Integrating wing morphometrics and mitochondrial DNA analysis to assess the filaria vector Mansonia uniformis (Diptera: Culicidae) populations in Thailand.

Mansonia uniformis (Diptera: Culicidae) is recognized as a vector of Brugia malayi and has been reported to transmit Wuchereria bancrofti, both causing lymphatic filariasis in humans. This study employed geometric morphometrics (GM) to investigate wing shape variation and analyzed genetic diversity through cytochrome c oxidase subunit 1 (COI) gene analyses in Ma. uniformis populations across Thailand. Wing GM analyses indicated significant differences in wing shape based on Mahalanobis distances among nearly all population pairs (p < 0.05), with no significant correlation between wing shape and geographic distance (r = 0.210, p > 0.05). Genetic analyses identified 63 haplotypes and 49 polymorphic sites, with the overall population exhibiting a nucleotide diversity of 0.006 (± 0.001) and a haplotype diversity of 0.912 (± 0.017). Deviations from neutrality, as indicated by Tajima's D and Fu's FS tests for the overall Ma. uniformis populations in Thailand, were statistically significant and negative, suggesting population expansion (both p < 0.05). Analysis of molecular variance revealed no significant genetic structure when all populations were categorized based on collection sites and geographic regions. However, significant differences in FST values were observed between some populations. These findings enhance our understanding of the geographical and genetic factors influencing Ma. uniformis populations, which are crucial for developing effective control strategies in Thailand.

Midgut morphology of the predator mosquito Lutzia bigoti (Diptera: Culicidae) and its implications for feeding behavior.

Lutzia mosquitoes (Theobald, 1903) are predaceous during their larval stages, but the adult feeding is not clearly understood, especially in relation to blood feeding. In case these mosquitoes are harmless to humans and related animals, they can be useful in biological control of mosquito vectors of pathogens. Investigating the midgut morphology is a good strategy to understand the feeding behavior of this species. The midgut in Lutzia bigoti Bellardi, 1862 displays two distinct portions, a thin anterior midgut and a more dilated posterior midgut. Digestive cells form a single epithelium in the midgut. These cells have long and packed microvilli at their apex and membrane infoldings at their basal portion, the basal labyrinth. The epithelium is supported by a basal lamina. Regarding their cytoplasm, it is noteworthy the abundance of mitochondria, distributed in an apical-basal fashion, and also a whirl-shaped endoplasmic reticulum in the posterior midgut. Basal cells are also found in the midgut of L. bigoti, resembling regenerative cells. The general organization of L. bigoti's midgut closely resembles that of numerous hematophagous mosquitoes previously documented. However, it diverges due to the presence of a peritrophic matrix even when exclusively fed on sugary solutions. Peculiar aspects of L. bigoti's midgut are discussed and compared to those of other mosquito species.

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.

Influence of insular conditions on wing phenotypic variation in two dominant mosquito vectors, Aedes albopictus and Armigeres subalbatus (Diptera: Culicidae), in the border archipelagos of Thailand.

Insects geographically separated into island and mainland populations often exhibit phenotypic variations, a phenomenon known as insular conditions. These conditions can lead to rapid evolutionary changes that affect the morphological characteristics of mosquito vectors. Nevertheless, studies that specifically examine phenotype differences between island and mainland mosquito populations have been limited. In this study, wing variation in size and shape was investigated using the geometric morphometric (GM) technique in two dominant mosquito vectors, Aedes albopictus and Armigeres subalbatus, in the Ranong and Trat archipelagos of Thailand. Significant differences in average wing centroid size (CS) were found in 6 out of 15 population pairs for Ae. albopictus (p < 0.05) and in 5 population pairs for Ar. subalbatus (p < 0.05). After removing the allometric effect, canonical variate analyses (CVA) based on wing shape analysis revealed overlap across all populations for both Ae. albopictus and Ar. subalbatus. However, the statistical analysis indicated that Ar. subalbatus exhibited wing shape differences across all populations (p < 0.05), and most Ae. albopictus populations also displayed distinct wing shapes (p < 0.05), except for the populations from Chang Island and the mainland of Ranong, which showed no significant differences (p > 0.05). These findings enhance our understanding of mosquito adaptability in island regions and provide valuable data for the surveillance and monitoring of vector evolution.

