (Westham Co., Hod-Hasharon, Israel) has demonstrated high feeding rates and significant reductions in vector density, human biting rate, and overall entomological inoculation rate for Anopheles gambiae sensu lato (s.l.) in the tropical savannah of western Mali. The study reported here was conducted in the more temperate, rainier region of Western Province, Zambia and was designed to confirm the primary vector species in region and to estimate corresponding rates of feeding from prototype attractive sugar bait (ASB) Sarabi Bait Stations.MethodsThe product evaluated was the Sarabi v1.1.1 ASB station, which did not include insecticide but did include 0.8% uranine as a dye allowing for the detection, using UV fluorescence light microscopy, of mosquitoes that have acquired a sugar meal from the ASB. A two-phase, crossover study design was conducted in 10 village-based clusters in Western Province, Zambia. One study arm initially received 2 ASB stations per eligible structure while the other initially received 3. Primary mosquito sampling occurred via indoor and outdoor CDC Miniature UV Light Trap collection from March 01 through April 09, 2021 (Phase 1) and from April 19 to May 28, 2021 (Phase 2).ResultsThe dominant vector in the study area is Anopheles funestus s.l., which was the most abundant species group collected (31% of all Anophelines; 45,038/144,5550), had the highest sporozoite rate (3.16%; 66 positives out of 2,090 tested), and accounted for 94.3% (66/70) of all sporozoite positive specimens. Of those An. funestus specimens further identified to species, 97.2% (2,090/2,150) were An. funestus sensu stricto (s.s.). Anopheles gambiae s.l. (96.8% of which were Anopheles arabiensis) is a likely secondary vector and Anopheles squamosus may play a minor role in transmission. Overall, 21.6% (9,218/42,587) of An. funestus specimens and 10.4% (201/1,940) of An. gambiae specimens collected were positive for uranine, translating into an estimated daily feeding rate of 8.9% [7.7–9.9%] for An. funestus (inter-cluster range of 5.5% to 12.7%) and 3.9% [3.3–4.7%] for An. gambiae (inter-cluster range of 1.0–5.2%). Feeding rates were no different among mosquitoes collected indoors or outdoors, or among mosquitoes from clusters with 2 or 3 ASBs per eligible structure. Similarly, there were no correlations observed between feeding rates and the average number of ASB stations per hectare or with weekly rainfall amounts.ConclusionsAnopheles funestus and An. gambiae vector populations in Western Province, Zambia readily fed from the prototype Sarabi v1.1.1 ASB sugar bait station. Observed feeding rates are in line with those thought to be required for ATSB stations to achieve reductions in malaria transmission when used in combination with conventional control methods (IRS or LLIN). These results supported the decision to implement a large-scale, epidemiological cluster randomized controlled trial of ATSB in Zambia, deploying 2 ATSB stations per eligible structure.

Distance from human dwellings differentially affects the efficacy of a synthetic cattle urine odour lure to trap malaria vectors

BackgroundCost–effective outdoor–based devices for surveillance and control of outdoor mosquito vector populations can substantially improve their efficacy when baited with synthetic human and animal odours. This study aimed at assessing the dose–dependent efficacy of a previously developed synthetic cattle urine odour to lure malaria vectors, and other mosquito species, to traps placed at different distances from human dwellings outdoors.MethodsThe efficacy of the cattle urine odour lure was assessed through a 5 × 5 Latin square design, using two sets of 5 Suna traps placed at either 1.5 m or 5 m from an adjacent human dwelling, in the rural village of Sagamaganga, Tanzania. Each trap was deployed with one of four doses of the synthetic cattle urine odour blend or a solvent control (heptane). Traps were rotated daily so that each dose and control visited each position twice over a period of 20 experimental nights. The relative attractiveness of each treatment dose and control was compared using a generalized linear mixed model for each species caught.ResultsA total of 1568 mosquitoes were caught, of which 783 were anophelines and 785 were culicines. Of the anophelines, 41.6 and 58.3% were primary and secondary vector species, respectively. Unfed and fed females of the primary vector, Anopheles arabiensis, were caught dose–dependently, close to human dwellings (1.5 m), whereas unfed, fed and gravid secondary vector Anopheles pharoensis females were caught dose–dependently, but at a farther distance from the dwellings (5 m). Females of Culex spp. were caught dose–dependently in similar numbers irrespective of the distance from human dwellings.ConclusionsThis study further clarifies the factors to be considered for the implementation of outdoor trapping using the synthetic cattle urine lure to target exophilic and exophagic malaria vectors, for which efficient surveillance and control tools are currently lacking. The findings resulting from this study make significant progress in providing the needed information to overcome the regulatory obstacles to make this tool available for integrated vector management programs, including registration, as well as evaluation and regulation by the World Health Organization.

