Effect of spatiotemporal variables on abundance, biting activity and parity of Nyssorhynchus darlingi (Diptera: Culicidae) in peri-Iquitos, Peru.

BACKGROUND: In malaria endemic regions of the Peruvian Amazon, rainfall together with river level and breeding site availability drive fluctuating vector mosquito abundance and human malaria cases, leading to temporal heterogeneity. The main variables influencing spatial transmission include location of communities, mosquito behaviour, land use/land cover, and human ecology/behaviour. The main objective was to evaluate seasonal and microgeographic biting behaviour of the malaria vector Nyssorhynchus (or Anopheles) darlingi in Amazonian Peru and to investigate effects of seasonality on malaria transmission. METHODS: We captured mosquitoes from 18:00 to 06:00 h using Human Landing Catch in two riverine (Lupuna, Santa Emilia) and two highway (El Triunfo, Nuevo Horizonte) communities indoors and outdoors from 8 houses per community, during the dry and rainy seasons from February 2016 to January 2017. We then estimated parity rate, daily survival and age of a portion of each collection of Ny. darlingi. All collected specimens of Ny. darlingi were tested for the presence of Plasmodium vivax or Plasmodium falciparum sporozoites using real-time PCR targeting the small subunit of the 18S rRNA. RESULTS: Abundance of Ny. darlingi varied across village, season, and biting behaviour (indoor vs outdoor), and was highly significant between rainy and dry seasons (p < 0.0001). Biting patterns differed, although not significantly, and persisted regardless of season, with peaks in highway communities at ~ 20:00 h in contrast to biting throughout the night (i.e., 18:00-06:00) in riverine communities. Of 3721 Ny. darlingi tested for Plasmodium, 23 (0.62%) were infected. We detected Plasmodium-infected Ny. darlingi in both community types and most (20/23) were captured outdoors during the rainy season; 17/23 before midnight. Seventeen Ny. darlingi were infected with P. vivax, and 6 with P. falciparum. No infected Ny. darlingi were captured during the dry season. Significantly higher rates of parity were detected in Ny. darlingi during the rainy season (average 64.69%) versus the dry season (average 36.91%) and by community, Lupuna, a riverine village, had the highest proportion of parous to nulliparous females during the rainy season. CONCLUSIONS: These data add a seasonal dimension to malaria transmission in peri-Iquitos, providing more evidence that, at least locally, the greatest risk of malaria transmission is outdoors during the rainy season mainly before midnight, irrespective of whether the community was located adjacent to the highway or along the river.

Mutations Linked to Insecticide Resistance Not Detected in the Ace-1 or VGSC Genes in Nyssorhynchus darlingi from Multiple Localities in Amazonian Brazil and Peru.

Indoor residual spray (IRS), mainly employing pyrethroid insecticides, is the most common intervention for preventing malaria transmission in many regions of Latin America; the use of long-lasting insecticidal nets (LLINs) has been more limited. Knockdown resistance (kdr) is a well-characterized target-site resistance mechanism associated with pyrethroid and DDT resistance. Most mutations detected in acetylcholinesterase-1 (Ace-1) and voltage-gated sodium channel (VGSC) genes are non-synonymous, resulting in a change in amino acid, leading to the non-binding of the insecticide. In the present study, we analyzed target-site resistance in Nyssorhynchus darlingi, the primary malaria vector in the Amazon, in multiple malaria endemic localities. We screened 988 wild-caught specimens of Ny. darlingi from three localities in Amazonian Peru and four in Amazonian Brazil. Collections were conducted between 2014 and 2021. The criteria were Amazonian localities with a recent history as malaria hotspots, primary transmission by Ny. darlingi, and the use of both IRS and LLINs as interventions. Fragments of Ace-1 (456 bp) and VGSC (228 bp) were amplified, sequenced, and aligned with Ny. darlingi sequences available in GenBank. We detected only synonymous mutations in the frequently reported Ace-1 codon 280 known to confer resistance to organophosphates and carbamates, but detected three non-synonymous mutations in other regions of the gene. Similarly, no mutations linked to insecticide resistance were detected in the frequently reported codon (995) at the S6 segment of domain II of VGSC. The lack of genotypic detection of insecticide resistance mutations by sequencing the Ace-1 and VGSC genes from multiple Ny. darlingi populations in Brazil and Peru could be associated with low-intensity resistance, or possibly the main resistance mechanism is metabolic.

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.

Vector role and human biting activity of Anophelinae mosquitoes in different landscapes in the Brazilian Amazon.

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.

Evidence for family-level variation of phenotypic traits in response to temperature of Brazilian Nyssorhynchus darlingi.

