Mosquito Collections Research Articles

Survey of malaria vectors on the Cambodia, Thailand and China-Laos Borders

BackgroundAnopheles maculatus, Anopheles minimus and Anopheles dirus are the major vectors of malaria transmission in the Greater Mekong Subregion (GMS). The malaria burden in this region has decreased significantly in recent years as all GMS countries progress towards malaria elimination. It is necessary to investigate the Anopheles diversity and abundance status and assess the Plasmodium infection rates to understand the malaria transmission potential of these vector species in GMS countries to guide the development of up-to-date vector control strategies and interventions.MethodsA survey of mosquitoes was conducted in Stung Treng, Sainyabuli and Phongsaly Provinces on the Cambodia-Laos, Thailand-Laos and China-Laos borders, respectively. Mosquito collection was done by overnight trapping at sentinel sites in each province. After morphological identification, the 18S rRNA-based nested-PCR was performed to detect malaria parasites in the captured Anopheles mosquitoes.ResultsA total of 18 965 mosquitoes comprising of 35 species of 2 subgenera (Subgenus Anopheles and Subgenus Cellia) and 4 tribes (Tribes Culicini, Aedini, Armigerini and Mansoniini) were captured. Tribe Culicini accounted for 85.66% of captures, followed by Subgenus Anopheles (8.15%). Anopheles sinensis dominated the Subgenus Anopheles by 99.81%. Plasmodium-infection was found in 25 out of the 1 683 individual or pooled samples of Anopheles. Among the 25 positive samples, 19, 5 and 1 were collected from Loum, Pangkhom and Siem Pang village, respectively. Eight Anopheles species were found infected with Plasmodium, i.e., An. sinensis, Anopheles kochi, Anopheles vagus, An. minimus, Anopheles annularis, Anopheles philippinensis, Anopheles tessellatus and An. dirus. The infection rates of Plasmodium falciparum, Plasmodium vivax and mixture of Plasmodium parasite species were 0.12% (2/1 683), 1.31% (22/1 683) and 0.06% (1/1 683), respectively.ConclusionsOverall, this survey re-confirmed that multiple Anopheles species carry malaria parasites in the international border areas of the GMS countries. Anopheles sinensis dominated the Anopheles collections and as carriers of malaria parasites, therefore may play a significant role in malaria transmission. More extensive investigations of malaria vectors are required to reveal the detailed vector biology, ecology, behaviour, and genetics in GMS regions in order to assist with the planning and implementation of improved malaria control strategies.Graphical

Seasonal variation in abundance and blood meal sources of primary and secondary malaria vectors within Kilombero Valley, Southern Tanzania

BackgroundExisting control tools have significantly reduced malaria over the past two decades. However, progress has been stalled due to increased resistance in primary vectors and the increasing role of secondary vectors. This study aimed to investigate the impact of seasonal change on primary and secondary vector abundance and host preference. Understanding the impact of seasonal dynamics of primary and secondary vectors on disease transmission will inform effective strategies for vector management and control.MethodsVector abundance was measured through longitudinal collection of mosquitoes, conducted monthly during the wet and dry seasons, in Sagamaganga, a village in the Kilombero Valley, Tanzania. Mosquitoes were collected indoors using CDC light traps and backpack aspirators, and outdoors using resting buckets baited with cattle urine. In addition, a direct measure of host preference was taken monthly using human- and cattle-baited mosquito electrocuting traps. A host census was conducted to provide an indirect measure of host preference together with monthly blood meal source analysis. All collected mosquitoes were assayed for Plasmodium sporozoites.ResultsA total of 2828 anophelines were collected, of which 78.5% and 21.4%, were primary and secondary vectors, respectively. The abundance of the primary vectors, Anopheles arabiensis and Anopheles funestus, and of the secondary vectors varied seasonally. Indirect measures of host preference indicated that all vectors varied blood meal choice seasonally, with the direct measure confirming this for An. arabiensis. All anopheline mosquitoes tested negative for sporozoites.ConclusionsAt the study location, the abundance of both primary and secondary vectors changed seasonally. Indirect and direct measures of host preference demonstrated that An. arabiensis varied from being zoophilic to being more opportunistic during the wet and dry seasons. A similar trend was observed for the other vectors.Graphical

Impact of different mosquito collection methods on indicators of Anopheles malaria vectors in Uganda

