A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

Mosquito Species Diversity and Abundance Patterns in Plots with Contrasting Land Use and Land Cover in Bloomington, Indiana.

Land use and land cover (LULC) gradients are associated with differences in mosquito species composition and the entomological risk of mosquito-borne disease. Here, we present results from a season-long study of mosquito species richness and abundance with samples collected at 9 locations from 2 plots with contrasting LULC, an urban farm and a forest preserve, in Bloomington, IN, a city in the midwestern USA. With a total sampling effort of 234 trap-nights, we collected 703 mosquitoes from 9 genera and 21 species. On the farm, we collected 15 species (285 mosquitoes). In the preserve, we collected 19 species (418 mosquitoes). Thirteen species were common in both study plots, 2 were exclusive to the farm, and 6 were exclusive to the forest preserve. In both plots, we collected Aedes albopictus and Ae. japonicus. In the farm, the most common mosquito species were Culex restuans/Cx. pipiens and Coquillettidia perturbans. In the preserve, Ae. japonicus and Ae. triseriatus were the 2 most common mosquito species. Time series analysis suggests that weather factors differentially affected mosquito species richness and mosquito abundance in the plots. Temperature, relative humidity (RH), and precipitation were positively associated with richness and abundance at the farm, while increases in the SD of RH decreased both richness and abundance at the preserve. Our results highlight the importance that LULC has for mosquito species diversity and abundance and confirm the presence of Ae. albopictus and Ae. japonicus in southwestern Indiana.

Comparative evaluation of two lures to attract female mosquitoes in an urban natural reserve during daytime.

The most widely used attractant to capture adult female mosquitoes is CO2. However, there are also baits available on the market that emit a scent resembling human skin. These baits were specifically designed to attract highly anthropophilic species such as Aedes albopictus and Aedes aegypti. In this study, we compare the effectiveness of CDC traps baited either with CO2 or with a commercial blend simulating skin odor, BG-Sweetscent, for trapping female mosquitoes during daylight hours in an urban reserve in the City of Buenos Aires. We employed a hurdle generalized linear mixed model to analyze trap capture probability and the number of mosquitoes captured per hour, considering the effects of attractant, mosquito species, and their interaction. Traps baited with CO2 captured ten mosquito species, while those baited with BG-Sweetscent captured six in overall significantly lower abundance. The odds of capturing mosquitoes were 292% higher for the CO2-baited traps than for those baited with BG-Sweetscent. No evidence of a combined effect of attractant type and species on female mosquito captures per hour was found. Results indicated that CDC traps baited with CO2 were more effective than those baited with BG-Sweetscent in capturing more mosquito species and a higher number of mosquitoes within each species, even if the species captured with CO2 exhibited a certain level of anthropophilia. This result has practical implications for mosquito surveillance and control in urban natural reserves.

Island mosquitoes of Thailand: an update on species diversity and DNA barcoding.

Mosquitoes (Diptera: Culicidae) are among the most medically significant insects, with several species acting as vectors for human pathogens. Although there are frequent reports of mosquito-borne diseases in the border island areas of Thailand, comprehensive data on the diversity and DNA barcoding of these mosquito species remain limited. This study investigated mosquito diversity in two main archipelagos in Thailand-the Trat archipelago (comprising Chang Island and Kood Island) and the Ranong archipelago (comprising Chang Island and Phayam Island)-and generated DNA barcode data from the mosquitoes found there. The survey across these islands discovered a total of 41 species, highlighting the presence of several species known to be vectors for human diseases. Thirty-seven mosquito species from the island areas were documented to provide reference DNA barcode sequences for mosquitoes in Thailand's island regions. Two species, Aedes fumidus and Finlaya flavipennis, have been added as new COI sequence records in the database. DNA barcoding was highly effective in classifying almost all species by identifying barcoding gaps, except for Anopheles baimaii and Anopheles dirus, which could not be distinguished. Additionally, the study noted that geographical variations might influence certain mosquito species, such as Anopheles barbirostris A3 and Mansonia dives, causing them to be split into two distinct subgroups. The findings of this study are crucial, as they aid in classifying mosquito species using molecular techniques and expand our knowledge of disease vectors in these biodiverse regions.