BACKGROUND: Nyssorhynchus darlingi (also known as Anopheles darlingi) is the primary malaria vector in the Amazon River Basin. In Brazil, analysis of single nucleotide polymorphisms (SNPs) previously detected three major population clusters, and a common garden experiment in a laboratory setting revealed significant population variation in life history traits. Increasing temperatures and local level variation can affect life history traits, i.e. adult longevity, that alter vectorial capacity with implications for malaria transmission in Ny. darlingi. METHODS: We investigated the population structure of Ny. darlingi from 7 localities across Brazil utilizing SNPs and compared them to a comprehensive Ny. darlingi catalog. To test the effects of local level variation on life history traits, we reared F1 progeny from the 7 localities at three constant temperatures (20, 24 and 28 °C), measuring key life history traits (larval development, food-starved adult lifespan, adult size and daily survival). RESULTS: Using nextRAD genotyping-by-sequencing, 93 of the field-collected Ny. darlingi were genotyped at 33,759 loci. Results revealed three populations (K = 3), congruent with major biomes (Amazonia, Cerrado and Mata Atlântica), with greater FST values between biomes than within. In the life history experiments, increasing temperature reduced larval development time, adult lifespan, and wing length in all localities. The variation of family responses for all traits within four localities of the Amazonia biome was significant (ANOVA, P < 0.05). Individual families within localities revealed a range of responses as temperature increased, for larval development, adult lifespan, wing length and survival time. CONCLUSIONS: SNP analysis of several Brazilian localities provided results in support of a previous study wherein populations of Ny. darlingi were clustered by three major Brazilian biomes. Our laboratory results of temperature effects demonstrated that population variation in life history traits of Ny. darlingi exists at the local level, supporting previous research demonstrating the high plasticity of this species. Understanding this plasticity and inherent variation between families of Ny. darlingi at the local level should be considered when deploying intervention strategies and may improve the likelihood of successful malaria elimination in South America.

Higher risk of malaria transmission outdoors than indoors by Nyssorhynchus darlingi in riverine communities in the Peruvian Amazon.

BACKGROUND: Malaria remains an important public health problem in Peru where incidence has been increasing since 2011. Of over 55,000 cases reported in 2017, Plasmodium vivax was the predominant species (76%), with P. falciparum responsible for the remaining 24%. Nyssorhynchus darlingi (previously Anopheles darlingi) is the main vector in Amazonian Peru, where hyperendemic Plasmodium transmission pockets have been found. Mazán district has pronounced spatial heterogeneity of P. vivax malaria. However, little is known about behavior, ecology or seasonal dynamics of Ny. darlingi in Mazán. This study aimed to gather baseline information about bionomics of malaria vectors and transmission risk factors in a hyperendemic malaria area of Amazonian Peru. METHODS: To assess vector biology metrics, five surveys (two in the dry and three in the rainy season), including collection of sociodemographic information, were conducted in four communities in 2016-2017 on the Napo (Urco Miraño, URC; Salvador, SAL) and Mazán Rivers (Visto Bueno, VIB; Libertad, LIB). Human-biting rate (HBR), entomological inoculation rate (EIR) and human blood index (HBI) were measured to test the hypothesis of differences in entomological indices of Ny. darlingi between watersheds. A generalized linear mixed effect model (GLMM) was constructed to model the relationship between household risk factors and the EIR. RESULTS: Nyssorhynchus darlingi comprised 95% of 7117 Anophelinae collected and its abundance was significantly higher along the Mazán River. The highest EIRs (3.03-4.54) were detected in March and June in URC, LIB and VIB, and significantly more Ny. darlingi were infected outdoors than indoors. Multivariate analysis indicated that the EIR was >12 times higher in URC compared with SAL. The HBI ranged from 0.42-0.75; humans were the most common blood source, followed by Galliformes and cows. There were dramatic differences in peak biting time and malaria incidence with similar bednet coverage in the villages. CONCLUSIONS: Nyssorhynchus darlingi is the predominant contributor to malaria transmission in the Mazán District, Peru. Malaria risk in these villages is higher in the peridomestic area, with pronounced heterogeneities between and within villages on the Mazán and the Napo Rivers. Spatiotemporal identification and quantification of the prevailing malaria transmission would provide new evidence to orient specific control measures for vulnerable or at high risk populations.

Brazil's first free-mating laboratory colony of Nyssorhynchus darlingi

Abstract INTRODUCTION: The lack of highly-productive Nyssorhynchus darlingi laboratory colonies limits some studies. We report the first well-established laboratory colony of Ny. darlingi in Brazil. METHODS: Mosquitoes were collected from Porto Velho and were reared at the Laboratory of Fiocruz/RO. After induced mating by light stimulation in the F1 to F6, the subsequent generations were free mating. Larvae were reared in distilled water and fed daily until pupation. RESULTS: In 11 generations, the colony produced a high number of pupae after the F5 generation. CONCLUSIONS: These results demonstrate the potential for permanently establishing Ny. darlingi colonies for research purposes in Brazil.