BackgroundMethods used to sample mosquitoes are important to consider when estimating entomologic metrics. Human landing catches (HLCs) are considered the gold standard for collecting malaria vectors. However, HLCs are labour intensive, can expose collectors to transmission risk, and are difficult to implement at scale. This study compared alternative methods to HLCs for collecting Anopheles mosquitoes in eastern Uganda.MethodsBetween June and November 2021, mosquitoes were collected from randomly selected households in three parishes in Tororo and Busia districts. Mosquitoes were collected indoors and outdoors using HLCs in 16 households every 4 weeks. Additional collections were done indoors with prokopack aspirators, and outdoors with pit traps, in these 16 households every 2 weeks. CDC light trap collections were done indoors in 80 households every 4 weeks. Female Anopheles mosquitoes were identified morphologically and Anopheles gambiae sensu lato were speciated using PCR. Plasmodium falciparum sporozoite testing was done with ELISA.ResultsOverall, 4,891 female Anopheles were collected, including 3,318 indoors and 1,573 outdoors. Compared to indoor HLCs, vector density (mosquitoes per unit collection) was lower using CDC light traps (4.24 vs 2.96, density ratio [DR] 0.70, 95% CIs 0.63–0.77, p < 0.001) and prokopacks (4.24 vs 1.82, DR 0.43, 95% CIs 0.37–0.49, p < 0.001). Sporozoite rates were similar between indoor methods, although precision was limited. Compared to outdoor HLCs, vector density was higher using pit trap collections (3.53 vs 6.43, DR 1.82, 95% CIs 1.61–2.05, p < 0.001), while the sporozoite rate was lower (0.018 vs 0.004, rate ratio [RR] 0.23, 95% CIs 0.07–0.75, p = 0.008). Prokopacks collected a higher proportion of Anopheles funestus (75.0%) than indoor HLCs (25.8%), while pit traps collected a higher proportion of Anopheles arabiensis (84.3%) than outdoor HLCs (36.9%).ConclusionIn this setting, the density and species of mosquitoes collected with alternative methods varied, reflecting the feeding and resting characteristics of the common vectors and the different collection approaches. These differences could impact on the accuracy of entomological indicators and estimates of malaria transmission, when using the alternative methods for sampling mosquitos, as compared to HLCs.

Community efforts to monitor and manage Aedes mosquitoes (Diptera: Culicidae) with ovitraps and litter reduction in east Tennessee

BackgroundEast Tennessee (USA) is burdened by mosquito-borne La Crosse virus disease, but minimal resources for mosquito surveillance, management, or related community education exist in the region. To address these needs, we developed a program to train middle and high school educators in basic medical entomology. The educators then used their skills in the classroom to teach students about La Crosse virus disease and conduct mosquito collection experiments. As a case study of a potential application of classroom-collected data, we also partnered with a local non-profit organization to assess the potential for a volunteer litter cleanup to reduce mosquito populations in a Tennessee neighborhood.MethodsOur first objective was to investigate the ability for educators and their students (schools) to collect high-quality mosquito surveillance data. In 2019 and 2020, we collected Aedes (Diptera: Culicidae) eggs during the same study period as schools and assessed whether data collected by schools reflected the same findings as our own data. Our second objective was to investigate the impact of a volunteer litter cleanup event on Aedes mosquito abundance. In 2021, we collected Aedes eggs before and after a neighborhood trash cleanup while schools conducted their own mosquito egg collections. Using the school collections as non-treatment sites, we used a Before-After-Control-Impact analysis to determine if there was a significant decline in egg abundance after the cleanup.ResultsIn 2019, mosquito abundance trends were similar between our data and school data but differed significantly during some weeks. After refining our protocols in 2020, school data was highly similar to our data, indicating that schools consistently collected high-quality surveillance data in the program’s second year. In 2021, we found a significant decline in Aedes egg abundance after the litter cleanup event in comparison to the schools, but the number of adults reared from those eggs did not differ between sites after the cleanup.ConclusionThe results of our work demonstrate the potential for community-driven programs to monitor mosquito abundance trends and for volunteer-based cleanup events to reduce the burden of Aedes mosquitoes. In the absence of infrastructure and resources, academic-community partnerships like the ones evaluated here, provide opportunities to help resource limited areas.

Arbovirus persistence in North-Western Europe: Are mosquitoes the only overwintering pathway?