Entomological Characteristics of Malaria Transmission across Benin: An Essential Element for Improved Deployment of Vector Control Interventions.

Entomological surveillance in Benin has historically been limited to zones where indoor residual spraying was performed or where long-standing sentinel surveillance sites existed. However, there are significant country-wide gaps in entomological knowledge. The National Malaria Control Program (NMCP) assessed population dynamics of Anopheles vectors and malaria transmission in each of Benin's 12 departments to create an entomological risk profile. Two communes per department (24/77 communes) were chosen to reflect diverse geographies, ecologies and malaria prevalence. Two villages per commune were selected from which four households (HH) per village were used for human landing catches (HLCs). In each HH, an indoor and outdoor HLC occurred between 7 p.m. and 7 a.m. on two consecutive nights between July-September 2017. Captured Anopheles were identified, and ovaries were dissected to determine parous rate. Heads and thoraces were tested for Plasmodium falciparum sporozoites by ELISA. The Entomological Inoculation Rate (EIR) was calculated as the product of mosquito bite rate and sporozoite index. Bite rates from An. gambiae&amp;nbsp;s.l., the primary vector species complex, differed considerably between communes; average sporozoite infection index was 3.5%. The EIR ranged from 0.02 infectious bites (ib) per human per night in the departments of Ouémé and Plateau to 1.66 ib/human/night in Collines. Based on transmission risk scales, Avrankou, Sakété and Nikki are areas of low transmission (0 &lt; EIR &lt; 3 ib/human/year), Adjarra, Adja Ouèrè, Zè, Toffo, Bopa, Pehunco, Pèrèrè and Kandi are of medium transmission (3 &lt; EIR &lt; 30 ib/human/year), and the other remaining districts are high transmission (EIR &gt; 30 ib/human/year). The heterogeneous and diverse nature of malaria transmission in Benin was not readily apparent when only assessing entomological surveillance from sentinel sites. Prospectively, the NMCP will use study results to stratify and deploy targeted vector control interventions in districts with high EIRs to better protect populations most at-risk.

Challenges in Understanding the Bionomics of Indian Malaria Vectors.

Many factors influence the success or failure of malaria vector control program such as political will, leadership, sustained funding, robustness of healthcare system and others. In addition, updated knowledge and information about the triad of host, parasite, and vector is of paramount importance. Vector bionomics studies that determine mosquito behavior in terms of feeding, resting, biting, mating, breeding, longevity, vectorial capacity, and response to different insecticides are a step towards enhancing our understanding. In the present work, we have compiled studies conducted in India over the past two decades (2000-2020) to identify gaps in our knowledge of malaria vector bionomics and the research that needs to be done in the future. We retrieved district-level data of India's six primary malaria vector species. According to our findings, vector bionomics studies have been undertaken in ∼50% and ∼15% of the country's high (annual parasite index >1) and low (annual parasite index <1) malaria-endemic districts respectively. Most of the research studies focused on mosquito density, insecticide susceptibility status, and parasite detection, whereas other vital bionomics parameters were neglected. Surveys conducted were incomplete, and vector bionomics data were not captured sufficiently. The absence of vector bionomics data can be a blind spot and the lack or inadequate understanding of vector bionomics can lead to use of inappropriate vector control tools. Thus, there is an urgent need to initiate comprehensive bionomics studies on India's primary and secondary malaria vectors.

