New Iflavirus Species Characterized from Mosquitoes Captured in the Sao Paulo Zoological Facilities.

Metagenomic studies of mosquito viromes demonstrated a more diverse composition than just an exclusive composition of pathogenic arboviruses transmitted to humans. In our study, the virome of 866 female mosquitoes collected throughout 2020 at the São Paulo Zoo, located in the city of São Paulo/SP-Brazil, was obtained. Specifically, in this paper, we describe a new virus found by viral RNA extraction and next-generation MiSeq sequencing of a group of 23 specimens of Anopheles (Nys.) strodei. The complete genome with a length of 9709 nucleotides was characterized by a positive orientation and a single strand, with a single large ORF, which encodes a polyprotein of 2987 amino acids. The phylogenetic analysis showed an association with the viral family Iflaviridae and the Riboviria realm. We carried out comparisons with translated sequences of the capsid regions of other iflavirus, and the identities in relation to our sequence were below the minimum limit of 90%, indicating that possibly it is a new species of iflavirus. Our findings contribute to expanding knowledge of virome composition among mosquito species in Brazil and globally. Moreover, we provide a viral genome reference specific to this geographic region and Culicidae family of mosquitoes. This resource facilitates future in silico recognition and assembly of viral genomes within metagenomic datasets.

COI DNA barcoding to differentiate Haemagogus janthinomys and Haemagogus capricornii (Diptera: Culicidae) mosquitoes.

The genus Haemagogus (Diptera: Culicidae) includes species that are important vectors of pathogens such as the yellow fever virus. The accurate identification of these species is essential for the control of zoonoses. Females of Hg. capricornii and Hg. janthinomys are morphologically indistinguishable, which makes the use of alternative identification techniques desirable. This study aimed to obtain sequences of the mitochondrial cytochrome c oxidase I (COI) gene, in the region widely used for DNA barcoding, of Haemagogus specimens from the state of São Paulo, Brazil, to evaluate the effectiveness of these sequences in the molecular identification of the species. A total of 37 female and 2 male mosquitoes were collected in various locations in the state of São Paulo, using methods such as hand-nets, Shannon traps, CDC light traps with CO2 bait and Nasci aspirators. The sequences of a 710 bp fragment of the COI gene were amplified by PCR and sequenced. A phylogenetic tree reconstruction was conducted using the Bayesian approach implemented in MrBayes v3.2.2, providing support values for taxa where genetic clusters may indicate the presence of new or cryptic species. We obtained 39 COI sequences representing three species: Haemagogus capricornii, Haemagogus leucocelaenus, and Haemagogus janthinomys. Bayesian analysis of the sequences produced clades that corroborate the morphological identification of the species. The separation of Hg. capricornii and Hg. janthinomys received 100 % statistical support and the Hg. capricornii was very well supported (91 %). The two sequences from male specimens, morphologically identified as Hg. capricornii, were grouped in the same clade, a sister clade of Hg. janthinomys. It is important to highlight that the Hg. janthinomys were positioned in several subclades, showing a polymorphism of this species within the state, a situation not observed for Hg. capricornii. For the first time, sequences of the mtCOI gene from Hg. capricornii were obtained and related to morphologically identified specimens. COI sequences proved effective in the molecular identification of Haemagogus species. This study contributes to the expansion of the GenBank database, providing the first sequences of Hg. capricornii and new sequences for Hg. janthinomys and Hg. leucocelaenus.

Tracking arboviruses, their transmission vectors and potential hosts by nanopore sequencing of mosquitoes.

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.

DNA Barcoding of Morphologically Characterized Mosquitoes Belonging to the Genus Mansonia from the Atlantic Forest and Brazilian Savanna.

The identification of mosquito species is necessary for determining the entomological components of disease transmission. However, identification can be difficult in species that are morphologically similar. The cytochrome c oxidase subunit I (COI) DNA barcode region is considered a valuable and reliable diagnostic tool for mosquito species recognition, including those that belong to species complexes. Mansonia mosquitoes are found in forests near swampy areas. They are nocturnal and are highly attracted to light. Hematophagous adult females exhibit aggressive biting behavior and can become infected with and transmit pathogens during their feeding, including some epizootic viruses and avian malaria. In Brazil, twelve Mansonia species have been reported. In a recent study from the São Paulo Zoo in Brazil, three morphologically distinct species were collected and identified, namely: Mansonia (Mansonia) indubitans, Ma. (Man.) pseudotitillans and Ma. (Man.) titillans. However, confirmation of these species by molecular identification was unsuccessful due to a lack of COI sequences in the GenBank database. Thus, this research aimed to describe the COI DNA barcode sequences of some morphologically characterized Mansonia (Man.) species from Brazil and to determine their utility in delimiting species collected from the Atlantic Forest and Brazilian Savanna. Accordingly, we provide tools for the genetic identification of species that play a significant role in pathogen transmission in wildlife and potentially humans. We show that the delimitation of Mansonia species via five different approaches based on COI DNA sequences (BI, NJ, ASAP, bPTP and GMYC) yield basically the same groups identified by traditional taxonomy, and we provide the identification of specimens that were previously identified only up to the subgenus level. We also provide COI sequences from two Mansonia species that were not previously available in sequence databases, Ma. wilsoni and Ma. pseudotitillans, and thus contribute to the ongoing global effort to standardize DNA barcoding as a molecular means of species identification.