Characterization of artificial larval habitats of Anopheles darlingi (Diptera: Culicidae) in the Brazilian Central Amazon

Mosquitoes belonging to the Anopheles genus are of great relevance in the epidemiology and transmission of malaria, with their larval phase developing in clean waters in the presence of organic matter. However, the human presence in the Amazon has increasingly influenced the emergence of new breeding sites and larval habitats, such as clay pits, fish ponds and dams, among others. The objective of the study was to characterize mosquito larval habitats using the biotic and abiotic parameters in the metropolitan area of Manaus. We collected in 23 artificial larval habitats in Manaus, classified in dams, fish ponds and clay pits. Water samples, Anopheles larvae, aquatic macrophytes and limnological parameters were collected from each artificial larval habitat. The Larvae Index per Man/Hour and canonical correspondence analysis were used for data analysis. Results indicate that artificial larval habitats with characteristics similar to natural sites present higher larval density, displaying a high abundance of An. triannulatus and An. darlingi. More than 90% of the determined limnological parameters were in agreement with the environmental resolution stipulated by the Brazilian environmental resolution, while pH, dissolved oxygen and phosphorus levels were below the established limits at some of the larval habitats. Conductivity, total suspended solids and phosphorus were positively correlated to the presence of An. albitarsis, An. peryassui and An. nuneztovari in fish ponds, and An. trianulatus and An. braziliensis in dams. Thus, the evaluated limnological variables and habitat structure explain Anopheles species distribution in artificial larval habitats in the metropolitan Manaus region.     

Population genetic structure of the major malaria vector Anopheles darlingi (Diptera: Culicidae) from the Brazilian Amazon, using microsatellite markers

The population genetic structure of Anopheles darlingi, the major human malaria vector in the Neotropics, was examined using seven microsatellite loci from nine localities in central and western Amazonian Brazil. High levels of genetic variability were detected (5-25 alleles per locus; H E = 0.519-0.949). There was deviation from Hardy-Weinberg Equilibrium for 59.79% of the tests due to heterozygote deficits, while the analysis of linkage disequilibrium was significant for only two of 189 (1.05%) tests, most likely caused by null alleles. Genetic differentiation (F ST = 0.001-0.095; Nm = 4.7-363.8) indicates that gene flow is extensive among locations < 152 km apart (with two exceptions) and reduced, but not absent, at a larger geographic scale. Genetic and geographic distances were significantly correlated (R(2) = 0.893, P < 0.0002), supporting the isolation by distance (IBD) model. The overall estimate of Ne was 202.4 individuals under the linkage disequilibrium model, and 8 under the heterozygote excess model. Analysis of molecular variance showed that nearly all variation (approximately 94%) was within sample locations. The UPGMA phenogram clustered the samples geographically, with one branch including 5/6 of the state of Amazonas localities and the other branch the Acre, Rond?nia, and remaining Amazonas localities. Taken together, these data suggest little genetic structure for An. darlingi from central and western Amazonian Brazil. These findings also imply that the IBD model explains nearly all of the differentiation detected. In practical terms, populations of An. darlingi at distances < 152 km should respond similarly to vector control measures, because of high gene flow.     

Location of ribosomal genes in the chromosomes of Anopheles darlingi and Anopheles nuneztovari (Diptera,Culicidae) from the Brazilian Amazon

Fluorescence in situ hybridization of Anopheles darlingi and A. nuneztovari demonstrated nucleolar organizer region activity at the end of the fourth larval instar, when the nucleolar organizer regions underwent gradual condensation. The heteromorphic sex chromosomes showed intraindividual size variation in the rDNA blocks located in the pericentromeric region and this coincided with the location of constitutive heterochromatin (C-banding).     

Multiple blood meals in Anopheles darlingi (Diptera: Culicidae)

Anopheles darlingi is an important vector of human malaria in the Amazon. Adult females of this mosquito species require a blood meal to develop eggs, preferring humans to other blood sources. Although gonotrophic concordance has been described as the norm for An. darlingi, here we report An. darlingi female mosquitoes taking two or more blood meals within their first gonotrophic cycle. Only half of field‐captured adult females fed one blood meal developed follicles to Christophers' stage V. This outcome is dependent on larval nutrition, as 88% of laboratory‐raised well‐nourished females completed the first gonotrophic cycle with only one blood meal, while less nourished females needed additional blood meals. Half of the field‐captured blood‐seeking An. darlingi females had follicles in intermediate (IIIa and IIIb) and final (V) stages of the gonotrophic cycle, supporting the conclusion that An. darlingi blood feed more than once during a gonotrophic cycle. Additionally, we observed females attempting to blood feed a second time during the same day. Additional studies of An. darlingi biting behavior are necessary to accurately estimate Plasmodium sp. entomologic inoculation rates throughout the An. darlingi vast geographical distribution.     