Geometric morphometrics to differentiate species and explore seasonal variation in three Mansonia species (Diptera: Culicidae) in central Thailand and their association with meteorological factors.

Mansonia mosquito species are recognised as a significant vector of human pathogens, primarily transmitting the filarial nematode, Brugia malayi. In central Thailand, the three most prevalent Mansonia species are Mansonia annulifera, Mansonia indiana and Mansonia uniformis. This study explored the influence of seasonal changes on the phenotypic variation of these Mansonia species in central Thailand using the geometric morphometrics (GM). To ensure accurate species identification, we integrated GM techniques with DNA barcoding, examining distinctions in both phenotype and genotype among the species. The intraspecific genetic divergence ranged from 0.00% to 1.69%, whereas the interspecific genetic divergence ranged from 10.52% to 16.36%. The clear distinction between intra- and interspecific distances demonstrated the presence of a barcoding gap, confirming the successful differentiation of the three Mansonia mosquito species through DNA barcoding. Similarly, the interspecies GM assessment for classifying Mansonia species demonstrated a high degree of accuracy, with an overall performance of 98.12%. Exploring seasonal variation in the three Mansonia species revealed wing variations across different seasons, and pronounced variations appearing in the cool season. Regarding their association with meteorological factors, Ma. annulifera and Ma. uniformis showed significant positive correlations with temperature (p < 0.05), and Ma. uniformis also displayed a significant negative correlation with atmospheric pressure (p < 0.05). The insights from this study will deepen our understanding of the adaptive patterns of Mansonia mosquitoes in Thailand's central region, paving the way for enhanced disease surveillance related to these vectors.

Morphological characterization of antennal sensilla in Toxorhynchites theobaldi, Toxorhynchites violaceus, and Lutzia bigoti adults: a comparative study using scanning electron microscopy.

Some mosquitoes, including species of the genus Toxorhynchites, are known for actively preying on other mosquito larvae, making these predators valuable allies in the fight against vector-borne diseases. A comprehensive understanding of the anatomy and physiology of these potential biological control agents is helpful for the development of effective strategies for controlling vector populations. This includes the antennae, a crucial component in the search for hosts, mating, and selection of oviposition sites. This study utilized scanning electron microscopy to characterize the sensilla on the antennae of adult mosquitoes from two species that are exclusively phytophagous, including Toxorhynchites theobaldi and Toxorhynchites violaceus, as well as Lutzia bigoti, which females are allegedly hematophagous. The types of sensilla in each species were compared, and five basic types of antennal sensilla were identified: trichoid, chaetic, coeloconic, basiconic, and ampullacea. The analysis also found that they were morphologically similar across the three species, regardless of feeding habits or sex. The identification and characterization of basic types of antennal sensilla in T. theobaldi, T. violaceus, and L. bigoti suggest that these structures, which play a crucial role in the behavior and ecology, have common functions across different mosquito species, despite differences in feeding habits or sex.

A study of Anopheles monticola of the Baileyi Complex, Culex sasai, and Lutzia vorax (Diptera: Culicidae) in Yunnan Province, China.

This article deals with the morphological and molecular assessments of 3 species of mosquitoes collected in Yunnan Province, China, i.e Anopheles monticola Somboon, Namgay & Harbach, the only species of the Baileyi Complex identified, Culex sasai Kano, Nitahara & Awaya, and Lutzia vorax Edwards. Interestingly, some adult specimens of An. monticola exhibited a variant form in having pale fringe spots, as found in An. simlensis James, another species of the Baileyi Complex. Culex sasai was recorded for the first time in Yunnan and mainland China. The prevalence of Lt. vorax was confirmed, and previous records of Lt. halifaxii in China are regarded as misidentifications of Lt. vorax.

