Bacterial community associated with Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) from an urban area in the Amazon, Brazil

ABSTRACT The emergence of insecticide resistance in different mosquito populations underscores the pressing need for alternative approaches to control vector-borne diseases. Among several technological strategies, the employment of bacterial symbionts, such as the Wolbachia pipientis strains wMel and wAlbB to inhibit the ability of Aedes aegypti (Linnaeus, 1762) to transmit dengue and Zika viruses in endemic regions worldwide is promising. This investigation examines both the bacterial diversity associated with Culex quinquefasciatus and the genetic diversity of Wolbachia in females collected in Coari, Amazonas State, Brazil. Both 16S rRNA and Wolbachia surface protein (wsp) gene sequences were generated and examined. Proteobacteria was the dominant phylum. Wolbachia was the predominant genus, followed by Providencia, unclassified Erwiniaceae, and Acinetobacter. The presence of Delftia in Cx. quinquefasciatus need further investigations to identify the strains and if any of them can inhibit the transmission of arboviruses by this mosquito. Wolbachia 16S rRNA sequences were detected in all samples analyzed. The wsp sequences from Coari specimens were identified as Wolbachia wPip strain of the supergroup B. These sequences are identical and share 100% similarity with those of other Cx. quinquefasciatus populations from Brazil. Our findings suggest the hypothesis of previous studies that the Wolbachia invasion in Cx. quinquefasciatus was recent.

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.

Bacterial diversity associated with the abdomens of naturally Plasmodium-infected and non-infected Nyssorhynchus darlingi.

BACKGROUND: The bacterial community present in the abdomen in Anophelinae mosquitoes can influence mosquito susceptibility to Plasmodium infection. Little is known about the bacteria associated with Nyssorhynchus darlingi, a primary malaria vector in the Amazon basin. We investigated the abdominal bacterial community compositions of naturally Plasmodium-infected (P-positive, n = 9) and non-infected (P-negative, n = 7) Ny. darlingi from the Brazilian Amazon region through massive parallel sequencing of the bacterial V4 variable region of the 16S rRNA gene. RESULTS: Bacterial richness of Ny. darlingi encompassed 379 operational taxonomic units (OTUs), the majority of them belonging to the Proteobacteria, Firmicutes and Bacteroides phyla. Escherichia/Shigella and Pseudomonas were more abundant in the P-positive and P-negative groups, respectively, than in the opposite groups. Enterobacter was found only in the P-negative group. The results of statistical analyses conducted to compare bacterial abundance and diversity between Plasmodium-infected and Plasmodium-non-infected mosquitoes were not significant. CONCLUSIONS: This study increased knowledge about bacterial composition in Ny. darlingi and revealed that Plasmodium-positive and Plasmodium-negative groups share a common core of bacteria. The genera Prevotella 9, Sphingomonas, Bacteroides, and Bacillus were reported for the first time in Ny. darlingi.

Influência de determinantes entomológicos e bacterianos no contexto da malária em áreas rurais e periurbanas da Amazônia brasileira / Influence of entomological and bacterial determinants in the context of malaria in rural and peri-urban areas of the Brazilian Amazon

Introdução - Nyssorhynchus darlingi e outros mosquitos da subfamília Anophelinae atuam como vetores dos parasitos da malária humana. Alterações na paisagem podem alterar a distribuição, abundância e comportamento desses mosquitos. Bactérias presentes no intestino médio de vetores de Plasmodium são capazes de modular a infecção por Plasmodium spp. no mosquito. Objetivos - Ampliar o conhecimento sobre os mosquitos que atuam na dinâmica de transmissão da malária em áreas rurais e periurbanas da Amazônia brasileira e investigar a diversidade bacteriana associada ao abdômen de Ny. darlingi e Nyssorhynchus braziliensis. Métodos - Capturas de mosquitos foram realizadas em áreas rurais e periurbanas da Amazônia brasileira. Testes de detecção de Plasmodium spp. foram realizados nos mosquitos. Análises de correlação e de regressão foram realizadas entre métricas de paisagem e as variáveis: incidência acumulada de malária, número de Ny. darlingi, mosquitos infectados e taxa de picada humana. Sequenciamento da região V4 do gene 16S rRNA foi realizado para explorar a diversidade bacteriana associada ao abdômen de Ny. braziliensis e de Ny. darlingi naturalmente infectado e não infectado por Plasmodium spp. Resultados - Nyssorhynchus darlingi, Nyssorhynchus rangeli, Nyssorhynchus benarrochi B e Nyssorhynchus konderi B foram encontrados infectados com Plasmodium. Plasmodium vivax e Plasmodium falciparum foram as espécies do parasito encontradas nos vetores. Foram encontradas correlações positivas entre incidência acumulada de malária e as variáveis taxa de picada humana, densidade de borda e número de Ny. darlingi. Porcentagem de cobertura florestal e taxa de picada humana apresentaram correlação negativa. O período entre 0 h:00 e 3 h:00 foi o que apresentou maior número de mosquitos infectados. Não houve diferença estatística entre a diversidade bacteriana de Ny. darlingi infectado e não infectado. Asaia e Serratia estavam presentes em ambas as espécies de Nyssochynchus. Enterobacter foi encontrado apenas em abdômen de Ny. darlingi não infectado e Pseudomonas foi o gênero mais abundante. Conclusão - Este estudo reporta pela primeira vez a infecção natural por Plasmodium em Ny. konderi B e Ny. benarrochi B em regiões da Amazônia brasileira. Os resultados obtidos sugerem que mudanças na paisagem podem favorecer a ocorrência de novos vetores e consequentemente, aumentar o número de casos de malária em regiões endêmicas. O encontro de maior número de mosquitos infectados após a meia-noite contribui para as medidas de controle do vetor e confirma a importância do uso de mosquiteiros impregnados com inseticida. A presença de Asaia e Serratia em ambas as espécies de Nyssorhynchus e a alta prevalência de Pseudomonas em Ny. darlingi indicam a necessidade de outros estudos sobre a possível utilização destas bactérias no controle da malária através da paratransgênese. A presença do gênero Enterobacter apenas em abdômen de Ny. darlingi não infectado sugere que bactérias deste gênero possam oferecer proteção a infecção por Plasmodium e outros estudos devem ser realizados para verificar essa hipótese.