Dynamics of Panstrongylus megistus infestation, the primary vector of Trypanosoma cruzi in Minas Gerais, Brazil

Panstrongylus megistus is considered one of the primary species of epidemiological importance for the transmission of Chagas disease in Brazil due to its wide geographical distribution throughout the national territory, good ability to invade and colonize houses, and high rates of natural infection by the Trypanosoma cruzi. The importance of this species in Minas Gerais has been recognized since the 80s. It is responsible for the high prevalence rates of Chagas disease in the west of the state. Studies conducted in the municipality of Jaboticatubas show that P. megistus is still the most captured vector in the region, even after 40 years of uninterrupted actions of the Chagas Disease Control Program in the municipality. Despite the importance of the species, its population dynamics is little studied. Consequently, crucial genetic information such as genetic diversity and gene flow among environments have not been well characterized yet. In this context, this work presents a population genetic analysis at the microgeographic level using microsatellite markers in samples of P. megistus obtained from Jaboticatubas to better understand the infestation dynamics of the primary vector species of T. cruzi in the region. The observed and expected heterozygosity ranged from 0.26 to 0.47 and 0.47 to 0.65, respectively. Most loci presented Hardy-Weinberg disequilibrium due to the excess of homozygotes. The pairwise Fst ranged from 0.05 to 0.35, with the p value significant for all comparisons, indicating the absence of gene flow between them. The values of Fis found ranged from 0.25 to 0.52, all values of p ≤ 0.05, probably due to structured populations, inbreeding, or null alleles. The results suggest an extended stay of this species in the colonized environments, with rare dispersal to other locations. Such results differ from that observed for Triatoma brasiliensis and Triatoma dimidiata, species that present constant movement, even after colonization of the artificial environment, and are similar to the behavior of Triatoma infestans. Thus, the study reinforces the importance of continuous entomological surveillance in the areas of occurrence of P. megistus to avoid the installation and formation of new foci of household infestation in the region.

Evaluation of Selective Deltamethrin Application with Household and Community Awareness for the Control of Chagas Disease in Southern Ecuador.

Chagas disease is endemic in ~70% of Ecuador. Rhodnius ecuadoriensis and Triatoma carrioni (Hemiptera: Reduviidae) are the primary vectors of Chagas disease in Southern Ecuador. This study tested the effectiveness of selective deltamethrin application of Domiciliary Units (DUs) infested with triatomines, coupled with community education activities and a community-based surveillance system. Ten communities were selected in Loja Province, 466 DUs were examined, of these, 5.6% were infested with R. ecuadoriensis (Density [D] = 4 triatomines/DUs searched, Crowding [CR] = 71 triatomines/infested house, Colonization Index [CI] = 77% infested DUs with nymphs) and 8% with T. carrioni (D = 0.6, CR = 7, CI = 64%). Infested DUs were sprayed with deltamethrin. Subsequent visits were conducted at 6 and 12 mo after spraying. At each time point, new entomological searches were carried out in all DUs. All entomological indexes dropped significantly for the primary vector species one year after the initial intervention (R. ecuadoriensis: I = 2%, D = 0.1, CR = 7, CI = 100%; T. carrioni: I = 1.6%, D = 0.1, CR = 5.5, CI = 50%). Fifteen min educational talks were conducted in every DUs and workshops for schoolchildren were organized. Community-based surveillance system was established. However, there is a high risk of DUs reinfestation, possibly from sylvatic habitats (especially of R. ecuadoriensis) and reinforcing educational and surveillance activities are necessary.

Evidence supporting deployment of next generation insecticide treated nets in Burkina Faso: bioassays with either chlorfenapyr or piperonyl butoxide increase mortality of pyrethroid-resistant Anopheles gambiae