Aedes pertinax, Anopheles perplexens, Culex declarator, and Cx. interrogator: An Update of Mosquito Species Records for Charlotte County, Florida.

Understanding the geographic occurrence of mosquito species is an important element to addressing public health and nuisance mosquito-related issues, particularly as changing climates and increased global connectivity is likely to facilitate changes in the distribution of mosquitoes and other species. In Charlotte County, FL, routine surveillance of mosquito species for public health in 2019-21 identified 4 mosquito species not previously documented in the county. Aedes pertinax, Anopheles perplexens, Culex declarator, and Cx. interrogator adults were collected and verified to species level. Aedes pertinax and Cx. declarator and were collected in 2019, whereas An. perplexens and Cx. interrogator were documented from collections in 2021. All 4 species were initially visually identified by external morphology and confirmed by sequencing the DNA barcoding region of the cytochrome c oxidase subunit I gene. Apart from native An. perplexens, in which only 1 specimen has been confirmed to date, the 3 newly documented nonnative species are now recognized throughout the county.

Redescription of Wyeomyia (Decamyia) felicia (Dyar amp; Nez Tovar, 1927) and description of a new species of the subgenus from Trinidad (Diptera: Culicidae).

Mosquito species of the subgenus Decamyia Dyar, 1919 of genus Wyeomyia Theobald, 1901 are known to have immature stages mostly associated with Heliconia plants. Currently, the subgenus includes five species, some of which are poorly characterized. Here, we redescribe and illustrate the larval, pupal and adult life stages of Wy. felicia (Dyar Nez Tovar, 1927). In addition, Wyeomyia monticola, a new species of Decamyia from Trinidad, close to Wy. felicia, is described. The new species can be distinguished by morphological characters of the male genitalia, larva and pupa.

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.

First description of the mitogenome and phylogeny:Aedes vexansand Ochlerotatus caspius of the Tribe Aedini (Diptera: Culicidae).

Culicidae, the mosquito family, includes more than 3600 species subdivided into the subfamilies Anophelinae and Culicinae. One-third of mosquitoes belong to the Aedini tribe, which is subordinate to the subfamily Culicinae, which comprises common vectors of viral zoonoses. The tribe of Aedini is extremely diverse in morphology and geographical distribution and has high ecological and medical significance. However, knowledge about the systematics of the Aedini tribe is still limited owing to its large population and the similar morphological characteristics of its species. This study provides the first description of the complete mitochondrial (mt) genome sequence of Aedes vexans and Ochlerotatus caspius belonging to the Aedini tribe. The mt genomes of A. vexans and O. caspius are circular molecules that are 15,861 bp and 15,954 bp in size, with AT contents of 78.54% and 79.36%, respectively. Both the circular mt genomes comprise 37 functional subunits, including 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes (tRNAs), and a control region (also known as the AT-rich region). The most common start codons are ATT/ATG, apart from cox1 (TCG) and nad5 (GTG), while TAA is the termination codon for all PCGs. All tRNAs have a typical clover leaf structure, except tRNA Ser1. Phylogenetic analysis of the concatenated, aligned amino acid sequences of the 13 PCGs showed that A. vexans gathered with Aedes sp. in a sister taxon, and O. caspius gathered with Ochlerotatus sp. in a sister taxon. The findings from the present study support the concept of monophyly of all groups, ratify the current taxonomic classification, and provide vital molecular marker resources for further studies of the taxonomy, population genetics, and systematics of the Aedini tribe.

Influence of seasonality on wing morphological variability in populations of Mansonia amazonensis (Theobald) (Diptera: Culicidae).

Mansonia amazonensis (Theobald, 1901) is one of 15 species of the subgenus Mansonia Blanchard, 1901. It is essentially a Neotropical species, recorded in Bolivia, Peru and Brazil. In the highly seasonal Amazon region, Mansonia species find ideal environmental conditions for reproduction, development and establishment. Considering that climate significantly influences the size and behavior of mosquitoes, and insects in general, we analyzed the influence of seasonality on wing morphological variability in populations of Ma. amazonensis. Captures were carried out near the banks of the Madeira River in Rondnia State, Brazil during the dry, rainy and transition periods between seasons during 2018 and 2019. Morphometric characters of 181 wings were analyzed using morphological methods. The results show that wing size of Ma. amazonensis increases following a relatively gradual trend, from smaller wings during the dry period to larger ones in the rainy season. This study provides the first evidence, detected using geometric morphometry, of seasonally associated phenotypic variability in the wing conformation of Ma. amazonensis.