Vegetation structure drives mosquito community composition in UK's largest managed lowland wetland.

PURPOSE: The rising burden of mosquito-borne diseases in Europe extends beyond urban areas, encompassing rural and semi-urban regions near managed and natural wetlands evidenced by recent outbreaks of Usutu and West Nile viruses. While wetland management policies focus on biodiversity and ecosystem services, few studies explore the impact on mosquito vectors. METHODS: Our research addresses this gap, examining juvenile mosquito and aquatic predator communities in 67 ditch sites within a South England coastal marsh subjected to different wetland management tiers. Using joint distribution models, we analyse how mosquito communities respond to abiotic and biotic factors influenced by wetland management. RESULTS: Of the 12 mosquito species identified, Culiseta annulata (Usutu virus vector) and Culex pipiens (Usutu and West Nile virus vector) constitute 47% of 6825 larval mosquitoes. Abundant predators include Coleoptera (water beetles) adults, Corixidae (water boatmen) and Zygoptera (Damselfy) larvae. Models reveal that tier 3 management sites (higher winter water levels, lower agricultural intensity) associated with shade and less floating vegetation are preferred by specific mosquito species. All mosquito species except Anopheles maculipennis s.l., are negatively impacted by potential predators. Culiseta annulata shows positive associations with shaded and turbid water, contrary to preferences of Corixidae predators. CONCLUSIONS: Tier 3 areas managed for biodiversity, characterised by higher seasonal water levels and reduced livestock grazing intensity, provide favourable habitats for key mosquito species that are known vectors of arboviruses, such as Usutu and West Nile. Our findings emphasise the impact of biodiversity-focused wetland management, altering mosquito breeding site vegetation to enhance vector suitability. Further exploration of these trade-offs is crucial for comprehending the broader implications of wetland management.

Application of computer vision and deep learning models to automatically classify medically important mosquitoes in North Borneo, Malaysia.

Mosquito-borne diseases have emerged in North Borneo in Malaysia due to rapid changes in the forest landscape, and mosquito surveillance is key to understanding disease transmission. However, surveillance programmes involving sampling and taxonomic identification require well-trained personnel, are time-consuming and labour-intensive. In this study, we aim to use a deep leaning model (DL) to develop an application capable of automatically detecting mosquito vectors collected from urban and suburban areas in North Borneo, Malaysia. Specifically, a DL model called MobileNetV2 was developed using a total of 4880 images of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus mosquitoes, which are widely distributed in Malaysia. More importantly, the model was deployed as an application that can be used in the field. The model was fine-tuned with hyperparameters of learning rate 0.0001, 0.0005, 0.001, 0.01 and the performance of the model was tested for accuracy, precision, recall and F1 score. Inference time was also considered during development to assess the feasibility of the model as an app in the real world. The model showed an accuracy of at least 97%, a precision of 96% and a recall of 97% on the test set. When used as an app in the field to detect mosquitoes with the elements of different background environments, the model was able to achieve an accuracy of 76% with an inference time of 47.33 ms. Our result demonstrates the practicality of computer vision and DL in the real world of vector and pest surveillance programmes. In the future, more image data and robust DL architecture can be explored to improve the prediction result.

Surveillance of mosquitoes harnessing their buzzing sound.