In some areas in temperate Europe, genomic analyses of mosquito-borne virus outbreaks have revealed the presence of similar virus strains over several years, indicating local overwintering of these viruses. However, it remains unclear how mosquito-borne viruses can persist in winter, when conditions are generally unfavourable for virus circulation. One of the presumed routes of virus persistence is via diapausing mosquitoes. Here, we set out to study whether arbovirus persistence of West Nile virus (WNV), Usutu virus (USUV) and Sindbis virus (SINV) occurs in diapausing mosquitoes in the Netherlands. To this end, mosquito collections were carried out in the winter of 2020 and 2021, in hibernacula located in two areas with previously observed WNV and/or USUV activity. In total, we collected 4200 mosquitoes belonging to four species (Culex pipiens, Culiseta annulata, Anopheles maculipennis s.l., and Culex territans), which were pooled in 490 monospecific pools. These pools were subjected to WNV-, USUV- and SINV-screening using a multiplex real-time RT-PCR assay. All mosquito pools tested negative for the presence of WNV, USUV and SINV RNA. Consequently, we did not find evidence of arbovirus persistence in diapausing mosquitoes in the Netherlands, even though USUV and WNV have re-appeared in birds and/or mosquitoes during the summer seasons of 2020–2022. Concluding, given the persistence of USUV and WNV in the Netherlands and SINV in other temperate regions, this study highlights the importance of further research on (alternative) arbovirus overwintering routes.

Malaria vector feeding, peak biting time and resting place preference behaviors in line with Indoor based intervention tools and its implication: scenario from selected sentinel sites of Ethiopia

In Ethiopia, malaria incidence has significantly reduced in the past decade through the combined use of conventional vector control approaches and treatment using antimalarial drugs. However, the sustainability of this achievement is threatened by the shift in biting and resting behaviors and emergence of insecticide resistance by the primary malaria vector. Therefore, continuous monitoring of the behaviour of malaria mosquitoes in different sentinel sites is crucial to design effective prevention and control methods in the local context. Entomological investigations were conducted in three sentinel sites for five consecutive months during the major malaria transmission season. The species composition, population dynamics, biting and resting behaviours of malaria vectors were determined using center for disease control and prevention (CDC) light trap, human landing catch (HLC), pyrethrum spray catch (PSC) and Pitfall shelter collection (PFS). Accordingly, 10 households for CDC, 10 households for PSC, 10 households for PFS and 5 households for HLC from each site were randomly enrolled for mosquito collection. A total of 8,297 anopheline mosquitoes were collected from the three sites, out of which 4,525 (54.5 %) were An. gambiae, s.l. 2,028 (24.4 %) were An. pharoensis, 160 (1.9 %) were An. funestus and the rest 1,584 (19 %) were other anophelines (An. coustani, An. cinerus and An. tenebrosus). No significant variation (P = 0.476) was observed between indoor (25.2/trap-night and outdoor collections (20.1/trap-night). Six hundred seventy six (43.3%) of An. gambiae s.l. (primary vector) were collected between 18:00 and 22:00 h. Biting activity declined between 00:00 and 02:00 h. The national malaria control program should pay close attention to the shifting behavior of vector mosquitoes as the observed outdoor feeding tendency of the vector population could pose challenges to the indoor intervention tools IRS and LLINs.

Mosquito Fauna and Spatial Distribution in an Atlantic Forest Area in Rio de Janeiro State, Brazil, Reveal a High Risk of Transmission of Yellow Fever and Other Arboviruses.

In 2017-2019, Brazil recorded its most severe outbreak of yellow fever due to the spread of the virus (YFV) in the country's southeast. Here, we investigated mosquito fauna and the spatial distribution of species in a primatology center in the Atlantic Forest bioregion in Rio de Janeiro state to evaluate the risk of YFV transmission in distinct environments. Fortnightly mosquito collections were performed from December 2018 to December 2019 at 12 sites along a disturbance gradient from a modified environment to 400 m inside the forest. We used ovitraps, BG-Sentinel, and protected human attraction (PHA). A total of 9349 mosquitoes of 21 species were collected. The collection method strongly influenced the captured fauna, with species such as Anopheles cruzii, Psorophora ferox, Runchomyia cerqueirai, Wyeomyia incaudata, Wy. theobaldi, Sabethes chloropterus, and Sa. albiprivus only collected via PHA. Collections with ovitraps resulted in low diversity and richness, with Haemagogus leucocelaenus and Hg. janthinomys/capricornii predominating. The diverse local fauna and the abundance and ubiquity of the latter species, which are the primary vectors of YFV, indicated that this area was highly vulnerable to arbovirus transmission, especially yellow fever, highlighting the need for improved surveillance and vaccination coverage in human and captive endangered non-human primates.