Spatial expansion and population structure of the neotropical malaria vector, Anopheles darlingi (Diptera: Culicidae)

Extensive population structuring is known to occur in Anopheles darlingi , the primary malaria vector of the Neotropics. We analysed the phylogeographic structure of the species using the mitochondrial cytochrome oxidase I marker. Diversity is divided into six main population groups in South America: Colombia, central Amazonia, southern Brazil, south-eastern Brazil, and two groups in north-east Brazil. The ancestral distribution of the taxon is hypothesized to be central Amazonia, and there is evidence of expansion from this region during the late Pleistocene. The expansion was not a homogeneous front, however, with at least four subgroups being formed due to geographic barriers. As the species spread, populations became isolated from each other by the Amazon River and the coastal mountain ranges of south-eastern Brazil and the Andes. Analyses incorporating distances around these barriers suggest that the entire South American range of An. darlingi is at mutation–dispersal–drift equilibrium. Because the species is distributed throughout such a broad area, the limited dispersal across some landscape types promotes differentiation between otherwise proximate populations. Moreover, samples from the An. darlingi holotype location in Rio de Janeiro State are substantially derived from all other populations, implying that there may be additional genetic differences of epidemiological relevance. The results obtained contribute to our understanding of gene flow in this species and allow the formulation of human mosquito health protocols in light of the potential population differences in vector capacity or tolerance to control strategies.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 854–866.     

Parity and age composition for Anopheles darlingi root (Diptera: Culicidae) and Anopheles albitarsis Lynch-Arribálzaga (Diptera: Culicidae) of the northern Amazon Basin, Brazil.

Parity and age composition for Anopheles darlingi and Anopheles albitarsis in the northern Amazon Basin, Brazil, were investigated. Anopheline ovaries and ovarioles were examined in order to determine whether hourly and seasonal parity status for the vectors An. albitarsis and An. darlingi would vary in two different landscapes (forest and savanna/forest) where malaria is endemic in the northern Amazon Basin. A total of 1,199 anophelines (535 An. darlingi and 664 An. albitarsis) was dissected for parity status, ovariole dilatations, and follicular stages. The total number of nulliparous and parous females for both species varied by time of collection, locality, and season. During the rainy season for the first two h of collection, more nulliparous An. albitarsis and An. darlingi females were collected in the first hour (18:00-19:00), but during the second hour (19:00-20:00) more parous females of both species were captured. During the dry season in Copaíbas, more parous females of An. albitarsis were observed in the first hour while more nulliparous females were observed in the second hour. Nulliparous and parous females of both species for both hours were not significantly different at Road 19 in the dry season. This location was characterized by a forest malaria pattern of transmission with higher numbers of parous females and population stability in the dry season. In Copaíbas, the density and parity of An. darlingi increased during the rainy season, and it could be classified as an alluvial malaria pattern of transmission. For Copaíbas, control measures would be more successful if adopted at the transition from dry to rainy season. Further investigation on longitudinal spatio-temporal change in longevity and survival rates would help us to clarify differences in vector competence for An. darlingi and An. albitarsis and add to the understanding of differences regarding prevailing landscapes in malaria epidemiology in the northern Amazon Basin.     

Karyotype of Brazilian Anopheles albitarsis sensu lato (Diptera:Culicidae)

Anopheles (Nyssorhynchus) albitarsis sensu lato is an important malaria vector in Brazil, especially in the Brazilian Amazon region. Chromosome preparations of fourth-instar larvae of A. albitarsis from Iranduba and Coari (AM) and Ilha Comprida (SP) were analyzed for karyotype determination and to improve cytogenetic identification of this species. Anopheles albitarsis possesses 2n = 6 chromosomes, with two pairs (submetacentric and metacentric) of autosomes and one pair of sex chromosomes, with X-Y dimorphism. The sex pair is homomorphic and acrocentric in females and heteromorphic in males, with a punctiform Y chromosome. Somatic pairing was detected in the prometaphase and metaphase chromosomes of the three A. albitarsis populations. Apparently, sex chromosome evolution in the Culicidae does not function as does evolution in the Culicidae, since it occurs in the subfamily Anophelinae, which possesses heteromorphic sex chromosomes and is regarded as primitive, based on several criteria. These karyotype data on the albitarsis complex reinforce the hypothesis that sex chromosome evolution in the subfamily Anophelinae is conserved, and the variation revealed in the mean size of chromosomes in three populations indicates that selective pressure in these populations is occurring only at a genetic level.     

piggyBac-mediated germline transformation of the malaria mosquito Anopheles sinensis (Diptera: Culicidae)

Dear Editor, Anopheles sinensis is the primary vector of vivax malaria in China.Given the risks posed by An.sinen-sis,many methods have been used to control thi...     

