The mosquito Aedes aegypti is the primary vector of dengue and is common throughout tropical and subtropical regions. Its distribution is modulated by environmental factors, such as temperature. This study aimed to evaluate the influence of temperature on the life cycle and expansion of Ae. aegypti populations in the cities of Campina Grande, João Pessoa, and Patos. Samples of Ae. aegypti were collected in the three cities and raised in the laboratory. We assessed the life cycles of the three Ae. aegypti populations under six constant temperatures (16, 22, 28, 33, 36, and 39° C), selected on the basis of historical temperature tendencies of each city. We also used existing climate data to calculate projected temperature increases for all three areas. Our results suggest that Campina Grande, João Pessoa, and Patos will experience, respectively, maximum temperature increases of 0.030° C/year, 0.069° C/year, and 0.061° C/year, and minimum temperature increases of 0.019° C/year, -0.047° C/year, and -0.086° C/year. These projected increases will result in temperatures favorable to the Ae. aegypti life cycle, causing rapid population growth. Therefore, Ae. aegypti populations are likely to expand in the mesoregions represented by these cities.

Aedes albopictus (Skuse) is an invasive mosquito species found across the southern U.S. with range expansion into many northern states. Intra- and interspecific larval competition have been evaluated for Ae. albopictus with respect to subsequent adult size, immature and adult survivability, and its capacity to vector pathogens as an adult. However, limited data are available on egg production as related to larval rearing conditions. Because Ae. albopictus is a container-inhabiting mosquito that oviposits in resource-limited habitats, it is found under variable density-dependent conditions. Therefore, we examined the impact of specific rearing conditions on Ae. albopictus clutch size and adult body size; comparing the egg production values and wing lengths from known developmental densities to those from field-collected populations. Field populations varied significantly among collection sites in mean clutch size (23 to 46). These clutch sizes were comparable to the mean clutch sizes of females reared at the larval densities of nine (20 eggs) and three (53 eggs) larvae per 3 ml of water in the laboratory. Field populations experienced density-dependent effects impacting adult mosquito size. Mosquitoes from the four sample sites had mean wing lengths of 1.99, 2.47, 2.51, and 2.54 mm, which were less than the mean wing length of mosquitoes reared at larval densities of three larvae per 3 ml of water (2.57 mm).

This study aimed to assess the vertical patterns of oviposition and temporal changes in the distribution of mosquito species in an area of the Atlantic Forest in Rio de Janeiro State, Brazil, and in particular, the behavior and oviposition of potential yellow fever virus vectors. Mosquito samples were collected from the Ecological Reserve Guapiaçu (REGUA, Brazil), which includes a somewhat disturbed forest, with a large diversity of plants and animals. In all, 5,458 specimens (ten species from seven genera) were collected. Haemagogus leucocelaenus was the most frequently captured species, representing 73% of the specimens collected. Species richness and diversity were the highest in the samples collected from the ground-level ovitraps and decreased with height. Species composition also differed significantly among heights. The largest species differences were detected between ovitraps set at the ground level and those set at 7 m and 9 m; Hg. leucocelaenus, Limatus durhamii, and Limatus paraensis contributed most to these differences. Sampling month and climatic variables had significant effects on species richness and diversity. Species diversity and richness decreased with height, suggesting that the conditions for mosquito breeding are more favorable closer to the ground. Species composition also showed vertical differences.

The phrase, “T. rangeli is pathogenic to its insect vector,” is commonly found in peer-reviewed publications on the matter, such that it has become the orthodox view of this interaction. In a literature survey, we identified over 20 papers with almost the exact phrase and several others alluding to it. The idea is of particular importance in triatomine population dynamics and the study of vector-borne T. cruzi transmission, as it could mean that triatomines infected with T. rangeli have lower fitness than uninfected insects. Trypanosoma rangeli pathogenicity was first observed in a series of studies carried out over fifty years ago using the triatomine species Rhodnius prolixus. However, there are few studies of the effect of T. rangeli on its other vector species, and several of the studies were carried out with R. prolixus under non-physiological conditions. Here, we re-evaluate the published studies that led to the conclusion that T. rangeli is pathogenic to its vector, to determine whether or not this indeed is the “true” effect of T. rangeli on its triatomine vector.

