Integrated Approaches for the Identification of Mosquitoes (Diptera: Culicidae) from the Volcanoes of Central America Physiographic Subprovince of the State of Chiapas, Mexico.

Nowadays, there is a lack of information on the mosquito's fauna and DNA barcoding sequence reference library from many areas in Mexico, including the Volcanoes of Central America physiographic subprovince in the state of Chiapas. Consequently, a survey was undertaken to delineate the mosquito (Diptera: Culicidae) fauna in this region across different seasons using different collecting techniques. All species were identified by morphology and DNA barcoding, and their ecological features were also defined. In total, 62 taxa were morphologically examined, 60 of these were successfully identified based on morphological characteristics, but two were unable to be identified at the species level. The genera Aedes, Anopheles, Culex, and Wyeomyia are the most diverse among mosquito genera collected and include several species of medical and veterinary importance. Ecological characteristics of the immature habitats indicated that they were grouped into four categories namely, (1) large water bodies at ground level, (2) small and shady phytotelmata (e.g., tree holes and bamboo internodes), (3) large phytotelmata (e.g., plant leaves and axis bromeliad), and (4) artificial containers. The cytochrome c oxidase subunit I (COI) DNA barcoding sequences successfully separated the majority of these species, although specific species showed >2% intraspecific genetic divergences.

DNA Barcoding of Mosquitoes from the Pantanos de Centla Biosphere Reserve, Southeastern Mexico.

Accurate identification of mosquito species is essential to support programs that involve the study of distribution and mosquito control. Numerous mosquito species are difficult to identify based only on morphological characteristics, due to the morphological similarities in different life stages and large numbers of some species that are members of morphologically similar species complexes. In the present study, the mosquitoes collected in the Pantanos de Centla Biosphere Reserve, southeastern Mexico, were evaluated using a combination of morphological and molecular approaches (mitochondrial cytochrome c oxidase subunit I [COI] DNA barcode). A total of 1,576 specimens of 10 genera and 35 species, mostly adult stages, were collected. A total of 225 COI DNA barcode sequences were analyzed; most species formed well-supported groups in the neighbor joining, maximum likelihood, and Bayesian inference trees. The intraspecific Kimura 2-parameter (K2P) genetic distance averaged 1.52%. An intraspecific K2P distance of 6.20% was observed in Anopheles crucians s.l., while a deep split was identified in Culex erraticus and Cx. conspirator. This study showed that COI DNA barcodes offer a reliable approach to support mosquito species identification in Mexico.

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.

Integrated Approaches in Support of Taxonomic Identification of Mosquitoes (Diptera: Culicidae) in Vector Surveillance in Spain.

The pandemic of Zika virus in 2016 and other arboviruses prompted La Rioja Government in Spain to implement an entomological surveillance program of mosquitoes (Diptera; Culicidae) in the region of La Rioja. The morphological identification was supported by genetic analysis using the COI (cytochrome c oxidase subunit I) and the ITS2 (internal transcribed spacer 2) genes. In total, we identified 24 species arranged in 6 genera: Aedes (7 species), Anopheles (4 species), Coquillettidia (1 species), Culex (7 species), Culiseta (4 species), and Uranotaenia (1 species). Aedes sticticus and Aedes geniculatus are newly reported for La Rioja region. In total, 465 COI sequences were analyzed for Culicinae and Anophelinae and 54 ITS2 sequences for Anophelinae; all individuals identified as the same species clustered together in the Neighbor Joining trees. The levels of sequence divergence based on COI ranged between 0% and 2.62%, while the interspecific genetic divergence ranged from 3.05% to 20.07%. Within the genus Culiseta, certain specimens of Culiseta annulata, Culiseta litorea, and Culiseta subochrea were morphologically misidentified due to variation in the main diagnostic characters. The interspecific genetic divergence based on the ITS2 ranged from 0% to 2.98%. An accurate identification of mosquito vectors is the first step to establish a vector surveillance program for preventing pathogen transmission.

Ticks on the Channel Islands and implications for public health.

The Channel Islands are British Crown dependencies located in the English Channel to the west of the Normandy coast in northern France. Whilst there have been studies investigating tick occurrence and distribution in different habitats on the mainland of the UK and in France, the Channel Islands have been relatively understudied. As such, little is known about whether the sheep tick, Ixodes ricinus, is present, and whether there is a potential risk of Lyme borreliosis on the Channel Islands. To ascertain the presence of I. ricinus on the three largest islands in the archipelago: Jersey, Guernsey and Alderney, surveys of ticks questing in the vegetation and ticks feeding on hosts were undertaken during April and May 2016. Across all three islands, the highest numbers of ticks were found in woodland habitats. Ixodes ricinus was the predominant questing tick species found on Jersey, and Ixodes ventalloi the most common questing tick species on Alderney and Guernsey, with little or no evidence of questing I. ricinus on either island. During field studies on small mammals, I. ricinus was the predominant tick species feeding on Jersey bank voles (Myodes glareolus caesarius), with Ixodes hexagonus the most common species infesting hedgehogs on Guernsey. We propose that the greater diversity of small mammals on Jersey may be important in supporting immature stages of I. ricinus, in contrast to Guernsey and Alderney. Morphological identification of tick species was confirmed by PCR sequencing based on amplification of the cytochrome c oxidase subunit one (cox1) gene (COI DNA barcoding). To date, there have been few records of human tick bites in the Channel Islands, suggesting that the current risk from tick-borne disease may be low, but continued reporting of any human tick bites, along with reporting of cases of Lyme borreliosis will be important for continued assessment of the impact of tick-borne diseases in the Channel Islands.