Rediscovering the mosquito fauna (Diptera: Culicidae) of Estonia: an annotated checklist with distribution maps and DNA evidence.

Female mosquitoes (Diptera: Culicidae) affect their hosts in numerous negative ways and are crucial to the spread of vector-borne pathogens. It is, therefore, important to have a detailed overview of regional mosquitoes, to be able to detect changes in species diversity and identify possible health threats. The aim of this study was to update the checklist of the mosquito fauna of Estonia for the first time since 1957. For this purpose, 24,344 adult mosquitoes (94% females) were collected in Estonia from 2008 to 2020 using various trapping methods. Specimens were primarily identified by morphological characteristics, but DNA barcoding based on the partial cytochrome c oxidase subunit 1 gene (COI) was also used. Species were included in the checklist based on historical records as well as new collections, while also considering reports from neighboring countries. Species records are supplemented with voucher specimens, distribution maps and DNA evidence. The updated checklist includes 34 species, 27 of which were confirmed with recently collected material. All in all, Aedes communis (de Geer, 1776) proved to be the most common mosquito in Estonia, accounting for 30.1% of the specimens collected. This is noteworthy, as this species has been implicated in the transmission of multiple disease agents present in the area. New evidence revealed the presence of Ae. hexodontus Dyar, 1916, Ae. sticticus (Meigen, 1838), Anopheles messeae Falleroni, 1926 and Culiseta bergrothi (Edwards, 1921) in Estonia.

Geometric morphometric wing analysis as a tool to discriminate female mosquitoes from different suburban areas of Chiang Mai province, Thailand.

Mosquitoes are hematophagous insects that transmit parasites and pathogens with devastating effects on humans, particularly in subtropical regions. Different mosquito species display various behaviors, breeding sites, and geographic distribution; however, they can be difficult to distinguish in the field due to morphological similarities between species and damage caused during trapping and transportation. Vector control methods for controlling mosquito-borne disease epidemics require an understanding of which vector species are present in the area as well as the epidemiological patterns of disease transmission. Although molecular techniques can accurately distinguish between mosquito species, they are costly and laborious, making them unsuitable for extensive use in the field. Thus, alternative techniques are required. Geometric morphometrics (GM) is a rapid and inexpensive technique that can be used to analyze the size, shape, and shape variation of individuals based on a range of traits. Here, we used GM to analyze the wings of 1,040 female mosquitoes from 12 different species in Thailand. The right wing of each specimen was removed, imaged microscopically, and digitized using 17 landmarks. Wing shape variation among genera and species was analyzed using canonical variate analysis (CVA), while discriminant function analysis was used to cross-validate classification reliability based on Mahalanobis distances. Phenetic relationships were constructed to illustrate the discrimination patterns for genera and species. CVA of the morphological variation among Aedes, Anopheles, Armigeres, Culex, and Mansonia mosquito genera revealed five clusters. In particular, we demonstrated a high percentage of correctly-distinguished samples among Aedes (97.48%), Armigeres (96.15%), Culex (90.07%), and Mansonia (91.67%), but not Anopheles (64.54%). Together, these findings suggest that wing landmark-based GM analysis is an efficient method for identifying mosquito species, particularly among the Aedes, Armigeres, Culex, and Mansonia genera.

Mosquito species identification using convolutional neural networks with a multitiered ensemble model for novel species detection.