We investigated the possible relationship between the agricultural use of insecticides and the emergence of insecticide resistance. Bioassays were conducted using simulated mosquito larval habitats and well known Anopheles gambiae strains. Soil samples were collected from vegetable production areas in Benin, including one site with insecticide use, one site where insecticides had not been used for two months, and a third where insecticides had not been used. Pupation and emergence rates were very low in pyrethroid-susceptible strains when exposed to soil that had been recently exposed to insecticides. Pupation and emergence rates in strains with the kdr mutation alone or both the kdr and Ace-1 mutations were much higher. Overall, strains with the kdr mutation survived at higher rates compared to that without kdr mutation. Although this study is observational, we provide indirect evidence indicating that soils from agricultural areas contain insecticide residues that can play a role in the emergence of insecticide resistance in Anopheles. This aspect should be taken into account to better utilize the insecticide in the context of integrated pest management programs.

Cervids host multiple species of ixodid ticks, other ectoparasites, and a variety of rickettsiae. However, diagnostic test cross-reactivity has precluded understanding the specific role of deer in rickettsial ecology. In our survey of 128 Columbian black-tailed deer (Odocoileus hemionus columbianus (Richardson)) and their arthropod parasites from two northern Californian herds, combined with reports from the literature, we identified four distinct Anaplasma spp. and one Ehrlichia species. Two keds, Lipoptena depressa (Say) and Neolipoptena ferrisi Bequaert, and two ixodid ticks, Ixodes pacificus Cooley and Kohls and Dermacentor occidentalis Marx, were removed from deer. One D. occidentalis was PCR-positive for E. chaffeensis; because it was also PCR-positive for Anaplasma sp., this is an Anaplasma/Ehrlichia co-infection prevalence of 4.3%. 29% of L. depressa, 23% of D. occidentalis, and 14% of deer were PCR-positive for Anaplasma spp. DNA sequencing confirmed A. bovis and A. ovis infections in D. occidentalis, A. odocoilei in deer and keds, and Anaplasma phagocytophilum strain WI-1 in keds and deer. This is the first report of Anaplasma spp. in a North America deer ked, and begs the question whether L. depressa may be a competent vector of Anaplasma spp. or merely acquire such bacteria while feeding on rickettsemic deer.

We conducted a lab-based comparative study on vector capacity features of two species of triatomines: Triatoma brasiliensis and T. melanica. Both are members of the T. brasiliensis species complex. The former is the most important Chagas disease vector in the northeastern region of Brazil. To date, no transmission via T. melanica has been recorded. Immature insects exhibited distinct intermoult periods without a direct relationship to a given species. Females of T. brasiliensis consumed an average of 1.9 times more meals (mean = 12.92 vs 6.63) and survived for a shorter period (mean =330.8 days) than T. melanica (mean = 365.2 days), probably due to the cost of reproduction (all significant at P<0.05). These data support the idea that T. brasiliensis is more adapted to lab conditions and is more able to infest domiciles than T. melanica. We also found significant distinctions in other features between these species, such as the elapsed time without eating before molting, which was higher for the second, third, and fifth nymph stages of T. melanica. Regarding features analyzed related to vector capacity, insects of all life stages of both species were considered competent to transmit Trypanosoma cruzi because they needed many feedings (mean =1.5–3.82) to moult and because a high proportion (>39%) of insects defecated rapidly (<30 s) after feeding. Overall, results highlight the need to extend vector surveillance to T. melanica.

The Australian native mosquito Aedes (Finlaya) notoscriptus (Skuse) is closely associated with natural and artificial water holding receptacles. Eggs are laid in habitats where they are exposed to drying conditions as water levels fluctuate. Withstanding desiccation enables survival in challenging environments and increases the potential for establishment in non-native habitats. Until now, the desiccation resistance of Ae. notoscriptus eggs has been unknown despite the historical invasive success of this important dog heartworm and arbovirus vector. Viability and mean survival times of eggs from two Ae. notoscriptus populations (metropolitan areas of Sydney, NSW and Adelaide, SA) were evaluated, with eggs stored under three dryness conditions for up to 367 days. Our results revealed that Ae. notoscriptus eggs can withstand desiccation for extended periods, under a variety of conditions, with approximately 9–13% egg viability recorded after one year. This prolonged egg survival reflects the widespread distribution of this mosquito in Australia and its history of incursions and subsequent establishment in non-native habitats. Differences in mean egg volume were recorded in addition to significantly different egg length to width ratios for the two populations, which may reflect adaptation to biotope of origin and an associated likelihood of drought and drying conditions. The results of this study suggest that the desiccation resistant eggs of Ae. notoscriptus make this species highly adaptable, increasing the risk of movement to non-endemic regions of the world.