BackgroundPyrethroid resistance poses a major threat to the efficacy of insecticide-treated nets (ITNs) in Burkina Faso and throughout sub-Saharan Africa, particularly where resistance is present at high intensity. For such areas, there are alternative ITNs available, including the synergist piperonyl butoxide (PBO)-based ITNs and dual active ingredient ITNs such as Interceptor G2 (treated with chlorfenapyr and alpha-cypermethrin). Before deploying alternative ITNs on a large scale it is crucial to characterize the resistance profiles of primary malaria vector species for evidence-based decision making.MethodsLarvae from the predominant vector, Anopheles gambiae sensu lato (s.l.) were collected from 15 sites located throughout Burkina Faso and reared to adults for bioassays to assess insecticide resistance status. Resistance intensity assays were conducted using WHO tube tests to determine the level of resistance to pyrethroids commonly used on ITNs at 1×, 5 × and 10 × times the diagnostic dose. WHO tube tests were also used for PBO synergist bioassays with deltamethrin and permethrin. Bottle bioassays were conducted to determine susceptibility to chlorfenapyr at a dose of 100 µg/bottle.ResultsWHO tube tests revealed high intensity resistance in An. gambiae s.l. to deltamethrin and alpha-cypermethrin in all sites tested. Resistance intensity to permethrin was either moderate or high in 13 sites. PBO pre-exposure followed by deltamethrin restored full susceptibility in one site and partially restored susceptibility in all but one of the remaining sites (often reaching mortality greater than 80%). PBO pre-exposure followed by permethrin partially restored susceptibility in 12 sites. There was no significant increase in permethrin mortality after PBO pre-exposure in Kampti, Karangasso-Vigué or Mangodara; while in Seguenega, Orodara and Bobo-Dioulasso there was a significant increase in mortality, but rates remained below 50%. Susceptibility to chlorfenapyr was confirmed in 14 sites.ConclusionHigh pyrethroid resistance intensity in An. gambiae s.l. is widespread across Burkina Faso and may be a predictor of reduced pyrethroid ITN effectiveness. PBO + deltamethrin ITNs would likely provide greater control than pyrethroid nets. However, since susceptibility in bioassays was not restored in most sites following pre-exposure to PBO, Interceptor G2 may be a better long-term solution as susceptibility was recorded to chlorfenapyr in nearly all sites. This study provides evidence supporting the introduction of both Interceptor G2 nets and PBO nets, which were distributed in Burkina Faso in 2019 as part of a mass campaign.

Molecular Analysis of Targeted Insecticide Resistance Gene Mutations in Field-Caught Mosquitos of Medical Importance From Saudi Arabia.

Gene mutations on target sites can be a valuable indicator of the status of insecticide resistance. Jeddah, a global commercial and major port-of-entry city, is bearing the brunt of dengue disease burden in Saudi Arabia. In the current study, six genotypes of three codon combinations (989, 1016, and 1534) were observed on voltage-gated sodium channel (VGSC) gene in Jeddah’s Aedes aegypti population, with PGF/PGC as the dominant one. Two types of introns between exon 20 and 21 on VGSC have been identified for the first time in Ae. aegypti in Saudi Arabia. Statistical and phylogenetic analyses showed that the intron type was significantly associated with the 1016 allele and may reflect the history of insecticide treatment in different continents. In addition, fixation of the L1014F allele on VGSC and G119S on acetylcholinesterase 1 gene was detected in local Culex quinquefasciatus populations, with frequencies of 95.24 and 100%, respectively. To the best of our knowledge, this is the first report of resistant-associated mutations in field-caught Cx. quinquefasciatus in Saudi Arabia. The high prevalence of insecticide resistance gene mutations in local primary mosquito vector species highlights the urgent need to carry out comprehensive insecticide resistance surveillance in Saudi Arabia.

Insecticide susceptibility status of malaria vectors, Anopheles culicifacies, Anopheles fluviatilis and Anopheles minimus in the tribal districts of Jharkhand state of India.

Insecticide resistance in malaria vectors has been a major challenge to vector control programs and updated information to the commonly used insecticides is essential for planning appropriate vector control measures. Anopheles culicifacies and An. fluviatilis are the two main vectors prevalent in Jharkhand state of India and role of An. minimus is contemplated in the transmission of malaria in this state. All the districts in the state are predominantly inhabited by the tribal population and are endemic for malaria. A study was undertaken in 12 districts of Jharkhand state to determine the insecticide susceptibility status of the 3 prevalent primary vector species, An. culicifacies, An. fluviatilis, and An. minimus. Wild-caught adult female An. culicifacies, An. fluviatilis, and An. minimus, mosquitoes were collected from stratified ecotypes from different localities of 12 tribal districts of Jharkhand state during 2018 and 2019. Susceptibility tube tests were conducted following the WHO method using test kits. Mosquitoes were exposed to WHO impregnated papers with the prescribed discriminatory dosages of DDT - 4%, malathion - 5%, deltamethrin - 0.05%, permethrin - 0.75%, cyfluthrin - 0.15% and lambda cyhalothrin - 0.05%. Results indicated that An. culicifacies has developed multiple insecticide resistance in all the 12 districts of Jharkhand state. An. fluviatilis was reported resistant for the first time to DDT in all the districts but was susceptible to malathion, deltamethrin, and permethrin whereas in one district it showed possible resistance to malathion. An. minimus was studied in Noamundi CHC of West Singhbhum district, showed possible resistance against DDT but was susceptible to malathion, deltamethrin, and permethrin. The development of multiple insecticide resistance in An. culicifacies including to pyrethroids, has been a concern for malaria control programmes for effective vector management but a report of resistance to DDT for the first time in An. fluviatlis in all the districts in the state is alarming, An. minimus was found in possible resistance category to DDT in one district and both the species were reported susceptible to malathion, deltamethrin, and permethrin. The result of the present study indicates a need for regular monitoring to assess the insecticide susceptibility to formulate effective vector control measures and resistance management.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia.