Mosquito surveillance for vector-borne disease management relies on traditional morphological and molecular techniques, which are tedious, time-consuming, and costly. The present study describes a simple and efficient recording device that analyzes mosquito sound to estimate species composition, male-female ratio, fed-unfed status, and harmonic convergence interaction using fundamental frequency (F0) bandwidth, harmonics, amplitude, and combinations of these parameters. The study examined a total of 19 mosquito species, including 3 species of Aedes, 7 species of Anopheles, 1 species of Armigeres, 5 species of Culex, 1 species of Hulecoetomyia, and 2 species of Mansonia. Among them, the F0 ranges between 269.09 ± 2.96 Hz (Anopheles culiciformis) to 567.51 ± 3.82 Hz (Aedes vittatus) and the harmonic band (hb) number ranges from 5 (An. culiciformis) to 12 (Ae. albopictus). In terms of species identification, the success rate was 95.32 % with F0, 84.79 % with F0-bandwidth, 84.79 % with harmonic band (hb) diversity, and 49.7 % with amplitude (dB). The species identification rate has gone up to 96.50 % and 97.66 % with the ratio and multiplication of F0 and hb, respectively. This is because of the matrices that combine multiple sound attributes. Comparatively, combinations of the amplitude of the F0 and the higher harmonic frequency band were non-significant for species identification (60.82 %). The fed females have shown a considerable increase in F0 in comparison to the unfed. The males of all the species possessed significantly higher frequencies with respect to the females. Interestingly, the presence of male-female of Ae. vittatus together showed harmonic convergence between the 2nd and 3rd harmonic bands. In conclusion, the sound-based technology is simple, precise, and cost-effective and provides better resolution for species, sex, and fed-unfed status detection in comparison to conventional methods. Real-time surveillance of mosquitoes could potentially utilize this technology.

Vector mosquito distribution and richness are predicted by socio-economic, and ecological variables.

Mosquitoes of vectorial importance represent a ubiquitous and constant threat of potentially devastating arboviral outbreaks. Our ability to predict such outcomes is still restricted. To answer this, we have used an extensive data collection of 23 vector and 233 non-vector mosquito species distributed throughout the Mexican territory and linked them to social and environmental factors. Our aim was to predict vector and non-vector mosquitoes' distribution and species richness based on socioeconomic and environmental data. We found that lack of health services, human population variation, ecological degradation, and urban-rural categorization contributed significantly to explain the distribution of vector mosquitoes. mosquitoes. This phenomenon is probably attributed to the degradation of natural ecosystems as it creates favorable conditions for the proliferation of vector mosquitoes. The richness of vector mosquitoes was similarly explained by most of these variables as well as altitude. As for non-vector mosquitoes, social marginalization, ecological degradation, anthropogenic impact, and altitude explain species richness and distribution. These findings illustrate the complex interaction of environmental and socioeconomic factors behind the distribution of mosquitoes, and the potential for arboviral disease outbreaks. Areas with human populations at highest risk for mosquito-borne diseases should be primary targets for vector control.

Species diversity and spatial distribution of mosquitoes (Diptera: Culicidae) from La Isla Amazon Park, Napo Province, Ecuador / Diversidad de especies y distribución espacial de mosquitos (Diptera: Culicidae) del Parque Amazónico La Isla, provincia de Napo, Ecuador

Abstract Introduction: Vector-borne diseases are prevalent in the Amazon and Coastal regions of Ecuador. However, there is a scarcity of mosquito ecology studies in these areas. The most recent list of species reported for the country comprises 8 tribes, 22 genera, and 200 species. Objectives: To document the Culicidae species found in La Isla Amazon Park, Napo, Ecuador, including those with epidemiological significance; and to analyze their composition, abundance, and diversity, focusing on larval habitats during the dry and rainy periods. Methods: We evaluated different larval habitats, considering collection duration as the primary criterion. We used CDC and Shannon traps to collect adult mosquitoes during both rainy and dry periods. To assess sampling effort, we used accumulation curves and non-parametric estimators of species richness, while we employed Hill numbers to determine diversity. Additionally, we used the Berger-Parker and Pielou indices to evaluate species dominance and evenness. We conducted cluster analysis and ANOSIM tests to assess the similarity between habitats and the differences in taxonomic composition between periods. Results: We collected a total of 802 individuals from 15 species and 4 taxonomic units, 5 genera, and 4 tribes. Notably, this may be the first records of Wyeomyia felicia Dyar & Núñez Tovar and Culex derivator Dyar & Knab from Ecuador. Additionally, the presence of Culex dunni Dyar and Psorophora ferox von Humboldt (both recognized as vectors) was correlated with increased rainfall. Conclusions: The abundance of mosquitoes, including potential vector species, increased during the rainy season, indicating a higher risk of pathogen transmission. However, the relationship between rainfall amount and diversity patterns is not well-defined.