Introduction - Nyssorhynchus darlingi and other mosquitoes of the subfamily Anophelinae act as vectors of human malaria parasites. Landscape changes can alter the distribution, abundance and behavior of these mosquitoes. Bacteria present in the midgut of Plasmodium vectors are able to modulate infection by Plasmodium spp. in the mosquito. Objectives - To broaden the knowledge about mosquitoes that act on the dynamics of malaria transmission in rural and peri-urban areas of the Brazilian Amazon and to investigate the bacterial diversity associated with abdomen of Ny. darlingi and Nyssorhynchus braziliensis. Methods - Mosquito collections were carried out in rural and peri-urban areas of the Brazilian Amazon. Detection tests for Plasmodium spp. mosquitoes were carried out. Correlation and regression analyzes were performed between landscape metrics and the following variables: cumulative incidence of malaria, number of Ny. darlingi, infected mosquitoes, and human biting rate. Sequencing of the V4 region of the 16S rRNA gene was performed to explore the bacterial diversity associated with abdomen of Ny. braziliensis and Ny. darlingi naturally infected and not infected by Plasmodium spp. Results - Nyssorhynchus darlingi, Nyssorhynchus rangeli, Nyssorhynchus benarrochi B and Nyssorhynchus konderi B were found infected with Plasmodium. Plasmodium vivax and Plasmodium falciparum were the parasite species found in the vectors. Positive correlations were found between cumulative incidence of malaria and the variables human biting rate, edge density and number of Ny. darlingi. Percentage of forest cover and human biting rate presented negative correlation. The period from 0 h: 00 to 3 h: 00 was the one with the highest number of infected mosquitoes. There was no statistical difference between bacterial diversity in Ny. darlingi infected and uninfected. Asaia and Serratia were present in both species of Nyssochynchus. Enterobacter was found only in abdomen of Ny. darlingi uninfected and Pseudomonas was the genus most abundant. Conclusion - This study reports for the first time the natural infection by Plasmodium in Ny. konderi B and Ny. benarrochi B in regions of the Brazilian Amazon. The results suggest that changes in the landscape can favor the occurrence of new vectors and, consequently, increase the number of malaria cases in endemic regions. The finding of a greater number of infected mosquitoes after midnight contributes to vector control measures and confirms the importance of insecticide-treated nets. The presence of Asaia and Serratia in both species of Nyssorhynchus and the high prevalence of Pseudomonas in Ny. darlingi indicate the need for further studies on the possible use of these bacteria in the control of malaria through paratransgenesis. The presence of the Enterobacter only in abdomen Ny. darlingi uninfected suggests that bacteria of this genus can offer protection to Plasmodium infection and other studies should be performed to verify this hypothesis.

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.

Mosquitoes of the Caatinga: 1. Adults stage survey and the emerge of seven news species endemic of a dry tropical forest in Brazil.

The Caatinga is the least known Brazilian biome in terms of the diversity of Culicidae. No systematic study of the diversity or ecology of the mosquitoes of this biome is available, despite the importance of vector diseases in Brazil. The present study addressed the mosquito biodiversity in the Caatinga biome by sampling adult populations. Specimens were sampled monthly from March 2013 to September 2014 in a Caatinga conservation unit located in the Brazilian semiarid zone. Mosquito collections were carried out in Shannon traps from late afternoon to early evening, and manual aspiration was used to capture diurnal species as well. A total of 4,692 mosquitoes were collected. The most dominant and constant species were all undescribed species belonging to the genera Wyeomyia and Runchomyia, which together represented 80% of the specimens. The most abundant species of epidemiological importance was Haemagogus (Con.) leucocelaenus. The abundance of mosquitoes was positively associated with the relative humidity and temperature recorded during the month preceding the collection date. In the Caatinga, the diversity of adult mosquitoes was associated with the availability (quantity and diversity) of natural larval habitats found in the different phytophysiognomies of the biome, which vary according to temperature and humidity. The number of species unknown to science reflects the levels of endemism that exist in the study area, and reinforces the need to further taxonomic investigation in the biome.

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.