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI’s nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.

Impact of prior and projected climate change on US Lyme disease incidence.

Lyme disease is the most common vector-borne disease in temperate zones and a growing public health threat in the United States (US). The life cycles of the tick vectors and spirochete pathogen are highly sensitive to climate, but determining the impact of climate change on Lyme disease burden has been challenging due to the complex ecology of the disease and the presence of multiple, interacting drivers of transmission. Here we incorporated 18years of annual, county-level Lyme disease case data in a panel data statistical model to investigate prior effects of climate variation on disease incidence while controlling for other putative drivers. We then used these climate-disease relationships to project Lyme disease cases using CMIP5 global climate models and two potential climate scenarios (RCP4.5 and RCP8.5). We find that interannual variation in Lyme disease incidence is associated with climate variation in all US regions encompassing the range of the primary vector species. In all regions, the climate predictors explained less of the variation in Lyme disease incidence than unobserved county-level heterogeneity, but the strongest climate-disease association detected was between warming annual temperatures and increasing incidence in the Northeast. Lyme disease projections indicate that cases in the Northeast will increase significantly by 2050 (23,619±21,607 additional cases), but only under RCP8.5, and with large uncertainty around this projected increase. Significant case changes are not projected for any other region under either climate scenario. The results demonstrate a regionally variable and nuanced relationship between climate change and Lyme disease, indicating possible nonlinear responses of vector ticks and transmission dynamics to projected climate change. Moreover, our results highlight the need for improved preparedness and public health interventions in endemic regions to minimize the impact of further climate change-induced increases in Lyme disease burden.

4524 Fighting Malaria, One Image at a Time: Using Computer Vision to Develop an Automated Vector Speciation Tool

OBJECTIVES/GOALS: Rapid and accurate identification of primary malaria vector species from collected specimens is the most critical aspect of effective vector surveillance and control. This interdisciplinary team of engineers aims to automate identification using a deep learning computer vision algorithm. METHODS/STUDY POPULATION: The team spent August of 2019 observing and participating in control and surveillance activities in Zambia and Uganda. They conducted &gt;65 interviews with key stakeholders across 9 malaria control and surveillance sites, ranging from field and community health workers, to malaria researchers and Ministry of Health employees. Stakeholder feedback validated the need for a more accurate and efficient method of vector identification in order to more effectively deploy targeted malaria interventions. The team set forth in designing and prototyping a portable, automated field tool that could speciate mosquito vectors to the complex level using artificial intelligence. RESULTS/ANTICIPATED RESULTS: The team’s research demonstrated that accuracy, cost effectiveness, and ease of use would be critical to the successful adoption of the tool. Results of initial prototyping, usability studies, and stakeholder surveys were used to determine the tool’s minimal user specifications: 1) the ability to distinguish between Anopheles Gambiae and Anopheles Funestus, the two principal malaria vectors in the countries visited, 2) achieving an identification accuracy of ≥90% to the complex level, and 3) accessibility to the speciation data 3-7 days following vector collection. Next steps include optimizing the tool to deploy a minimal viable product for testing in Kenya by the summer of 2020. DISCUSSION/SIGNIFICANCE OF IMPACT: The accurate, high-quality surveillance enabled by this device would allow malaria control programs to scale surveillance to remote regions where an entomologist may not be available, allowing malaria programs to deploy effective interventions, monitor results, and prevent disease.

Mosquito abundance in a Dirofilaria immitis hotspot in the eastern state of Rio de Janeiro, Brazil.