Resumen Introducción: Las enfermedades vectoriales son prevalentes en las regiones amazónica y costera de Ecuador. Sin embargo, hay una escasez de estudios de ecología de mosquitos en estas áreas. En el país se ha reportado 8 tribus, 22 géneros y 200 especies. Objetivos: Documentar las especies de Culicidae encontradas en el Parque Amazónico La Isla, Napo, Ecuador, incluyendo aquellas con importancia epidemiológica; y analizar su composición, abundancia y diversidad, enfocándose en los hábitats de las larvas durante los períodos seco y lluvioso. Métodos: Evaluamos diferentes hábitats larvarios, con la duración de la recolecta como criterio. Las trampas CDC y Shannon recolectaron mosquitos adultos durante los períodos seco y lluvioso. Evaluamos la riqueza de especies con curvas de acumulación y estimadores no paramétricos, mientras que determinamos la diversidad con los números de Hill. Además, utilizamos los índices de Berger-Parker y Pielou para evaluar la dominancia y la uniformidad de las especies. Realizamos análisis de conglomerados y la prueba ANOSIM para evaluar la similitud entre hábitats y estaciones, así como las diferencias en la composición taxonómica, respectivamente. Resultados: Recolectamos un total de 802 individuos de 15 especies y 4 unidades taxonómicas, 5 géneros y 4 tribus. Este podría ser el primer registro de Wyeomyia felicia Dyar & Núñez Tovar y Culex derivator Dyar & Knab en Ecuador. Además, la presencia de Culex dunni Dyar y Psorophora ferox von Humboldt (ambos potenciales vectores) se correlacionó con el aumento de las precipitaciones. Conclusiones: El aumento de la abundancia de mosquitos durante el periodo lluvioso indica un mayor riesgo de transmisión de patógenos. Sin embargo, la relación entre la cantidad de precipitaciones y los patrones de diversidad no está bien definida.

Description of Larva and Pupa of Psorophora (Grabhamia) dimidiata (Diptera: Culicidae) and Redescription of the Adult Female and Male.

Psorophora (Grabhamia) dimidiata Cerqueira (1943) was described based on the adult female and male. Later, descriptions of the male and female genitalia were published by Lane (in Neotropical Culicidae, vols. I & II. Editora da Universidade de São Paulo, S. P., Brazil, 1953) and Guedes et al. (in Catalogo ilustrado dos mosquitos da coleção do Instituto Nacional de Endemias Rurais. I. Gênero Psorophora Robineau-Desvoidy, 1827. Rev. Bras. Malariol. Doencas Trop. 12: 3-24; 1965), respectively. Here we describe the pupa and fourth-instar larva and redescribe the adult male and female genitalia and female cibarium. All stages (except the egg) are illustrated. Unambiguous recognition is provided. Distinctions from Ps. cingulata (Fabricius) and possible phylogenetic relationships are discussed.

The olfactory gating of visual preferences to human skin and visible spectra in mosquitoes.

Mosquitoes track odors, locate hosts, and find mates visually. The color of a food resource, such as a flower or warm-blooded host, can be dominated by long wavelengths of the visible light spectrum (green to red for humans) and is likely important for object recognition and localization. However, little is known about the hues that attract mosquitoes or how odor affects mosquito visual search behaviors. We use a real-time 3D tracking system and wind tunnel that allows careful control of the olfactory and visual environment to quantify the behavior of more than 1.3 million mosquito trajectories. We find that CO2 induces a strong attraction to specific spectral bands, including those that humans perceive as cyan, orange, and red. Sensitivity to orange and red correlates with mosquitoes' strong attraction to the color spectrum of human skin, which is dominated by these wavelengths. The attraction is eliminated by filtering the orange and red bands from the skin color spectrum and by introducing mutations targeting specific long-wavelength opsins or CO2 detection. Collectively, our results show that odor is critical for mosquitoes' wavelength preferences and that the mosquito visual system is a promising target for inhibiting their attraction to human hosts.

Mosquito population structure, pathogen surveillance and insecticide resistance monitoring in urban regions of Crete, Greece.

