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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.

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