Coastal lowlands in the state of Rio de Janeiro, Brazil, support high numbers of heartworm-infected dogs. Microfilariae of heartworm need to be ingested by a potencial mosquito vector in order to develop into infective larvae and infect a new host. Ochlerotatus taeniorhynchus and Ochlerotatus scapularis are the primary vector species in the coastal lowlands of metropolitan Rio de Janeiro; thus, the aim of this study was to investigate whether these two species were abundant enough at the heartworm hotspot in the eastern area of the state to be important to the local parasite's life cycle. The study was conducted at the Massambaba sandbank (22°55'45″S;42°18'51″W), where canine heartworm prevalence was 53.1%. Mosquitoes were captured monthly using two traps with CO2, as well as eventual human landing. A total of 6118 mosquitoes of 16 species were collected. Ochlerotatus taeniorhynchus (37.5%), Culex quinquefasciatus (19.3%), Oc. scapularis (18.2%), and Coquillettidia sp. (10.5%) were the most abundant species. The monthly variation in frequency was marked for Oc. scapularis and Cx. quinquefasciatus and steadier for Oc. taeniorhynchus and Coquillettidia sp. The abundance of the two Ochlerotatus species reaffirms that they are the primary natural vectors for D. immitis in the coastal Rio de Janeiro state lowlands.

Spatial ecology of urban striped skunks (Mephitis mephitis) in the Northern Great Plains: a framework for future oral rabies vaccination programs

Few studies have investigated the ecology of urban striped skunks (Mephitis mephitis) despite their role as a primary rabies vector species paired with an ability to thrive in these landscapes. Information on home range, nightly movements, and habitat selection, is important for rabies management planning regarding the placement of oral rabies vaccine (ORV) baits and for management of skunk populations more generally. Our aim was to obtain baseline ecological information with an emphasis on spatial ecology of urban striped skunks in the Northern Great Plains region that is lacking in the literature. We used radio telemetry to track 22 (4 M, 18 F) skunks during September 2016 to November 2016 and March 2017 to November 2017. Size of home range using kernel density estimation with smoothing by least squares cross validation identified that male skunks ( $$ \overline{x} $$ = 4.36 km2, SE ± 0.79) had larger home ranges than females ( $$ \overline{x} $$ = 1.79 km2, SE ± 0.24). Female skunk home ranges varied by season with the largest home ranges detected during summer 2017. We found no evidence that use-availability was associated with habitat type or season among female skunks. Female skunks were more likely to be found closer to water; however, we found no association of use with road characteristics and geographic location within the study area. Targeting ORV efforts in areas near water may be warranted, but considering differences in habitat selection for skunk studies in urban areas, we caution that ORV baiting programs may not be one-size-fits-all, and a framework for effective bait placement would benefit from studies specific to the management area of interest.

Detection of Plasmodium falciparum infected Anopheles gambiae using near-infrared spectroscopy

BackgroundLarge-scale surveillance of mosquito populations is crucial to assess the intensity of vector-borne disease transmission and the impact of control interventions. However, there is a lack of accurate, cost-effective and high-throughput tools for mass-screening of vectors.MethodsA total of 750 Anopheles gambiae (Keele strain) mosquitoes were fed Plasmodium falciparum NF54 gametocytes through standard membrane feeding assay (SMFA) and afterwards maintained in insectary conditions to allow for oocyst (8 days) and sporozoite development (14 days). Thereupon, each mosquito was scanned using near infra-red spectroscopy (NIRS) and processed by quantitative polymerase chain reaction (qPCR) to determine the presence of infection and infection load. The spectra collected were randomly assigned to either a training dataset, used to develop calibrations for predicting oocyst- or sporozoite-infection through partial least square regressions (PLS); or to a test dataset, used for validating the calibration’s prediction accuracy.ResultsNIRS detected oocyst- and sporozoite-stage P. falciparum infections with 88% and 95% accuracy, respectively. This study demonstrates proof-of-concept that NIRS is capable of rapidly identifying laboratory strains of human malaria infection in African mosquito vectors.ConclusionsAccurate, low-cost, reagent-free screening of mosquito populations enabled by NIRS could revolutionize surveillance and elimination strategies for the most important human malaria parasite in its primary African vector species. Further research is needed to evaluate how the method performs in the field following adjustments in the training datasets to include data from wild-caught infected and uninfected mosquitoes.