BACKGROUND: In Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece's major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority. METHODOLOGY: Under the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018-2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species. PRINCIPAL FINDINGS: Important disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25-46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses. CONCLUSIONS/SIGNIFICANCE: The findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.

MALDI-TOF mass spectrometry identification of mosquitoes collected in Vietnam.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a tool that has revolutionised clinical microbiology and has recently been described as an innovative and effective approach to arthropod identification. METHODS: In this study, mosquitoes were captured in Vietnam using four different methods (human landing catch, CDC light traps, BG-Sentinel traps, animal-baited net traps). A total of 4215 mosquitoes were captured and morphologically identified as belonging to three genera: Aedes, Anopheles and Culex. We randomly selected 1253 mosquitoes, including 662 specimens of 14 Anopheles species, 200 specimens of two Aedes species and 391 morphologically unidentified Culex specimens, for molecular and MALDI-TOF MS analysis. The DNA from 98 mosquitoes (69 Anopheles specimens, 23 Culex specimens and six Aedes sp. specimens) was subjected to molecular analysis, either to confirm our morphological identification or the MALDI-TOF MS results, as well as to identify the Culex species that were morphologically identified at the genus level and to resolve the discrepancies between the morphological identification and the MALDI-TOF MS identification. RESULTS: High-quality MS spectra were obtained for 1058 of the 1253 specimens (84%), including 192/200 for Aedes, 589/662 for Anopheles and 277/391 for Culex. The blind test showed that 986/997 (99%) of the specimens were correctly identified by MALDI-TOF MS, with log score values ranging from 1.708 to 2.843. Eleven specimens of Culex could not be identified based on morphological features, MALDI-TOF MS or molecular analysis. CONCLUSIONS: This study enabled us to identify several species of mosquitoes from Vietnam using MALDI-TOF MS, and to enrich our database of MALDI-TOF MS reference spectra.

Seasonal Dynamics of Mosquito-Borne Viruses in the Southwestern Florida Everglades, 2016, 2017.

Mosquitoes were collected for 12 consecutive months beginning June 2016, from 11 locations in the Florida Everglades, Collier County, and tested for viruses by isolation in Vero cells and subsequent identification. One species complex and 31 species of mosquitoes were identified from 668,809 specimens. Ochlerotatus taeniorhynchus comprised 72.2% of the collection. Other notable species were Anopheles crucians complex, Culex nigripalpus, Cx. erraticus, and Cx. cedecei. Seven species of virus were identified from 110 isolations: Everglades, Gumbo Limbo, Mahogany Hammock, Pahayokee, Shark River, Tensaw, and West Nile viruses. Everglades, West Nile, Tensaw, and Mahogany Hammock viruses were most frequently isolated. Largest numbers of viruses were identified from Cx. cedecei, Cx. nigripalpus, and An. crucians complex. Five species of virus were isolated from Cx. cedecei. Viruses were isolated from mangrove, cypress swamp, hardwood hammock, and sawgrass habitats. West Nile virus was isolated August through October when Cx. nigripalpus was most abundant. Everglades virus was the most frequently isolated virus from nine species of mosquitoes collected from June through August. Tensaw virus was isolated primarily from Anopheles species. Isolations were made in July, August, January, February, and April, suggesting that this virus may be present in host-seeking mosquitoes throughout the year. Mahogany Hammock, Shark River, Gumbo Limbo, and Pahayokee viruses were isolated primarily from Cx. cedecei from June through December. Shotgun metagenomic sequencing was used to document that seven pools of Cx. cedecei were infected with two arboviruses. As communities expand into the Everglades, more humans will become exposed to arboviruses.

Improving the DNA Barcode Library of Mosquito Species With New Identifications and Discoveries in North-Central Argentina.

