DNA Barcodes of Mansonia (Mansonia) Blanchard, 1901 (Diptera, Culicidae).

Females of the genus Mansonia feed on the blood of humans, livestock, and other vertebrates to develop their eggs. The females' biting behavior may cause severe disturbance to blood hosts, with a negative impact on public health and economics. Certain species have been identified as potential or effective disease vectors. The accurate species identification of field-collected specimens is of paramount importance for the success of monitoring and control strategies. Mansonia (Mansonia) morphological species boundaries are blurred by patterns of intraspecific heteromorphism and interspecific isomorphism. DNA barcodes can help to solve taxonomic controversies, especially if combined with other molecular tools. We used cytochrome c oxidase subunit I (COI) gene 5' end (DNA barcode) sequences to identify 327 field-collected specimens of Mansonia (Mansonia) spp. The sampling encompassed males and females collected from three Brazilian regions and previously assigned to species based on their morphological characteristics. Eleven GenBank and BOLD sequences were added to the DNA barcode analyses. Initial morphospecies assignments were mostly corroborated by the results of five clustering methods based on Kimura two-parameter distance and maximum likelihood phylogeny. Five to eight molecular operational taxonomic units may represent taxonomically unknown species. The first DNA barcode records for Mansonia fonsecai, Mansonia iguassuensis, and Mansonia pseudotitillans are presented.

Host Feeding Patterns of Mansonia (Diptera, Culicidae) in Rural Settlements near Porto Velho, State of Rondonia, Brazil.

Mosquito females of the genus Mansonia (Blanchard) can be a nuisance to humans and animals since they are voraciously hematophagous and feed on the blood of a variety of vertebrates. Despite their relevance, there is a lack of investigation into the blood-feeding patterns of the Mansonia species. Knowledge of the host preference is crucial in establishing the public health importance of a mosquito species and its potential to be involved in the transmission dynamics of pathogens. Species that are primarily anthropophilic can be more effective in spreading vector-borne pathogens to humans. In this study, we used an Illumina Nextera sequencing protocol and the QIIME2 workflow to assess the diversity of DNA sequences extracted in the ingested blood of mosquito species to evaluate the overall and local host choices for three species: Ma. titillans, Ma. Amazonensis, and Ma. humeralis, in rural areas alongside the Madeira River in the vicinities of the Santo Antonio Energia (SAE) reservoir in the municipality of Porto Velho, Rondônia, Western Brazil. By performing our analysis pipeline, we have found that host diversity per collection site showed a significant heterogeneity across the sample sites. In addition, in rural areas, Ma. amazonensis present a high affinity for B. taurus, Ma. humeralis shows an overall preference for C. familiaris and B. taurus, but also H. sapiens and E. caballus in urban areas, and Ma. titillans showed more opportunistic behavior in rural areas, feeding on wild animals and G. gallus, though with an overall preference for H. sapiens.

Susceptibility of Field-Collected Nyssorhynchus darlingi to Plasmodium spp. in Western Amazonian Brazil.

Mosquito susceptibility to Plasmodium spp. infection is of paramount importance for malaria occurrence and sustainable transmission. Therefore, understanding the genetic features underlying the mechanisms of susceptibility traits is pivotal to assessing malaria transmission dynamics in endemic areas. The aim of this study was to investigate the susceptibility of Nyssorhynchus darlingi-the dominant malaria vector in Brazil-to Plasmodium spp. using a reduced representation genome-sequencing protocol. The investigation was performed using a genome-wide association study (GWAS) to identify mosquito genes that are predicted to modulate the susceptibility of natural populations of the mosquito to Plasmodium infection. After applying the sequence alignment protocol, we generated the variant panel and filtered variants; leading to the detection of 202,837 SNPs in all specimens analyzed. The resulting panel was used to perform GWAS by comparing the pool of SNP variants present in Ny. darlingi infected with Plasmodium spp. with the pool obtained in field-collected mosquitoes with no evidence of infection by the parasite (all mosquitoes were tested separately using RT-PCR). The GWAS results for infection status showed two statistically significant variants adjacent to important genes that can be associated with susceptibility to Plasmodium infection: Cytochrome P450 (cyp450) and chitinase. This study provides relevant knowledge on malaria transmission dynamics by using a genomic approach to identify mosquito genes associated with susceptibility to Plasmodium infection in Ny. darlingi in western Amazonian Brazil.

A Multi-Gene Analysis and Potential Spatial Distribution of Species of the Strodei Subgroup of the Genus Nyssorhynchus (Diptera: Culicidae).