Comparison of diurnal biting activity, life table, and demographic attributes of Aedes albopictus (Asian tiger mosquito) from different urbanized settings in West Java, Indonesia

The Asian tiger mosquito, Aedes albopictus has well-adaptive behavior to environmental changes, including human urbanization, and has an essential role as the main vector of important pathogenic arboviruses. This study aims to analyze the biology and life table of the Ae. albopictus populations collected from urban and peri-urban areas of Bogor, West Java, Indonesia under laboratory conditions. Mosquito eggs collection was carried out in urban and peri-urban areas using ovitraps. The observation of the life table experiment that followed the development of Ae. albopictus started from the emergence of the first individual to the last surviving individual. Several biological parameters comparing Ae. albopictus from two collection sites based on life table analysis were shown to be significantly different. Biting activity of all mosquitoes from urban and peri-urban areas showed a clear bimodal activity with morning peak at 09:00-10.00 and evening peak at 16:00-17:00. Ae. albopictus from the urban area have higher fecundity, considerably longer lifespan, more gonotrophic cycles, and a higher net reproduction rate (R0) than Ae. albopictus from the peri-urban area. These findings will provide valuable information about the well-adapted Ae. albopictus in urban areas and assist in providing basic reproductive data to improve vector control and current surveillance strategies especially in the study area.

Outdoor and early hour human biting activities of malaria mosquitoes and the suitability of clay pot for outdoor resting mosquito collection in malaria endemic villages of southern Rift Valley, Ethiopia

BackgroundSampling adult Anopheles mosquitoes is important for assessing vector density, estimating the sporozoite infection rate, and quantifying the impact of vector control interventions. The objective of this study was to assess the Anopheles mosquito species composition, and their outdoor and indoor biting activities, and to evaluate the suitability of clay pots for indoor and outdoor resting mosquito collections. MethodsTwo malaria-endemic villages in the Gamo zone were purposely selected. Forty clay pots were deployed for outdoor resting mosquitoes sampling and another forty for indoor resting sampling. Twenty pit shelters were constructed for outdoor resting mosquito collection. The human landing catch (HLC) technique was employed to collect indoor and outdoor host-seeking mosquitoes in two households in each village. Morphological identification of the Anopheles mosquito was done using an identification key. Enzyme-linked immunosorbent assay technique was used for blood meal origin and circumsporozoite proteins (CSP) test. Speciation of An. gambiae complex was done using polymerase chain reaction. A Chi-square test was used to compare the effectiveness of clay pot and pit shelters for outdoor resting sampling. ResultsA total of 904 female Anopheles mosquitoes comprising An. gambiae complex, An. pharoensis, An. tenebrosus, An. dencalicus and An. demelloni were sampled. The majority (64%) of them were sampled by the HLC technique. There was a slight difference between the outdoor clay pot (19%) and pit shelter (17%) collection. No Anopheles mosquitoes were collected indoor using clay pots. All mosquitoes were tested for CSPs, but none of them were found to be positive. Anopheles mosquitoes were tending to bite humans outdoor than indoors, and their peak biting hours was 10–11 pm. The human blood meal index of Anopheles mosquitoes was 0.07 from pit shelters and it was 0.04 from clay pots. The bovine blood meal index was 0.45 for mosquitoes from both pit shelters and clay pot collections. ConclusionAnopheles arabiensis was the predominant species and it was tending to bite cattle more than humans. Clay pot could be suitable for outdoor resting mosquito collection, but not for indoor resting species.

International Research and Philanthropy

International Research and Philanthropy

Adapting field-mosquito collection techniques in a perspective of near-infrared spectroscopy implementation.