The family Culicidae is represented by 244 species in Argentina, many of them with epidemiological importance. DNA barcodes are effective tools for identifying mosquito species, for knowing genetic variability, and for establishing phylogenetic relationships. This work aims to explore mosquito diversity employing different species delimitation approaches and to establish formally a DNA barcode library for the Argentinian mosquito fauna. Barcode fragments of 80 specimens of Argentinian mosquitoes of 28 species of the genera Aedeomyia Theobald (Diptera: Culicidae), Anopheles Meigen (Diptera: Culicidae), Coquillettidia Dyar (Diptera: Culicidae), Culex L. (Diptera: Culicidae), Haemagogus Williston (Diptera: Culicidae), Mansonia Blanchard (Diptera: Culicidae), Nyssorhynchus Blanchard (Diptera: Culicidae), Ochlerotatus Lynch-Arribálzaga (Diptera: Culicidae), Psorophora Robinneau-Desvoidy (Diptera: Culicidae) and Uranotaenia Lynch-Arribálzaga (Diptera: Culicidae) were sequenced. Another 82 sequences were obtained from public databases to establish the phylogenetic relationships using Maximum Likelihood and Bayesian Inference, and the species boundaries based on three approaches (ABGD, GMYC, and mPTP). Sixteen of the 28 species sequenced were recovered as monophyletic, of which 12 were also recognized as molecular operational taxonomic units according to the three methodologies. The disparity between morphology and barcode-based identifications could be explained by synonymy, species complexes occurrence, hybridization, incomplete lineage sorting, or the effect of the geographical scale of sampling. Twenty of the 28 sequenced species are new barcodes for Argentina and 11 are the first for science. This increases from 31 to 52 (12.7 to 21.31%) and from six to 10 (28.57 to 47.62%) the number of species and genera, respectively, with barcode sequences in Argentina. New species records are provided.

Application of Deep Learning to Community-Science-Based Mosquito Monitoring and Detection of Novel Species.

Mosquito-borne diseases account for human morbidity and mortality worldwide, caused by the parasites (e.g., malaria) or viruses (e.g., dengue, Zika) transmitted through bites of infected female mosquitoes. Globally, billions of people are at risk of infection, imposing significant economic and public health burdens. As such, efficient methods to monitor mosquito populations and prevent the spread of these diseases are at a premium. One proposed technique is to apply acoustic monitoring to the challenge of identifying wingbeats of individual mosquitoes. Although researchers have successfully used wingbeats to survey mosquito populations, implementation of these techniques in areas most affected by mosquito-borne diseases remains challenging. Here, methods utilizing easily accessible equipment and encouraging community-scientist participation are more likely to provide sufficient monitoring. We present a practical, community-science-based method of monitoring mosquito populations using smartphones. We applied deep-learning algorithms (TensorFlow Inception v3) to spectrogram images generated from smartphone recordings associated with six mosquito species to develop a multiclass mosquito identification system, and flag potential invasive vectors not present in our sound reference library. Though TensorFlow did not flag potential invasive species with high accuracy, it was able to identify species present in the reference library at an 85% correct identification rate, an identification rate markedly higher than similar studies employing expensive recording devices. Given that we used smartphone recordings with limited sample sizes, these results are promising. With further optimization, we propose this novel technique as a way to accurately and efficiently monitor mosquito populations in areas where doing so is most critical.

Landscape Anthropization Affects Mosquito Diversity in a Deciduous Forest in Southeastern Mexico.

Mosquitoes (Diptera: Culicidae) are considered the group of insects that most impacts human health. Land use change, conversion of conserved sites into agricultural environments, urbanization, defaunation, and introduction of domestic animals can affect mosquito diversity positively or negatively, increasing the risk of transmission of zoonotic diseases. Here, we describe the diversity of adult mosquitoes in two environments (deciduous forest and anthropized zone) over 2 yr (2014-2016), using eight CDC traps at each site in three climatic seasons (rainy, cold, and dry). We captured 795 individuals belonging to 22 species. We constructed rank-abundance curves to determine spatial and temporal changes in the mosquito communities. We measured alpha diversity using the Shannon index (H'), Shannon exponential (eH) and Simpson dominance (Ds), and beta diversity using Jaccard's coefficient of similarity (Ij). The most abundant species were Culex quinquefasciatus (40.5%), Culex coronator (18.3%), and Anopheles pseudopunctipennis (12.4%). The highest mosquito diversity was in the deciduous forest during the rainy season. Beta diversity analysis showed that species overlap varied among climatic seasons, with the sites sharing 65% species during the rainy season, but only 33% of species during the dry season. We found differences in the diversity of mosquitoes at the two sites, and the mosquito assemblage of the anthropized zone was significantly different from that of the deciduous forest.