Esterases are enzymatic proteins known to play a role in insecticide resistance formation. To further our understanding of the development of insecticide resistance, we tested the gene expression level of a gene implicated in insecticide resistance (Temsha est-1) from Culex nigripalpus Theobald (Diptera: Culicidae) in field mosquitoes. We found that the level of expression of TE-1 differed depending on the frequency of exposure to organophosphate insecticide through expression studies. Temsha est-1 cDNA is 1,808 base pairs and fully sequenced with up to 96% nucleotide sequence identity to esterase B genes of other mosquito species. The genes from five different species, including TE-1, were closely related by genetic distance and phylogenetic analysis. Differential expression of this gene that is correlated to differences in susceptibility towards organophosphate would provide the ability to use Temsha est-1 as an indicator of the formation of tolerance/resistance. This would greatly enhance mosquito control efforts by allowing targeted application of insecticides to mosquito populations that are most susceptible. Also, it would provide resistance information so that a rational design could be used for insecticide rotation schedules.

The emerging vector of Chagas disease, Triatoma maculata (Hemiptera, Reduviidae), is one of the most widely distributed Triatoma species in northern South America. Despite its increasing relevance as a vector, no consistent picture of the magnitude of genetic and phenetic diversity has yet been developed. Here, several populations of T. maculata from eleven Colombia and Venezuela localities were analyzed based on the morphometry of wings and the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene sequences. Our results showed clear morphometric and genetic differences among Colombian and Venezuelan populations, indicating high intraspecific diversity. Inter-population divergence is suggested related to East Cordillera in Colombia. Analyses of other populations from Colombia, Venezuela, and Brazil from distinct eco-geographic regions are still needed to understand its systematics and phylogeography as well as its actual role as a vector of Chagas disease.

The developmental time and survival to eclosion of Anopheles hermsi Barr & Guptavanij fed monotypic and mixed diets of ten food types were examined in laboratory studies. Larvae fed monotypic diets containing animal detritus (freeze-dried rotifers, freeze-dried Daphnia pulicaria, and TetraMin® fish food flakes) and the mixotrophic protistan Cryptomonas ovata developed faster and survived better than larvae that were fed other monotypic diets. Survival to adulthood of larvae fed several concentrations of the diatom Planothidium (=Achnanthes) lanceolatum was poor (<13%) and larval development time was approximately twice that of larvae fed TetraMin® fish food flakes, the standard laboratory diet. Larvae fed monotypic diets containing prokaryotes (bacteria [Bacillus cereus] and cyanobacteria [Oscillatoria prolifera]) and brewer's yeast (Saccharomyces cerevisiae) failed to survive beyond the 1^st and 2^nd instar, respectively. Larvae fed only chlorophytes, single-celled Chlamydomonas reinhardtii and filamentous Spirogyra communis, failed to complete larval development, regardless of the concentration tested. Cohorts fed a combination of food types (mixed diets) usually developed better than cohorts fed monotypic diets. Food types that failed to support complete development when fed alone often facilitated development to adulthood when fed in combination with food types containing >1% C₂₀ polyunsaturated fatty acids as total fat, but regardless of essential fatty acid content, algae that produced mucilage and filaments that sank out of the feeding zone were poor quality diets.

Peridomestic structures are considered the main sites where Triatoma infestans (Hemiptera: Reduviidae) remain and disperse, representing the first risk factor for intradomestic invasion, even after vector control activities. This study analyzed T. infestans dispersal during vector control interventions in six rural houses of the arid Chaco (La Rioja, Argentina). Flying and walking dispersers were captured during five months of two consecutive warm seasons after insecticide spraying of intra- and peridomiciles. These data were compared with previous published data in the same scenario but without insecticide spraying in peridomiciles. Recorded climatic conditions were favorable for active dispersion during the study. Total number of T. infestans dispersers moving among domestic habitats decreased after insecticide spraying. Sylvatic triatomines T. guasayana, T. eratyrusiformis, T. garciabesi, and T. platensis, not targeted by insecticide spraying, were captured simultaneously within peridomestic areas and showed higher invasion pressure than T. infestans. Adult T. infestans peridomestic populations showed high nutritional status, indicating low dispersion probability. Some peridomiciles remained infested at the end of the study. However, no intradomiciles were recolonized. These results suggest that there is a low probability of intradomestic recolonization by active dispersion from peridomiciles during 15 months post-spraying.