BackgroundNear-infrared spectroscopy (NIRS) has the potential to be a useful tool for assessing key entomological parameters of malaria-transmitting mosquitoes, including age, infectious status and species identity. However, before NIRS can be reliably used in the field at scale, methods for killing mosquitoes and conserving samples prior to NIRS scanning need to be further optimized. Historically, mosquitoes used in studies have been killed with chloroform, although this approach is not without health hazards and should not be used in human dwellings. For the application of NIRS scanning it is also unclear which mosquito preservation method to use. The aim of the study reported here was to investigate the use of pyrethrum spray, a commercially available insecticide spray in Burkina Faso, for killing mosquitoesMethodsLaboratory-reared Anopheles gambiae and Anopheles coluzzii were killed using either a pyrethrum insecticide spray routinely used in studies involving indoor mosquito collections (Kaltox Paalga®; Saphyto, Bobo-Dioulasso, Burkina Faso) or chloroform (“gold standard”). Preservative methods were also investigated to determine their impact on NIRS accuracy in predicting the species of laboratory-reared Anopheles and wild-caught mosquito species. After analysis of fresh samples, mosquitoes were stored in 80% ethanol or in silica gel for 2 weeks and re-analyzed by NIRS. In addition, experimentally infected An. coluzzii and wild-caught An. gambiae sensu lato (s.l.) were scanned as fresh samples to determine whether they contained sporozoites, then stored in the preservatives mentioned above for 2 weeks before being re-analyzed.ResultsThe difference in the accuracy of NIRS to differentiate between laboratory-reared An. gambiae mosquitoes and An. coluzzii mosquitoes killed with either insecticide (90%) or chloroform (92%) was not substantial. NIRS had an accuracy of 90% in determining mosquito species for mosquitoes killed with chloroform and preserved in ethanol or silica gel. The accuracy was the same when the pyrethrum spray was used to kill mosquitoes followed by preservation in silica gel, but was lower when ethanol was used as a preservative (80%). Regarding infection status, NIRS was able to differentiate between infected and uninfected mosquitoes, with a slightly lower accuracy for both laboratory and wild-caught mosquitoes preserved in silica gel or ethanol.ConclusionsThe results show that NIRS can be used to classify An. gambiae s.l. species killed by pyrethrum spray with no loss of accuracy. This insecticide may have practical advantages over chloroform for the killing of mosquitoes in NIRS analysis.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-022-05458-6.

Mitochondrial DNA barcoding of mosquito species (Diptera: Culicidae) in Thailand

The correct identification of mosquito species is important for effective mosquito vector control. However, the standard morphological identification of mosquito species based on the available keys is not easy with specimens in the field due to missing or damaged morphological features during mosquito collections, often leading to the misidentification of morphologically indistinguishable. To resolve this problem, we collected mosquito species across Thailand to gather genetic information, and evaluated the DNA barcoding efficacy for mosquito species identification in Thailand. A total of 310 mosquito samples, representing 73 mosquito species, were amplified using mitochondrial cytochrome c oxidase subunit I (COI) primers. The average maximum intraspecific genetic variation of the 73 mosquito species was 1% ranged from 0-5.7%. While, average minimum interspecific genetic variation (the distance to the nearest neighbour) of the 73 mosquito species was 7% ranged from 0.3-12.9%. The identification of success rates based on the "Best Match," "Best Close Match," and "All Species Barcodes" methods were 97.7%, 91.6%, and 81%, respectively. Phylogenetic analyses of Anopheles COI sequences demonstrated a clear separation between almost all species (except for those between An. baimaii and An. dirus), with high bootstrap support values (97%-99%). Furthermore, phylogenetic analyses revealed potential sibling species of An. annularis, An. tessellatus, and An. subpictus in Thailand. Our results indicated that DNA barcoding is an effective molecular approach for the accurate identification of mosquitoes in Thailand.

Overlaying human and mosquito behavioral data to estimate residual exposure to host-seeking mosquitoes and the protection of bednets in a malaria elimination setting where indoor residual spraying and nets were deployed together.

Characterizing persistent malaria transmission that occurs after the combined deployment of indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) is critical to guide malaria control and elimination efforts. This requires a detailed understanding of both human and vector behaviors at the same temporal and spatial scale. Cross-sectional human behavior evaluations and mosquito collections were performed in parallel in Magude district, Mozambique. Net use and the exact time when participant moved into each of five environments (outdoor, indoor before bed, indoor in bed, indoor after getting up, and outdoor after getting up) were recorded for individuals from three different age groups and both sexes during a dry and a rainy season. Malaria mosquitoes were collected with CDC light traps in combination with collection bottle rotators. The percentage of residual exposure to host-seeking vectors that occurred in each environment was calculated for five local malaria vectors with different biting behaviors, and the actual (at observed levels of LLIN use) and potential (i.e. if all residents had used an LLIN) personal protection conferred by LLINs was estimated. Anopheles arabiensis was responsible for more than 74% of residents’ residual exposure to host-seeking vectors during the Magude project. The other four vector species (An. funestus s.s., An. parensis, An. squamosus and An. merus) were responsible for less than 10% each. The personal protection conferred by LLINs prevented only 39.2% of the exposure to host-seeking vectors that survived the implementation of both IRS and LLINs, and it differed significantly across seasons, vector species and age groups. At the observed levels of bednet use, 12.5% of all residual exposure to host-seeking vectors occurred outdoor during the evening, 21.9% indoor before going to bed, almost two thirds (64%) while people were in bed, 1.4% indoors after getting up and 0.2% outdoor after leaving the house. Almost a third of the residual exposure to host-seeking vectors (32.4%) occurred during the low transmission season. The residual bites of An. funestus s.s. and An. parensis outdoors and indoor before bedtime, of An. arabiensis indoors when people are in bed, and of An. squamosus both indoors and outdoors, are likely to have sustained malaria transmission throughout the Magude project. By increasing LLIN use, an additional 24.1% of exposure to the remaining hosts-seeking vectors could have been prevented. Since An. arabiensis, the most abundant vector, feeds primarily while people are in bed, increasing net use and net feeding inhibition (through e.g. community awareness activities and the selection of more effective LLINs) could significantly reduce the exposure to remaining host-seeking mosquitoes. Nonetheless, supplementary interventions aiming to reduce human-vector contact outdoors and/or indoors before people go to bed (e.g. through larval source management, window and eave screening, eave tubes, and spatial repellents) will be needed to reduce residual exposure to the outdoor and early biting An. funestus s.s. and An. parensis.