Spatial clustering and longitudinal variation of Anopheles darlingi (Diptera: Culicidae) larvae in a river of the Amazon: the importance of the forest fringe and of obstructions to flow in frontier malaria

Deforestation has been linked to a rise in malaria prevalence. In this paper, we studied longitudinally 20 spots, including forested and deforested portions of a temporary river in a malarigenous frontier zone. Larval habitat parameters influencing distribution of Anopheles darlingi (Diptera: Culicidae) larvae were studied. We observed that larvae were clustered in forested-deforested transitions. For the first time in the literature, it was verified that parameters determining larval distribution varied from deforested to forested areas. The proximity to human dwellings was also a significant factor determining distribution, but larvae was most importantly associated with a previously undescribed parameter, the presence of small obstructions to river flow, such as tree trunks within the river channel, which caused pooling of water during the dry season ('microdams'). In deforested areas, the most important factor determining distribution of larvae was shade (reduced luminance). Larvae were absent in the entire studied area during the wet season and present in most sites during the dry season. During the wet-dry transition, larvae were found sooner in areas with microdams, than in other areas, suggesting that flow obstruction prolongs the breeding season of An. darlingi. Adult mosquito densities and malaria incidence were higher during the dry season. Our data correlate well with the published literature, including the distribution of malaria cases near the forest fringes, and has permitted the creation of a model of An. darlingi breeding, where preference for sites with reduced luminance, human presence and microdams would interact to determine larval distribution.     

Isolation of polymorphic microsatellite markers from the malaria vector Anopheles marajoara (Diptera: Culicidae)

Microsatellite‐containing regions were isolated and characterized in Anopheles (Nyssorhynchus) marajoara, a primary vector of malaria parasites in northeastern Amazonia, Brazil. An enrichment protocol yielded 500 positive clones. We designed primers to amplify 40 unique microsatellites, 11 of which amplified cleanly and were polymorphic. A survey of 323 individuals showed that these loci are highly variable with the number of alleles ranging from 11 to 52, and expected heterozygosity ranging from 0.64 to 0.95. These markers will be useful for studies of population structure and intraspecific variation in A. marajoara.     

Heat shock response during development of the malaria vector Anopheles stephensi (Culicidae: Diptera)

The pattern of synthesis of heat shock proteins (HSP) and thermotolerance to elevated temperatures during the development of the malaria vector Anopheles stephensi normally reared at 28 +/- 2 degrees C was studied using SDS-PAGE. In total twelve heat shock proteins (i.e. 31, 33, 38, 43, 44, 51, 57, 62, 69, 71, 113 and 121 kD were induced by heat shock during various stages of development. Eight polypeptides (HSP during one or other of the instars) appeared during normal development of the adult, which showed very little response towards heat shock. Only two polypeptides (57 and 69 kD) were induced while the 22.5 kD protein disappeared during adult life. The HSP 62 and 71 kD induced during the larval stages showed a sharp decline in quantity in male and female adults upon heat shock. Three HSP (31, 43 and 44 kD) were induced in pupae due to heat shock. The synthesis of HSP in A. stephensi was correlated with the various morphological and physiological events occurring during development.     

Role of Anopheles (Kerteszia) bellator as malaria vector in southeastern Brazil (Diptera: Culicidae)

New research concerning Anopheles bellator in the southeast of the State of S?o Paulo, Brazil, are reported. Adult females of this mosquito showed remarkable endophily and endophagy which was even greater than An. cruzii. The epidemiological role of this anopheline as a malaria vector is discussed.     

Asymptomatic Plasmodium vivax malaria in the Brazilian Amazon: Submicroscopic parasitemic blood infects Nyssorhynchus darlingi

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria.     

Molecular comparison of topotypic specimens confirms Anopheles (Nyssorhynchus) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.     