First Evidence of West Nile Virus Overwintering in Mosquitoes in Germany.

Mosquitoes collected from mid-December 2020 to early March 2021 from hibernacula in northeastern Germany, a region of West Nile virus (WNV) activity since 2018, were examined for WNV-RNA. Among the 6101 mosquitoes tested in 722 pools of up to 12 specimens, one pool of 10 Culex pipiens complex mosquitoes collected in early March 2021 in the cellar of a medieval castle in Rosslau, federal state of Saxony-Anhalt, tested positive. Subsequent mosquito DNA analysis produced Culex pipiens biotype pipiens. The pool homogenate remaining after nucleic acid extraction failed to grow the virus on Vero and C6/36 cells. Sequencing of the viral NS2B-NS3 coding region, however, demonstrated high homology with virus strains previously collected in Germany, e.g., from humans, birds, and mosquitoes, which have been designated the East German WNV clade. The finding confirms the expectation that WNV can overwinter in mosquitoes in Germany, facilitating an early start to the natural transmission season in the subsequent year. On the other hand, the calculated low infection prevalence of 0.016-0.20%, depending on whether one or twelve of the mosquitoes in the positive pool was/were infected, indicates a slow epidemic progress and mirrors the still-hypoendemic situation in Germany. In any case, local overwintering of the virus in mosquitoes suggests its long-term persistence and an enduring public health issue.

Diversidad, riqueza de especies y abundancia relativa de mosquitos adultos (Diptera: Culicidae) en localidades del municipio Gran Sabana, edo. Bolívar / Diversity, species richness and relative abundance of adult mosquitoes (Diptera: Culicidae) in localities of the Gran Sabana municipality, Bolívar state

Este estudio forma parte de la primera etapa de una serie de estudios sobre la ecología, biodiversidad y biogeografía de los mosquitos de la familia Culicidae de Venezuela, principalmente en Bolívar y Delta Amacuro. El objetivo del estudio fue caracterizar la composición de especies, la abundancia y la biodiversidad de mosquitos (Diptera; Culicidae) en el municipio Gran Sabana del estado Bolívar. Las capturas de mosquitos adultos se realizaron con aspiradores bucales mediante capturas por atracción al cebo humano y con trampas de luz negra. Se determinó la riqueza de especies, su abundancia relativa e índices ecológicos de diversidad Shannon-Wiener (H'), equidad de Simpson (E) y dominancia-D, del área general de estudio y sitios o comunidades de muestreo. Los resultados del estudio, generaron el registro de 7.860 ejemplares adultos de Culicidae identificados a nivel de especie, pertenecientes a 69 especies y 17 géneros, de ellos 7.797 (99,19%) fueron hembras adultas capturadas picando o con trampas de luz y 63 (0,81%) fueron machos capturados con trampas de luz. El 16,6% (1.297) de las hembras fue atraído por trampas de luz y el 83,4% (6.500) por cebo humano. Entre las especies más abundantes del estudio están: Culex quinquefasciatus (26,67 %), Anopheles peryassui (12,32 %), Aedes aegypti (11,87 %), Coquilletidia juxtamansonia (8,28 %), Anopheles braziliensis (6,97 %) Anopheles triannulatus s.l. (6,39 %), Coquilletidia nigricans (2,88 %), Coquilletidia venezuelensis (2,51 %) y Anopheles albitarsis s.l. (2,44 %), las cuales en conjunto representan 80,33 % del total de adultos capturado. Se discuten algunos aspectos sobre su ecología, importancia médica y el riesgo epidemiológico que representan para el país algunas especies reportadas. La diversidad de Shannon-Wiener (H') del área general de estudio fue de 2,665, la equidad de Simpson (E) fue igual a 0,8787222 y la dominancia (D) fue de 0,1213. El mayor índice de diversidad (Shannon-Wiener) ocurrió en Chiririka (2,675), seguido de Betania (2,409), Santa Elena Capital (2,354), Manak-Krü (2,203) y Waramasén (2,181). En cuanto a la riqueza de especies por localidad, las comunidades con mayor riqueza de especies fueron: Santa Elena (Capital), Waramasén y Chiririka con 50, 35 y 30 especies respectivamente, seguidas por San Antonio del Morichal con 23 y Kinok-Pon Parú, con 22 especies(AU)