Nyssorhynchus strodei (Root) is an understudied species of potential epidemiological importance, having been found naturally infected in Brazil with Plasmodium falciparum Welch, Plasmodium vivax Grassi & Feletti, and Plasmodium malariae Feletti & Grassi. It belongs to the strodei subgroup that includes Nyssorhynchus albertoi (Unti), Nyssorhynchus arthuri (Unti), Nyssorhynchus rondoni (Neiva & Pinto), Nyssorhynchus striatus (Sant'Ana & Sallum), and three unnamed species, Nyssorhynchus arthuri B, Nyssorhynchus arthuri C, and Nyssorhynchus arthuri D. As the accurate identification of vector species is of fundamental importance for public health entomology, the aim of the study was to provide additional information for the presence of seven species that had been previously misidentified as Ny. strodei. Bayesian phylogenetic analyses using DNA sequences of the genes COI mtDNA, white, CAD and CAT nuclear genes confirmed Ny. albertoi, Ny. arthuri, Ny. strodei, and Ny. striatus as distinct groups within the strodei subgroup and corroborated the presence of three undescribed species under the name Ny. arthuri. Results of the GMYC model analysis corroborated Ny. arthuri B, Ny. arthuri C, and Ny. arthuri D; however, they did not distinguish between Ny. strodei and Ny. albertoi. Predicted distribution of seven species based on maximum entropy in MaxEnt showed that each species has its specific ecological niche suitability.

Phylogeny of Anophelinae using mitochondrial protein coding genes.

Malaria is a vector-borne disease that is a great burden on the poorest and most marginalized communities of the tropical and subtropical world. Approximately 41 species of Anopheline mosquitoes can effectively spread species of Plasmodium parasites that cause human malaria. Proposing a natural classification for the subfamily Anophelinae has been a continuous effort, addressed using both morphology and DNA sequence data. The monophyly of the genus Anopheles, and phylogenetic placement of the genus Bironella, subgenera Kerteszia, Lophopodomyia and Stethomyia within the subfamily Anophelinae, remain in question. To understand the classification of Anophelinae, we inferred the phylogeny of all three genera (Anopheles, Bironella, Chagasia) and major subgenera by analysing the amino acid sequences of the 13 protein coding genes of 150 newly sequenced mitochondrial genomes of Anophelinae and 18 newly sequenced Culex species as outgroup taxa, supplemented with 23 mitogenomes from GenBank. Our analyses generally place genus Bironella within the genus Anopheles, which implies that the latter as it is currently defined is not monophyletic. With some inconsistencies, Bironella was placed within the major clade that includes Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia, which were found to be monophyletic groups within Anophelinae. Our findings provided robust evidence for elevating the monophyletic groupings Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia to genus level; genus Anopheles to include subgenera Anopheles, Baimaia, Cellia and Christya; Anopheles parvus to be placed into a new genus; Nyssorhynchus to be elevated to genus level; the genus Nyssorhynchus to include subgenera Myzorhynchella and Nyssorhynchus; Anopheles atacamensis and Anopheles pictipennis to be transferred from subgenus Nyssorhynchus to subgenus Myzorhynchella; and subgenus Nyssorhynchus to encompass the remaining species of Argyritarsis and Albimanus Sections.

Plasmodium falciparum in the southeastern Atlantic forest: a challenge to the bromeliad-malaria paradigm?

BACKGROUND: Recently an unexpectedly high prevalence of Plasmodium falciparum was found in asymptomatic blood donors living in the southeastern Brazilian Atlantic forest. The bromeliad-malaria paradigm assumes that transmission of Plasmodium vivax and Plasmodium malariae involves species of the subgenus Kerteszia of Anopheles and only a few cases of P. vivax malaria are reported annually in this region. The expectations of this paradigm are a low prevalence of P. vivax and a null prevalence of P. falciparum. Therefore, the aim of this study was to verify if P. falciparum is actively circulating in the southeastern Brazilian Atlantic forest remains. METHODS: In this study, anophelines were collected with Shannon and CDC-light traps in seven distinct Atlantic forest landscapes over a 4-month period. Field-collected Anopheles mosquitoes were tested by real-time PCR assay in pools of ten, and then each mosquito from every positive pool, separately for P. falciparum and P. vivax. Genomic DNA of P. falciparum or P. vivax from positive anophelines was then amplified by traditional PCR for sequencing of the 18S ribosomal DNA to confirm Plasmodium species. Binomial probabilities were calculated to identify non-random results of the P. falciparum-infected anopheline findings. RESULTS: The overall proportion of anophelines naturally infected with P. falciparum was 4.4% (21/480) and only 0.8% (4/480) with P. vivax. All of the infected mosquitoes were found in intermixed natural and human-modified environments and most were Anopheles cruzii (22/25 = 88%, 18 P. falciparum plus 4 P. vivax). Plasmodium falciparum was confirmed by sequencing in 76% (16/21) of positive mosquitoes, whereas P. vivax was confirmed in only 25% (1/4). Binomial probabilities suggest that P. falciparum actively circulates throughout the region and that there may be a threshold of the forested over human-modified environment ratio upon which the proportion of P. falciparum-infected anophelines increases significantly. CONCLUSIONS: These results show that P. falciparum actively circulates, in higher proportion than P. vivax, among Anopheles mosquitoes of fragments of the southeastern Brazilian Atlantic forest. This finding challenges the classical bromeliad-malaria paradigm, which considers P. vivax circulation as the driver for the dynamics of residual malaria transmission in this region.