Triatomines are the vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease, the main endemic disease affecting five to seven million people in Latin America. Besides Triatoma infestans, the most important T. cruzi vector in the Gran Chaco region, other triatomine species associated with sylvatic birds and mammals are responsible for the maintenance of the wild cycle of T. cruzi. The present study aimed at evaluating the house invasion by sylvatic triatomine species in rural communities of the Los Llanos region (La Rioja, Argentina) and its association with environmental variables. House invasion by flying adult triatomines was recorded by trained collectors that surveyed over 377 houses distributed over 73 localities in a 56,600 km² study region, between October, 2014 and February, 2015. The result of the study showed the frequent house invasion by adult triatomines: 26.3% houses were infested in 53% of the localities. Seven sylvatic triatomine species were collected, with T. guasayana and T. garciabesi among the most abundant. House invasion by triatomine species showed no spatial aggregation and was not associated with temperature, precipitation, or vegetation cover at the spatial scale considered in the present study. House invasion by the epidemiologically important T. infestans is a concern of rural communities. Besides constituting a latent, although low, risk, the presence of these species negatively interferes with the vigilance activities of the provincial Chagas disease program.

Cutaneous leishmaniasis (CL) is a complex vector-borne disease caused by Leishmania parasites that are transmitted by the bite of several species of infected female phlebotomine sand flies. Monthly factor analysis of climatic variables indicated fundamental variables. Principal component-based regionalization was used for recognition of climatic zones using a clustering integrated method that identified five climatic zones based on factor analysis. To investigate spatial distribution of the sand fly species, the kriging method was used as an advanced geostatistical procedure in the ArcGIS modeling system that is beneficial to design measurement plans and to predict the transmission cycle in various regions of Khuzestan province, southwest of Iran. However, more than an 80% probability of P. papatasi was observed in rainy and temperate bio-climatic zones with a high potential of CL transmission. Finding P. sergenti revealed the probability of transmission and distribution patterns of a non-native vector of CL in related zones. These findings could be used as models indicating climatic zones and environmental variables connected to sand fly presence and vector distribution. Furthermore, this information is appropriate for future research efforts into the ecology of Phlebotomine sand flies and for the prevention of CL vector transmission as a public health priority.

Infestation of Ixodes vespertilionis Koch, 1844 on Myotis punicus Felten, 1977 from two sites (Trios Tunnel and Sidi Trad cave) in northeastern Algeria was studied. An overall infestation of 41.4% for all stages was found among bats collected from both sites. By stage, a total of eight females, 70 nymphs, and 107 larvae were recovered from both populations. The number of females recovered per bat at Sidi Trad ranged from 0-1, for nymphs 0-2, and for larvae 0-2. While no female ticks were collected at Trios Tunnel, the number of nymphs ranged from 0-2 and for larvae 0-2. At Trios Tunnel, the number of nymphs was significantly higher during April and June but not for July and September. On the other hand, the number of larvae increased from July to November, while at Sidi Trad cave, female ticks were recovered during April and May and then disappeared until the end of the study period. Significant differences were noted during all the months when compared with all stages. Nymphs infested bats significantly during April and May, declined in June and July, and then became steady until October. Larvae peaked in July, with low frequency in April, and then fluctuated from August to November.

Infectious diseases increasingly play a role in the decline of wildlife populations. Vector-borne diseases, in particular, have been implicated in mass mortality events and localized population declines are threatening some species with extinction. Transmission patterns for vector-borne diseases are influenced by the spatial distribution of vectors and are therefore not uniform across the landscape. Avian malaria is a globally distributed vector-borne disease that has been shown to affect endemic bird populations of North America. We evaluated shared habitat use between avian malaria vectors, mosquitoes in the genus Culex and a native grassland bird, the Greater Prairie-Chicken (Tympanuchus cupido), by (1) modeling the distribution of Culex spp. occurrence across the Smoky Hills of north-central Kansas using detection data and habitat variables, (2) assessing the occurrence of these vectors at nests of female Greater Prairie-Chickens, and (3) evaluating if shared habitat use between vectors and hosts is correlated with malarial infection status of the Greater Prairie-Chicken. Our results indicate that Culex occurrence increased at nest locations compared to other available but unoccupied grassland habitats; however the shared habitat use between vectors and hosts did not result in an increased prevalence of malarial parasites in Greater Prairie-Chickens that occupied habitats with high vector occurrence. We developed a predictive map to illustrate the associations between Culex occurrence and infection status with malarial parasites in an obligate grassland bird that may be used to guide management decisions to limit the spread of vector-borne diseases.