Biting activity and host preference of the malaria vectors Anopheles maculatus and Anopheles sawadwongporni (Diptera: Culicidae) in Thailand

Adult mosquitoes in the Anopheles maculatus group were surveyed from different regions of Thailand and five different species were morphologically identified, including Anopheles maculatus, Anopheles sawadwongporni, Anopheles notanandai, Anopheles dravidicus, and Anopheles willmori. Blood‐feeding activity and host preference of two species, Anopheles maculatus and Anopheles sawadwongporni, were observed during a one‐year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, west‐central Thailand. Both species were more prevalent during the wetter period of the year and each had a greater predilection to feed on cattle than humans. Primary feeding activity occurred between 20:00‐23:00 and a smaller peak at 01:00‐03:00. Findings are discussed relative to the importance of these two vectors for malaria transmission in Pu Teuy.     

Effect of acute and chronic exposure to ammonia on different larval instars of Anopheles darlingi (Diptera: Culicidae)

Anopheles darlingi (Diptera: Culicidae) is the most important vector of malaria in South America and has already been found in peri‐urban areas that commonly contain toxic nitrogenous compounds, such as ammonia. The adaptation of mosquitoes to polluted breeding sites can increase their distribution and affect the dynamics of vector‐borne diseases such as malaria. Therefore, the present study investigated the tolerance of larval instars of An. darlingi to ammonia under acute and chronic exposure conditions. Anopheles darlingi larval mortality, development time, and pupal and adult production using larvae of the 1^st (L1) and 3^rd (L3) instar were assessed as both acute and chronic effects of exposure to different concentrations of ammonia. Lethal concentrations (LCs) for L1 larvae were lower than LCs for L3 larvae. In general, higher ammonia concentrations caused an increase in larval mortality, especially in chronically exposed L1 larvae. The larval development time in L1 and L3 was longer with chronic treatment and decreased with increasing concentrations of ammonia. The number of pupae was very low for acutely exposed L1 and L3 larvae. Likewise, the probability of adult production decreased with increasing ammonia concentrations. This is the first report on the tolerance of An. darlingi to pollutants.     

A new species of Anopheles (Anopheles) from Namibia (Diptera: Culicidae)

Abstract. The adult, pupa, larva and egg of Anopheles {Anopheles) namibien-sis sp.n. from the Kavango district, Namibia (South West Africa), are described and comparisons made with Anopheles (Anopheles) ziemanni Griinberg. A photomap of the polytene chromosomes from the salivary glands of the fourth stage larvae is presented.     

Development of Exon-Primed Intron-Crossing (EPIC) PCR primers for the malaria vector Anopheles pseudopunctipennis (Diptera: Culicidae)

Using the Anopheles gambiae Giles genome as a template, we designed, screened and identified 14 novel Exon-Primed Intron-Crossing (EPIC) PCR primer pairs for Anopheles pseudopunctipennis Theobald 1901, a major vector of human Plasmodium sp. in South America. These primers were designed to target the conserved regions flanking consecutive exons of different genes and enabled the amplification of 17 loci of which nine were polymorphic. Polymorphisms at these loci ranged from two to four alleles. Intron length polymorphism analysis is a useful tool, which will allow the study of the population structure of this mosquito species, which remains poorly understood.     

Role of species composition in malaria transmission by the Anopheles funestus group (Diptera: Culicidae) in Ghana

Malaria remains a public health problem in Ghana, with Anopheles gambiae and Anopheles funestus as the predominant vectors. While much information exists on the species composition of An. gambiae, very little exists for An. funestus. This study was carried out to determine the species composition of An. funestus Giles populations from three ecological areas in Ghana and investigate their role in malaria transmission. Mosquitoes were collected using human landing and pyrethrum spray methods. A total of 10,254 Anopheles individuals were collected, out of which An. funestus constituted 53.6% (5,496). An. funestus sensu stricto (s.s.) and Anopheles lessoni were identified as the only members of the An. funestus group in all three ecological areas. All 62 sporozoite positive specimens that were identified as An. funestus s.s. were highly anthropophilic with a human blood index in the range of 80–96%, whereas more than 83% of the An. leesoni had fed on either bovine, goat, or sheep. Malaria transmission was higher in the Sahel savannah area than the rest of the ecological zones, with An. funestus s.s. being implicated as a vector of malaria in all ecological zones. Anopheles leesoni occurred in all the ecological areas but played no role in malaria transmission. The study established the importance of An. funestus s.s. in malaria transmission in Ghana.