Establishment of a colony of Anopheles darlingi from French Guiana for vector competence studies on malaria transmission

Anopheles darlingi is a major vector of both Plasmodium falciparum and Plasmodium vivax in South and Central America including French Guiana. However, the vector competence and physiology of this mosquito species have been scarcely studied due to difficulties in rearing it in the laboratory. Here, we report the successful establishment of a robust colony, from a mosquito collection in French Guiana. We describe our mosquito colonization procedure with relevant information on environmental conditions, mating ability, larval development, and survival, recorded over the first six critical generations. Experimental infection showed that our An. darlingi colony has a moderate permissiveness to in vitro produced gametocytes of the P. falciparum NF54 strain originating from Africa. This colony, which has reached its 21st generation, will allow further characterization of An. darlingi life-history traits and of Plasmodium–mosquito interactions with South American malaria parasites.

A Mark-Release-Recapture Study to Estimate Field Performance of Imported Radio-Sterilized Male Aedes albopictus in Albania.

The pathogen transmitting Aedes albopictus mosquito is spreading rapidly in Europe, putting millions of humans and animals at risk. This species is well-established in Albania since its first detection in 1979. The sterile insect technique (SIT) is increasingly gaining momentum worldwide as a component of area-wide-integrated pest management. However, estimating how the sterile males will perform in the field and the size of target populations is crucial for better decision-making, designing and elaborating appropriate SIT pilot trials, and subsequent large-scale release strategies. A mark-release-recapture (MRR) experiment was carried out in Albania within a highly urbanized area in the city of Tirana. The radio-sterilized adults of Ae. albopictus Albania strain males were transported by plane from Centro Agricoltura Ambiente (CAA) mass-production facility (Bologna, Italy), where they were reared. In Albania, sterile males were sugar-fed, marked with fluorescent powder, and released. The aim of this study was to estimate, under field conditions, their dispersal capacity, probability of daily survival and competitiveness, and the size of the target population. In addition, two adult mosquito collection methods were also evaluated: BG-Sentinel traps baited with BG-Lure and CO2, (BGS) versus human landing catch (HLC). The overall recapture rates did not differ significantly between the two methods (2.36% and 1.57% of the total male released were recaptured respectively by BGS and HLC), suggesting a similar trapping efficiency under these conditions. Sterile males traveled a mean distance of 93.85 ± 42.58 m and dispersed up to 258 m. Moreover, they were observed living in the field up to 15 days after release with an average life expectancy of 4.26 ± 0.80 days. Whether mosquitoes were marked with green, blue, yellow, or pink, released at 3.00 p.m. or 6.00 p.m., there was no significant difference in the recapture, dispersal, and survival rates in the field. The Fried competitiveness index was estimated at 0.28. This mark-release-recapture study provided important data for better decision-making and planning before moving to pilot SIT trials in Albania. Moreover, it also showed that both BG-traps and HLC were successful in monitoring adult mosquitoes and provided similar estimations of the main entomological parameters needed.