Mosquito egg traps, aquatic habitats baited with oviposition attractant and insecticide, are important tools for surveillance and control efforts in integrated vector management programs. The bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used as an environmentally friendly alternative to chemical insecticides and the combination of Bti with a simple oviposition attractant like leaf litter to create an effective egg trap seems appealing. However, previous research suggests that Bti may itself alter oviposition, and that leaf litter may dramatically reduce Bti toxicity. Here we present results from field experiment designed to link the effects of litter and Bti on mosquito oviposition habitat selection and post-colonization survival to production of adult mosquitoes. Tripling litter increased Culex spp. oviposition nearly nine-fold, while Bti had no effect on oviposition. Neither factor altered egg survival, thus larval abundance reflected the effects of litter on oviposition. Both Bti and litter reduced larval survival by ∼60%. We found no evidence that increased litter reduced Bti toxicity. Adult production was dependent upon both litter and Bti. In the absence of Bti, effects of litter on oviposition translated into three-fold more adults. However, in the presence of Bti, initial increases in oviposition were erased by the combined negative effects of Bti and litter on post-colonization survival. Thus, our study provides field evidence that combined litter and Bti application creates an effective ovitrap. This combined treatment had the highest oviposition and the lowest survival, and thus removed the greatest number of mosquitoes from the landscape.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a zoonotic virus transmitted by Hyalomma ticks, the immature stages of which may be carried by migratory birds. In this study, a total of 12 Hyalomma ticks were recovered from five of 228 migratory birds trapped in Spring, 2012 in southern Spain along the East Atlantic flyway. All collected ticks tested negative for CCHFV. While most birds had zero Hyalomma ticks, two individuals had four and five ticks each and the statistical distribution of Hyalomma tick counts per bird is over-dispersed compared to the Poisson distribution, demonstrating the need for intensive sampling studies to avoid underestimating the total number of ticks. Rates of tick exchange on migratory birds during their northwards migration will affect the probability that a Hyalomma tick entering Great Britain is positive for CCHFV. Drawing on published data, evidence is presented that the latitude of a European country affects the probability of entry of Hyalomma ticks on wild birds. Further data on Hyalomma infestation rates and tick exchange rates are required along the East Atlantic flyway to further our understanding of the origin of Hyalomma ticks (i.e., Africa or southern Europe) and hence the probability of entry of CCHFV into GB.

We investigated the morphology and morphometry of eggs from the colonies of the Entomology Laboratory at ENCB-IPN belonging to six species of the genus Meccus and Triatoma recurva, using both light and scanning electron microscopy. Egg ornamentation is an important parameter to consider in the differentiation of species. Samples were observed under a scanning electron microscope. Measurements of fifty eggs per species included length, width, and opercular diameter, which were used for the morphometric analysis. The results showed that the seven species display a polygonal ornamentation dominated by hexagons; the operculum shows also a polygonal ornamental characteristic in each species. Morphometry provided valuable information for discriminating between closely related species of the genus Meccus and Triatoma recurva, a species akin to this group, thereby facilitating the complete discrimination of these species.

The Asian tiger mosquito, Aedes albopictus (Diptera: Culicidae, SKUSE), is an important threat to public health due to its rapid spread and its potential as a vector. The eggs of Ae. albopictus are the most cold resistant life stage and thus, the cold hardiness of eggs is used to predict the future occurrence of the species in distribution models. However, the mechanism of cold hardiness has yet to be revealed. To address this question, we analyzed the layers of diapausing and cold acclimatized eggs of a temperate population of Ae. albopictus in a full factorial test design using transmission electron microscopy. We reviewed the hypotheses that a thickened wax layer or chorion is the cause of cold hardiness but found no evidence. As a result of the induced diapause, the thickness of the dark endochorion as a layer of high electron density and thus an assumed location for waxes was decreasing. We therefore hypothesized a qualitative alteration of the wax layer due to compaction. Cold acclimation was causing an increase in the thickness of the middle serosa cuticle indicating a detachment of serosa membrane from the endochorion as a potential adaptation strategy to isolate inoculating ice formations in the inter-membranous space.

