Un estudio de la distribución de los mosquitos del estado de Quintana Roo, México fue realizado por medio de colectas de estados inmaduros y adultos durante Septiembre y Octubre del 2006. Las colectas fueron realizadas en diferentes localidades de las tres provincias fisiográficas del estado: Carso Yucateco, Carso, Lomeríos de Campeche y Costa Baja de Quintana Roo. Un total de 420 larvas, 294 pupas y 726 adultos fueron colectados representando 13 géneros y 41 especies. Dos géneros, tres subgéneros y 11 especies son nuevos registros estatales para Quintana Roo.

Identifying novel viruses or assessing viral variation by NGS requires high sequencing coverage. More than 90% of total RNA is ribosomal (rRNA), making variant calling, virus discovery or transcriptomic profiling difficult. Current methods to increase informative reads suffer from drawbacks, either they cannot be used for some viruses, are optimized for a single species, or introduce bias. We describe a two-part approach combining reverse-transcription to create RNA/DNA hybrids which are then degraded with RNaseH/DNase sequentially that works for three medically relevant mosquito genera; Aedes, Anopheles, and Culex. We demonstrate depletion of rRNA from different samples, including whole mosquitoes and midgut contents from FTA cards. We describe novel insect-specific virus genomes from field collected mosquitoes. The protocol requires only common laboratory reagents and small oligonucleotides specific to rRNA. This approach can be adapted for other organisms, aiding virus diversity analyses, virus discovery and transcriptomics in both laboratory and field samples.

22Studies aimed at identifying novel viral sequences or assessing intrahost viral variation require sufficient 23 sequencing coverage to assemble contigs and make accurate variant calling at low frequencies. Many 24 samples come from host tissues where ribosomal RNA represents more than 90% of total RNA 25 preparations, making unbiased sequencing of viral samples inefficient and highly expensive, as many 26 reads will be wasted on cellular RNAs. In the presence of this amount of ribosomal RNA, it is difficult to 27 achieve sufficient sequencing depth to perform analyses such as variant calling, haplotype prediction, 28 virus population analyses, virus discovery or transcriptomic profiling. Many methods for depleting 29 unwanted RNA or enriching RNA of interest have been devised, including poly-A selection, RNase H 30 based specific depletion, duplex-specific nuclease treatment and hybrid capture selection, among 31 others. Although these methods can be efficient, they either cannot be used for some viruses (i.e. non-32 polyadenylated viruses), have been optimized for use in a single species, or have the potential to 33 introduce bias. In this study, we describe a novel approach that uses an RNaseH possessing reverse 34 transcriptase coupled with selective probes for ribosomal RNA designed to work broadly for three 35 medically relevant mosquito genera; Aedes, Anopheles, and Culex. We demonstrate significant depletion 36 of rRNA using multiple assessment techniques from a variety of sample types, including whole 37 mosquitoes and mosquito midgut contents from FTA cards. To demonstrate the utility of our approach, 38 we describe novel insect-specific virus genomes from numerous species of field collected mosquitoes 39 that underwent rRNA depletion, thereby facilitating their detection. The protocol is straightforward, 40 relatively low-cost and requires only common laboratory reagents and the design of several small 41 oligonucleotides specific to the species of interest. This approach can be adapted for use with other 42 organisms with relative ease, thus potentially aiding virus population genetics analyses, virus discovery 43 and transcriptomic profiling in both laboratory and field samples. 44 RNA:cDNA hybrid. The samples were then digested with DNase I (NEB) to remove the cDNA and residual 108 oligos. The RNA was then purified using RNAClean XP beads at a 1.8x ratio. 109

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.