The ability to successfully identify and incriminate pathogen vectors is fundamental to effective pathogen control and management. This task is confounded by the existence of cryptic species complexes. Molecular markers can offer a highly effective means of species identification in such complexes and are routinely employed in the study of medical entomology. Here we evaluate a multi-locus system for the identification of potential malaria vectors in the Anopheles strodei subgroup.

The Coronator Group currently encompasses six morphologically similar species (Culex camposi Dyar, Culex coronator Dyar and Knab, Culex covagarciai Forattini, Culex usquatus Dyar, Culex usquatissimus Dyar, and Culex ousqua Dyar). Culex coronator has been incriminated as a potential vector of West Nile Virus (WNV), Saint Louis Encephalitis Virus (SLEV), and Venezuelan Equine Encephalitis Virus (VEEV). The complete mitochondrial genome of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi was sequenced, annotated, and analyzed to provide genetic information about these species. The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx.usquatissimus, and Cx. camposi varied from 15,573 base pairs in Cx. usquatus to 15,576 in Cx. coronator. They contained 37 genes (13 protein-encoding genes, 2 rRNA genes, and 22 tRNA genes) and the AT-rich control region. Comparative analyses of the 37 genes demonstrated the mitochondrial genomes to be composed of variable and conserved genes. Despite the small size, the ATP8, ATP6 plus NADH5 protein-encoding genes were polymorphic, whereas tRNAs and rRNAs were conserved. The control region contained some poly-T stretch. The Bayesian phylogenetic tree corroborated that both the Coronator Group and the Culex pipens complex are monophyletic taxa. The mitochondrial genomes of Cx. coronator, Cx. usquatus, Cx. usquatissimus and Cx. camposi share the same gene composition and arrangement features that match to those reported for most Culicidae species. They are composed of the same 37 genes and the AT-rich control region, which contains poly-T stretches that may be involved in the functional role of the mitochondrial genome. Taken together, results of the dN/dS ratios, the sliding window analyses and the Bayesian phylogenetic analyses suggest that ATP6, ATP8 and NADH5 are promising genes to be employed in phylogenetic studies involving species of the Coronator Group, and probably other species groups of the subgenus Culex. Bayesian topology corroborated the morphological hypothesis of the Coronator Group as monophyletic lineage within the subgenus Culex.

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. 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. 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. 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.

Background: Environmental disturbance, deforestation and socioeconomic factors all affect malaria incidence in tropical and subtropical endemic areas. Deforestation is the major driver of habitat loss and fragmentation, which frequently leads to shifts in the composition, abundance and spatial distribution of vector species. The goals of the present study were to: (i) identify anophelines found naturally infected with Plasmodium; (ii) measure the effects of landscape on the number of Nyssorhynchus darlingi, presence of Plasmodium-infected Anophelinae, human biting rate (HBR) and malaria cases; and (iii) determine the frequency and peak biting time of Plasmodium-infected mosquitoes and Ny. darlingi. Methods: Anopheline mosquitoes were collected in peridomestic and forest edge habitats in seven municipalities in four Amazon Brazilian states. Females were identified to species and tested for Plasmodium by real-time PCR. Negative binomial regression was used to measure any association between deforestation and number of Ny. darlingi, number of Plasmodium-infected Anophelinae, HBR and malaria. Peak biting time of Ny. darlingi and Plasmodium-infected Anophelinae were determined in the 12-h collections. Binomial logistic regression measured the association between presence of Plasmodium-infected Anophelinae and landscape metrics and malaria cases. Results: Ninety-one females of Ny. darlingi, Ny. rangeli, Ny. benarrochi B and Ny. konderi B were found to be infected with Plasmodium. Analysis showed that the number of malaria cases and the number of Plasmodium-infected Anophelinae were more prevalent in sites with higher edge density and intermediate forest cover (30–70%). The distance of the drainage network to a dwelling was inversely correlated to malaria risk. The peak biting time of Plasmodium-infected Anophelinae was 00:00–03:00 h. The presence of Plasmodium-infected mosquitoes was higher in landscapes with > 13 malaria cases. Conclusions: Nyssorhynchus darlingi, Ny. rangeli, Ny. benarrochi B and Ny. konderi B can be involved in malaria transmission in rural settlements. The highest fraction of Plasmodium-infected Anophelinae was caught from midnight to 03:00 h. In some Amazonian localities, the highest exposure to infectious bites occurs when residents are sleeping, but transmission can occur throughout the night. Forest fragmentation favors increases in both malaria and the occurrence of Plasmodium-infected mosquitoes in peridomestic habitat. The use of insecticide-impregnated mosquito nets can decrease human exposure to infectious Anophelinae and malaria transmission. Graphical

Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, such as the Anopheles albitarsis complex. Except for a few studies that employed a limited number of nuclear or mitochondrial loci to address the genetic structure and species status of Anopheles cruzii, Anopheles bellator, and Anopheles homunculus, little is known about genetic markers that can be employed in studies focusing on Kerteszia species. The complete mitochondrial genomes of seven specimens of An. bellator, An. cruzii, An. homunculus, and Anopheles laneanus were sequenced using long-range polymerase chain reaction and Illumina sequencing. The mitochondrial genomes varied from 15,446 to 15,738 bp in length and contained 37 genes (13 protein-encoding genes, 2 rRNA genes [12S rRNA and 16S rRNA] and 22 tRNA genes), and the AT-rich control region, as all do other Anopheles mitochondrial genomes sequenced to date. Specimens from four populations of An. cruzii showed differences in codon composition.

Mosquitoes (Diptera: Culicidae) are found widely throughout the world. Several species can transmit pathogens to humans and other vertebrates. Mosquitoes harbor great amounts of bacteria, fungi, and viruses. The bacterial composition of the microbiota of these invertebrates is associated with several factors, such as larval habitat, environment, and species. Yet little is known about bacterial interaction networks in mosquitoes. This study investigates the bacterial communities of eight species of Culicidae collected in Vale do Ribeira (Southeastern São Paulo State) and verifies the bacterial interaction network in these species. Sequences of the 16S rRNA region from 111 mosquito samples were analyzed. Bacterial interaction networks were generated from Spearman correlation values. Proteobacteria was the predominant phylum in all species. Wolbachia was the predominant genus in Haemagogus leucocelaenus. Aedes scapularis, Aedes serratus, Psorophora ferox, and Haemagogus capricornii were the species that showed a greater number of bacterial interactions. Bacterial positive interactions were found in all mosquito species, whereas negative correlations were observed in Hg. leucocelaenus, Ae. scapularis, Ae. serratus, Ps. ferox, and Hg. capricornii. All bacterial interactions with Asaia and Wolbachia were negative in Aedes mosquitoes.