The Mariae species complex, consisting of Aedes mariae, Aedes phoeniciae, and Aedes zammitii, has a limited distribution worldwide. All three species are found in rocky habitats on the coastal areas of Mediterranean countries. Aedes phoeniciae and Ae. zammitii are two members of the Mariae complex that exist in Turkey. The aim of this study was to determine the distribution pattern and genetic structure of Ae. zammitii along the Mediterranean and Aegean regions. For this purpose, larval and adult samples of Ae. zammitii were collected from 19 different rocky habitats along the coastal regions of Antalya, Muğla, Aydın, Izmir, Balikesir, and Çanakkale provinces. DNA isolation was performed primarily from collected samples, and mitochondrial NADH dehydrogenase 4 (ND4) gene was amplified by polymerase chain reaction. Based on ND4 sequence analyses, 21 haplotypes were detected along the distribution range of the species. Analyses of molecular variance (AMOVA) and spatial analyses of molecular variance (SAMOVA) indicated six groups, and most of the variation was among groups, demonstrating the population structuring at group level. Isolation by distance analyses (IBD) showed a correlation between geographic and genetic distances.

The floodwater mosquito Aedes vexans can be a massive nuisance in the flood plain areas of mainland Europe, and is the vector of Tahyna virus and a potential vector of Dirofilaria immitis. This epidemiologically important species forms three subspecies worldwide, of which Aedes vexans arabiensis has a wide distribution in Europe and Africa. We quantified the genetic and phenotypic variation in Ae. vexans arabiensis in populations from Sweden (northern Europe), Hungary, and Serbia (central Europe). A landscape genetics approach (F_ST, STRUCTURE, BAPS, GENELAND) revealed significant differentiation between northern and southern populations. Similar to genetic data, wing geometric morphometrics revealed two different clusters, one made by Swedish populations, while another included Hungarian and Serbian populations. Moreover, integrated genetic and morphometric data from the spatial analysis suggested groupings of populations into three clusters, one of which was from Swedish and Hungarian populations. Data on spatial analysis regarding an intermediate status of the Hungarian population was supported by observed Isolation-by-Distance patterns. Furthermore, a low proportion of interpopulation vs intrapopulation variance revealed by AMOVA and low-to-moderate F_ST values on a broader geographical scale indicate a continuous between-population exchange of individuals, including considerable gene flow on the regional scale, are likely to be responsible for the maintenance of the observed population similarity in Aе. vexans. We discussed data considering population structure in the light of vector control strategies of the mosquito from public health importance.

Two different genetic loci, flaB and ospC, were employed to assign genospecies and OspC phylogenetic type to 18 strains isolated from ticks collected in Pisárky, a suburban park in the city of Brno, Czech Republic. The RFLP analysis revealed three different genospecies (B. afzelii, B. garinii, and B. valaisiana). Three samples from the collection contained more than one genospecies. In the other 15 strains, nucleotide sequences of flaB and ospC were determined. The following phylogenetic analysis assigned 12 isolates to genospecies B. garinii and three to B. afzelii. These isolates were further subdivided into seven distinct ospC groups. The most related OspC types were G2, G4, and G5 (B. garinii) and A3 and A8 (B. afzelii).

African horse sickness (AHS) is an infectious, non-contagious arthropod-borne disease of equids, caused by the African horse sickness virus (AHSV), an orbivirus of the Reoviridae family. It is endemic in sub-Saharan Africa and thought to be the most lethal viral disease of horses. This study focused on detection of AHSV in Culicoides imicola (Diptera: Ceratopogonidae) pools by the application of a RT-qPCR. Midges were fed on AHSV-infected blood. A single blood-engorged female was allocated to pools of unfed nulliparous female midges. Pool sizes varied from 1 to 200. RNA was extracted and prepared for RT-qPCR. The virus was successfully detected and the optimal pool size for the limit of detection of the virus was determined at a range between 1 to 25. Results from this investigation highlight the need for a standardized protocol for AHSV investigation in Culicoides midges especially for comparison among different studies and for the determination of infection rate.