This is the first part of a series of studies related to biodiversity, ecological and biogeographic aspects of the mosquitoes of Culicidae family in Venezuela. The objective of this study was the ecological characterization of species composition, diversity and abundance of Culicidae mosquitoes (Diptera: Culicidae) in the Gran Sabana Municipality, Bolívar State. Mosquitoes were captured in adult collections, with aspirators on human landing catches and with black light traps. A total of 7.860 adult's mosquitoes were collected and 69 species and 17 genera of Culicidae were identified. A total of 7.797 mosquitoes (99.19%) were females and 63 (0,81%) were males. Some 16,6 % (1.297) of females mosquitoes were attracted to the light traps and 83,4% (6.500) to human bait. The most abundant species in adult stage were: Culex quinquefasciatus (26,67%), Anopheles peryassui (12,32 %), Aedes aegypti (11,87 %), Coquilletidia juxtamansonia (8,28%), Anopheles braziliensis (6,97%), Anopheles triannulatus s.l. (6,39%), Coquilletidia nigricans (2,88%), Coquilletidia venezuelensis (2,51%) and Anopheles albitarsis s.l. (2,44%). Their medical importance of some mosquito species is commented, and some ecological and epidemiological aspects of the mosquito reported are discussed. The diversity index (Shannon-Wiener) of the estudy area was 2,665, the Simpson equity index was 0, 8787222 and the index of dominance was 0, 1213. The higher diversity index was registered in Chiririka (2,675), Betania (2,409), Santa Elena Capital (2,354), Manak-Krü (2,203) and Waramasén (2,181). The higher species richness was registered in Santa Elena (50 species), Waramasén (35 species), Chiririka (30 species), San Antonio del Morichal (23 species) and Kinok-Pon Parú (22 species) (AU)

Phylogenetic analysis of the Neotropical Albitarsis Complex based on mitogenome data.

BACKGROUND: Some of the most important malaria vectors in South America belong to the Albitarsis Complex (Culicidae; Anophelinae; Anopheles). Understanding the origin, nature, and geographical distribution of species diversity in this important complex has important implications for vector incrimination, control, and management, and for modelling future responses to climate change, deforestation, and human population expansion. This study attempts to further explore species diversity and evolutionary history in the Albitarsis Complex by undertaking a characterization and phylogenetic analysis of the mitogenome of all 10 putative taxa in the Albitarsis Complex. METHODS: Mitogenome assembly and annotation allowed for feature comparison among Albitarsis Complex and Anopheles species. Selection analysis was conducted across all 13 protein-coding genes. Maximum likelihood and Bayesian inference methods were used to construct gene and species trees, respectively. Bayesian methods were also used to jointly estimate species delimitation and species trees. RESULTS: Gene composition and order were conserved across species within the complex. Unique signatures of positive selection were detected in two species-Anopheles janconnae and An. albitarsis G-which may have played a role in the recent and rapid diversification of the complex. The COI gene phylogeny does not fully recover the mitogenome phylogeny, and a multispecies coalescent-based phylogeny shows that considerable uncertainty exists through much of the mitogenome species tree. The origin of divergence in the complex dates to the Pliocene/Pleistocene boundary, and divergence within the distinct northern South American clade is estimated at approximately 1 million years ago. Neither the phylogenetic trees nor the delimitation approach rejected the 10-species hypothesis, although the analyses could not exclude the possibility that four putative species with scant a priori support (An. albitarsis G, An. albitarsis H, An. albitarsis I, and An. albitarsis J), represent population-level, rather than species-level, splits. CONCLUSION: The lack of resolution in much of the species tree and the limitations of the delimitation analysis warrant future studies on the complex using genome-wide data and the inclusion of additional specimens, particularly from two putative species, An. albitarsis I and An. albitarsis J.