Rapid detection of kdr mutation F1534C in Aedes aegypti using recombinase polymerase amplification and lateral flow dipsticks

Insecticide resistance monitoring is essential in assessing the efficacy of vector control measures. However, gold standard PCR-based molecular analyses for insecticide resistance detection are often hindered by time-consuming sample processing, as well as considerable infrastructure and resourcing requirements. In this study, we combined a novel one-step sample preparation reagent with a rapid isothermal molecular test that detects a knock down resistance (kdr) mutation (F1534C) that enables pyrethroid resistance in Aedes aegypti mosquitoes. We trialled the rapid F1534C pyrethroid resistance test using insecticide resistant Ae. aegypti mosquito bodies and compared results to a conventional, allele-specific quantitative PCR (AS-qPCR) coupled with melt curve genotyping in corresponding mosquito heads. From a strain of Ae. aegypti established from an insecticide resistant population in Merida, Mexico (n = 27), all the mosquito bodies (n = 27) tested positive with the rapid F1534C test regardless of whether they were homozygous or heterozygous. To assess diagnostic test specificity, we confirmed that F1534 was not detected in laboratory-reared, fully susceptible Ae. aegypti mosquito bodies (n = 28) using the rapid F1534C test or the conventional AS-qPCR melt curve analysis. All corresponding mosquito heads (n = 28) were homozygous wild-type FF1534. The rapid F1534C test thus demonstrated 100% diagnostic sensitivity (95% CI: 87.23% to 100%) and 100% diagnostic specificity (95% CI: 87.66% to 100.00%) for detection of the F1534C pyrethroid resistant single nucleotide polymorphism (SNP) in both heterozygous and homozygous Ae. aegypti. In the collection of mutant mosquitoes from Mexico, CC1534 homozygous mutants occurred at a frequency of 74.1% (n = 20) and FC heterozygous mutants at a frequency of 25.9% (n = 7). The rapid F1534C test significantly reduced the sample processing and testing time from approximately 6 h for the AS-qPCR melt curve analysis to only 25 min. These results demonstrate significant potential for our approach to resistance testing as a field-based, low-resource, rapid alternative to time-consuming and expensive laboratory-based detection.

Honey-baited FTA cards in box gravid traps for the assessment of Usutu virus circulation in mosquito populations in Germany

Usutu virus (USUV) is becoming increasingly important to veterinary and human health in Germany. USUV has been implicated in mass die-off events of birds, especially of blackbirds (Turdus merula), and has experienced significant range expansion in the years since its first detection in 2010. Current detection methods rely primarily on dead bird surveillance or mass mosquito collection using CO2 as the main attractant. Dead bird surveillance can result in detection of disease circulation past the point at which control efforts would be most impactful. Vector surveillance offers the opportunity to detect disease circulation before significant outbreaks occur. However, current methods result in collections of extremely large numbers of predominantly nulliparous female mosquitoes who have not yet taken a blood meal. This study sought to test whether box gravid traps could successfully trap USUV infected gravid Culex mosquitoes, and if viral RNA could be successfully transferred and stabilised on an FTA card. During the month of August 2020, 18 Reiter-Cummings style box gravid traps with honey-baited FTA cards were set in a region of known USUV circulation around the southern border of Hesse, Germany. Four 48-hour trapping rounds were conducted. All mosquitoes and FTA cards were collected and stored during transport to the laboratory on dry ice. Samples and FTA cards were then transferred and stored in a freezer at -5 °C until identification. Identification was carried out on a chill plate before being sent with overnight courier in a styrofoam box with cooling elements for virus detection with a modified generic flavivirus RT-PCR. Mosquitoes were separated into pools by trap, date, species and feeding status. 2003 mosquitoes were caught in four rounds of trapping, 1834 or 88% of which were female Culex mosquitoes used for examination. 13 pools of mosquitoes and four FTA cards tested positive for USUV. No positive FTA cards were found in traps with positive mosquitoes and no positive mosquitoes were found in traps with positive FTA cards. Although fewer FTA cards than expected returned a positive result, this may have been a result of the extreme conditions experienced in the field and highlights the need to establish the temperature and humidity boundaries such a collection method can withstand. Box gravid traps however, provided a highly effective and targeted approach for capturing gravid female Culex mosquitoes, the most appropriate subpopulation for testing for USUV. Additionally, the simplicity and effectiveness of this trapping and surveillance method make it an attractive option for use as an early warning system, including for large scale surveillance programmes.

West Nile Virus Surveillance in Sentinel Chickens and Mosquitoes in Panama City Beach, Florida, from 2014 To 2020.