An Integrated Molecular Approach to Untangling Host-Vector-Pathogen Interactions in Mosquitoes (Diptera: Culicidae) From Sylvan Communities in Mexico.

There are ~240 species of Culicidae in Mexico, of which some are vectors of arthropod-borne viruses such as Zika virus, dengue virus, chikungunya virus, and West Nile virus. Thus, the identification of mosquito feeding preferences is paramount to understanding of vector-host-pathogen interactions that, in turn, can aid the control of disease outbreaks. Typically, DNA and RNA are extracted separately for animal (insects and blood meal hosts) and viral identification, but this study demonstrates that multiple organisms can be analyzed from a single RNA extract. For the first time, residual DNA present in standard RNA extracts was analyzed by DNA barcoding in concert with Sanger and next-generation sequencing (NGS) to identify both the mosquito species and the source of their meals in blood-fed females caught in seven sylvan communities in Chiapas State, Mexico. While mosquito molecular identification involved standard barcoding methods, the sensitivity of blood meal identification was maximized by employing short primers with NGS. In total, we collected 1,634 specimens belonging to 14 genera, 25 subgenera, and 61 morphospecies of mosquitoes. Of these, four species were new records for Mexico (Aedes guatemala, Ae. insolitus, Limatus asulleptus, Trichoprosopon pallidiventer), and nine were new records for Chiapas State. DNA barcode sequences for >300 bp of the COI gene were obtained from 291 specimens, whereas 130 bp sequences were recovered from another 179 specimens. High intraspecific divergence values (>2%) suggesting cryptic species complexes were observed in nine taxa: Anopheles eiseni (5.39%), An. pseudopunctipennis (2.79%), Ae. podographicus (4.05%), Culex eastor (4.88%), Cx. erraticus (2.28%), Toxorhynchites haemorrhoidalis (4.30%), Tr. pallidiventer (4.95%), Wyeomyia adelpha/Wy. guatemala (7.30%), and Wy. pseudopecten (4.04%). The study increased the number of mosquito species known from 128 species to 138 species for Chiapas State, and 239 for Mexico as a whole. Blood meal analysis showed that Aedes angustivittatus fed on ducks and chicken, whereas Psorophora albipes fed on humans. Culex quinquefasciatus fed on diverse hosts including chicken, human, turkey, and Mexican grackle. No arbovirus RNA was detected by reverse transcriptase-polymerase chain reaction in the surveyed specimens. This study demonstrated, for the first time, that residual DNA present in RNA blood meal extracts can be used to identify host vectors, highlighting the important role of molecular approaches in both vector identification and revealing host-vector-pathogen interactions.

Spiders (Araneae) of Churchill, Manitoba: DNA barcodes and morphology reveal high species diversity and new Canadian records.

BACKGROUND: Arctic ecosystems, especially those near transition zones, are expected to be strongly impacted by climate change. Because it is positioned on the ecotone between tundra and boreal forest, the Churchill area is a strategic locality for the analysis of shifts in faunal composition. This fact has motivated the effort to develop a comprehensive biodiversity inventory for the Churchill region by coupling DNA barcoding with morphological studies. The present study represents one element of this effort; it focuses on analysis of the spider fauna at Churchill. RESULTS: 198 species were detected among 2704 spiders analyzed, tripling the count for the Churchill region. Estimates of overall diversity suggest that another 10-20 species await detection. Most species displayed little intraspecific sequence variation (maximum <1%) in the barcode region of the cytochrome c oxidase subunit I (COI) gene, but four species showed considerably higher values (maximum = 4.1-6.2%), suggesting cryptic species. All recognized species possessed a distinct haplotype array at COI with nearest-neighbour interspecific distances averaging 8.57%. Three species new to Canada were detected: Robertus lyrifer (Theridiidae), Baryphyma trifrons (Linyphiidae), and Satilatlas monticola (Linyphiidae). The first two species may represent human-mediated introductions linked to the port in Churchill, but the other species represents a range extension from the USA. The first description of the female of S. monticola was also presented. As well, one probable new species of Alopecosa (Lycosidae) was recognized. CONCLUSIONS: This study provides the first comprehensive DNA barcode reference library for the spider fauna of any region. Few cryptic species of spiders were detected, a result contrasting with the prevalence of undescribed species in several other terrestrial arthropod groups at Churchill. Because most (97.5%) sequence clusters at COI corresponded with a named taxon, DNA barcoding reliably identifies spiders in the Churchill fauna. The capacity of DNA barcoding to enable the identification of otherwise taxonomically ambiguous specimens (juveniles, females) also represents a major advance for future monitoring efforts on this group.