With over 3500 mosquito species described, accurate species identification of the few implicated in disease transmission is critical to mosquito borne disease mitigation. Yet this task is hindered by limited global taxonomic expertise and specimen damage consistent across common capture methods. Convolutional neural networks (CNNs) are promising with limited sets of species, but image database requirements restrict practical implementation. Using an image database of 2696 specimens from 67 mosquito species, we address the practical open-set problem with a detection algorithm for novel species. Closed-set classification of 16 known species achieved 97.04 ± 0.87% accuracy independently, and 89.07 ± 5.58% when cascaded with novelty detection. Closed-set classification of 39 species produces a macro F1-score of 86.07 ± 1.81%. This demonstrates an accurate, scalable, and practical computer vision solution to identify wild-caught mosquitoes for implementation in biosurveillance and targeted vector control programs, without the need for extensive image database development for each new target region.

Oviposition behavior of wild yellow fever vector mosquitoes (Diptera: Culicidae) in an Atlantic Forest fragment, Rio de Janeiro state, Brazil.

Although there are many studies on the control of mosquito vectors of the yellow fever virus (YFV) in tropical forests, there are still few ecological studies regarding abiotic factors effect on these mosquitoes. Here we characterize these effects on oviposition behavior, abundance, and diversity of mosquito vectors of YFV. The study was conducted in Córrego da Luz Municipal Park, in Casimiro de Abreu, Rio de Janeiro state, Brazil, from July 2018 to December 2019. Ovitraps were placed at ground level and 3 m high. The data were tested for normality using the Shapiro-Wilk test, followed by an independent sample analysis, the Mann-Whitney test. The Shannon Diversity Index was used to evaluate the abundance of mosquitos' eggs collected at both ground level and 3 m high. We highlight the presence of Haemagogus janthinomys and Hg. leucocelaenus, primary YFV vectors in forest areas. The abundance of Hg. leucocelaenus (63%), Hg. janthinomys (75%), and Aedes terrens (58%) was higher at the height of 3 m, while Ae. albopictus (52%) was higher at ground level. Aedes albopictus was positively correlated with temperature. Culicidae monitoring is essential for assessing the YFV transmission cycle in Atlantic forest fragments.

Deep learning identification for citizen science surveillance of tiger mosquitoes.

Global monitoring of disease vectors is undoubtedly becoming an urgent need as the human population rises and becomes increasingly mobile, international commercial exchanges increase, and climate change expands the habitats of many vector species. Traditional surveillance of mosquitoes, vectors of many diseases, relies on catches, which requires regular manual inspection and reporting, and dedicated personnel, making large-scale monitoring difficult and expensive. New approaches are solving the problem of scalability by relying on smartphones and the Internet to enable novel community-based and digital observatories, where people can upload pictures of mosquitoes whenever they encounter them. An example is the Mosquito Alert citizen science system, which includes a dedicated mobile phone app through which geotagged images are collected. This system provides a viable option for monitoring the spread of various mosquito species across the globe, although it is partly limited by the quality of the citizen scientists' photos. To make the system useful for public health agencies, and to give feedback to the volunteering citizens, the submitted images are inspected and labeled by entomology experts. Although citizen-based data collection can greatly broaden disease-vector monitoring scales, manual inspection of each image is not an easily scalable option in the long run, and the system could be improved through automation. Based on Mosquito Alert's curated database of expert-validated mosquito photos, we trained a deep learning model to find tiger mosquitoes (Aedes albopictus), a species that is responsible for spreading chikungunya, dengue, and Zika among other diseases. The highly accurate 0.96 area under the receiver operating characteristic curve score promises not only a helpful pre-selector for the expert validation process but also an automated classifier giving quick feedback to the app participants, which may help to keep them motivated. In the paper, we also explored the possibilities of using the model to improve future data collection quality as a feedback loop.

Wyeomyia shannoni Lane amp; Cerqueira, 1942, a taxonomic puzzle (Diptera: Culicidae): synonymy, genus transfer, homonymy, and description of a new species of Sabethes Robineau-Desvoidy, 1827.