Over 20 years since its introduction, the West Nile virus (WNV) continues to be the leading cause of arboviral disease in the USA. In Panama City Beach (Bay County, FL), WNV transmission is monitored using sentinel chickens and testing mosquito pools for presence of viral RNA. In the current work, we monitored WNV transmission from 2014 to 2020 through weekly serology sampling of sentinel chickens; mosquito populations through biweekly mosquito collections by suction traps (1 m and 9 m) and weekly gravid trap collections; and mosquito infection rates using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Samples were sent to the Bureau of Public Health Laboratories (Tampa, FL) for testing presence/absence of WNV via RT-PCR assay. Our results indicated that canopy surveillance could augment ground collections, providing greater proportions of Culex mosquitoes with less bycatch compared with ground collections. Serology indicated 94 seroconversions to WNV in the study area from 2014 to 2020. The most active year was 2016, which accounted for 32% (n = 30) of all seroconversions reported during the study period. We detected 20 WNV-positive mosquito pools from Culex quinquefasciatus during 2014-17; mosquito infection rates ranged from 2.02 to 23.81 per thousand (95% CI). Climate data indicated anomalously high precipitation in 2014-19 preceding WNV transmission. Data analyzed herein indicate utility in year-round continuous and diversified surveillance methodologies. This information is needed to properly calibrate future models that could assist with predicting transmission events of WNV in Panama City Beach, FL.

The Distribution of Uranotaenia sapphirina and Ur. socialis in Tabasco, Southern Mexico.

In Mexico, the genus Uranotaenia includes 11 species distributed mainly in the tropical and subtropical regions in the southeast of the country. Uranotaenia sapphirina has been reported in 18 states in Mexico: Campeche, Coahuila, Colima, Chiapas, Guerrero, Hidalgo, Jalisco, Mexico City, Mexico State, Michoacán, Morelos, Oaxaca, Quintana Roo, Sinaloa, Tabasco, Tamaulipas, Veracruz, and Yucatán; whereas Ur. socialis has been reported in Chiapas and Quintana Roo. In recent surveillance studies of mosquito species in Tabasco, Ur. sapphirina and Ur. socialis were omitted due to the lack of recent collection records, but in historical records, the presence of Ur. sapphirina and one species consistent with the description of Ur. socialis were mentioned. During a mosquito survey collection, immature stages from ground-level natural habitats in conservation areas of Tabasco, Ur. sapphirina and Ur. socialis were collected in association with Anopheles albimanus, Culex erraticus, Mansonia titillans, and Ur. lowii. Additionally, 2 Mexican entomological collections were reviewed, searching additional records of those species. An identification key to separate larvae and adult females of Ur. sapphirina and Ur. socialis is provided. With the addition of Ur. sapphirina and Ur. socialis to the mosquito fauna of Tabasco, there are currently 107 species in the state, being the 3rd state in Mexico with the highest richness of mosquito species. Specimens collected during this study were deposited in the Collection of the Entomological and Bioassay Research Unit of Tabasco.

Diversity of mosquito species and potential arbovirus transmission in long-tailed macaque (Macaca fascicularis) breeding facilities

Background and Aim:Mosquito-borne viral infections are diseases that reduce human and animal health levels. Their transmission involves wildlife animals as reservoirs and amplifying hosts, including long-tailed macaques (Macaca fascicularis), and potentially transmits to humans and vice versa. This study aimed to determine the species diversity, richness, and biting activity of mosquitoes in a long-tailed macaque breeding area facility and discover the presence of Flavivirus and Alphavirus as the two main arboviruses reported to infect macaques.Materials and Methods:Human landing catch, light trap, and sweep net methods were used for mosquito collection around long-tailed macaques cages at parallel times for 12 h (18:00–06:00) for 12 nights. Mosquito species were identified to the species level based on the morphological identification key for Indonesian mosquitoes. Mosquito diversity was analyzed by several diversity indices. Mosquitoes caught using the human landing catch method were pooled based on mosquito species for viral ribonucleic acid extraction. Reverse transcription-polymerase chain reaction (RT-PCR) detected the non-structural protein 5 of the Flavivirus region and the non-structural protein 4 of the Alphavirus region. This study used the man-hour density and man-biting rate formulas for mosquito density.Results:Ten mosquito species were collected, namely, Aedes albopictus, Anopheles aconitus, Anopheles minimus, Anopheles vagus, Armigeres foliatus, Armigeres subalbatus, Culex gelidus, Culex hutchinsoni, Culex tritaeniorhynchus, and Culex quinquefasciatus. The number of mosquitoes caught using the light trap method had the highest abundance. In contrast, the number of mosquito species caught using the sweep net method had lower diversity than the other two methods. Seven mosquito species were obtained using the human landing catch method. The mosquito species with the highest density was Cx. quinquefasciatus within the observed densest period from 20:00 to 21:00. Negative results were obtained from RT-PCR testing on five species detected using universal Flavivirus and Alphavirus primers.Conclusion:The occurrence of mosquitoes in long-tailed macaque breeding facilities can be a source of transmission of zoonotic vector-borne diseases between animals and humans and vice versa.