We discovered the immature stages of Wyeomyia shannoni Lane Cerqueira, 1942, unknown for almost 80 years, and found that they, as well as the adult male, are identical with those previously described for Sabethes (Peytonulus) paradoxus Harbach, 2002. Consequently, the species described by Lane and Cerqueira is here considered a senior synonym of Sa. paradoxus and is transferred to the genus Sabethes, hereafter named Sabethes (Peytonulus) shannoni (Lane Cerqueira, 1942), comb. n. As the name shannoni is preoccupied in the genus Sabethes, the species known as Sabethes (Sabethes) shannoni Cerqueira, 1961 is here renamed Sabethes (Sabethes) cerqueirai Nascimento-Pereira, Neves, Loureno-de-Oliveira Motta, nom. n. We improved the morphological characterization of Sa. (Pey.) shannoni (Lane Cerqueira) by including an illustration of the female genitalia, larval mouthparts and the female genital lobe of the pupa. Finally, we describe and illustrate a new species of Sabethes closely related to Sa. (Pey.) shannoni, named Sabethes (Peytonulus) harbachi Nascimento-Pereira, Guimares, Loureno-de-Oliveira Motta, sp. n.

Is There a Minimum Number of Landmarks That Optimizes the Geometric Morphometric Analysis of Mosquito (Diptera, Culicidae) Wings?

Culicids are the most significant arthropods affecting human health. Thus, their correct identification is critical. The use of Geometric Morphometrics (GM) has been recently incorporated into mosquito taxonomy and has begun to complement classic diagnostic techniques. Since sampling size depends on the number of Landmarks (LMs) used, this study aimed to establish the minimum number of wing LMs needed to optimize GM analysis of mosquito species and/or genera from urban and peri-urban areas of Argentina. Female left wings were used for the optimization phase, in which 17 LMs were reduced to four by iterative LM exclusion. To verify its efficiency, Principal Component Analysis (PCA), Discriminant Analysis (DA), and Canonical Variate Analysis (CVA) were performed. Additionally, a phenogram was constructed to visualize the results. We observed that five LMs for the PCA, CVA, and phenogram and nine for the DA enabled discrimination and/or clustering of almost all species and genera. Therefore, we tested the LM selection by using nine LMs and adding new species. The resulting PCA showed little overlap between species and almost all species clustered as expected, which was also reflected in the phenogram. Significant differences were found between wing shape among all species, together with a low total error rate in the DA. In conclusion, the number of LMs can be reduced and still be used to effectively differentiate and cluster culicids. This is helpful for better exploitation of available material and optimization of data processing time when classic taxonomy methods are inadequate or the material is scarce.

Identification of mosquitoes (Diptera: Culicidae) from Mexico State, Mexico using morphology and COI DNA barcoding.

Mosquitoes are commonly identified to species level using morphological traits, but complementary methods for identification are often necessary when specimens are collected as immature stages, stored inadequately, or when delineation of species complexes is problematic. DNA-barcoding using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene is one such tool used for the morphological identification of species. A comprehensive entomological survey of mosquito species in Mexico State identified by COI DNA barcoding and morphology is documented in this paper. Specimens were collected from all the physiographic provinces in Mexico State between 2017 and 2019. Overall, 2,218 specimens were collected from 157 localities representing both subfamilies Anophelinae and Culicinae. A species checklist that consists of 6 tribes, 10 genera, 20 subgenera, and 51 species, 35 of which are new records for Mexico State, is provided. Three hundred and forty-two COI sequences of 46 species were analysed. Mean intraspecific and interspecific distances ranged between 0% to 3.9% and from 1.2% to 25.3%, respectively. All species groups were supported by high bootstraps values in a Neighbour-Joining analysis, and new COI sequences were generated for eight species: Aedes chionotum Zavortink, Ae. vargasi Schick, Ae. gabriel Schick, Ae. guerrero Berlin, Ae. ramirezi Vargas and Downs, Haemagogus mesodentatus Komp and Kumm, Culex restrictor Dyar and Knab, and Uranotaenia geometrica Theobald. This study provides a detailed inventory of the Culicidae from Mexico State and discusses the utility of DNA barcoding as a complementary tool for accurate mosquito species identification in Mexico.