Regional variation in life history traits and plastic responses to temperature of the major malaria vector Nyssorhynchus darlingi in Brazil.

The primary Brazilian malaria vector, Nyssorhynchus darlingi (formerly Anopheles darlingi), ranges from 0°S-23°S across three biomes (Amazonia, Cerrado, Mata Atlântica). Rising temperatures will increase mosquito developmental rates, and models predict future malaria transmission by Ny. darlingi in Brazil will shift southward. We reared F1 Ny. darlingi (progeny of field-collected females from 4 state populations across Brazil) at three temperatures (20, 24, 28 °C) and measured key life-history traits. Our results reveal geographic variation due to both genetic differences among localities and plastic responses to temperature differences. Temperature significantly altered all traits: faster larval development, shorter adult life and overall lifespan, and smaller body sizes were seen at 28 °C versus 20 °C. Low-latitude Amazonia mosquitoes had the fastest larval development at all temperatures, but at 28 °C, average development rate of high-latitude Mata Atlântica mosquitoes was accelerated and equivalent to low-latitude Amazonia. Body size of adult mosquitoes from the Mata Atlântica remained larger at all temperatures. We detected genetic variation in the plastic responses among mosquitoes from different localities, with implications for malaria transmission under climate change. Faster development combined with larger body size, without a tradeoff in adult longevity, suggests vectorial capacities of some Mata Atlântica populations may significantly increase under warming climates.

Species composition and distribution of adult Anopheles (Diptera: Culicidae) in Panama.

Anopheles (Diptera: Culicidae) species composition and distribution were studied using human landing catch data over a 35-yr period in Panama. Mosquitoes were collected from 77 sites during 228 field trips carried out by members of the National Malaria Eradication Service. Fourteen Anopheles species were identified. The highest average human biting rates were recorded from Anopheles (Nyssorhynchus) albimanus (Wiedemann) (9.8 bites/person/night) and Anopheles (Anopheles) punctimacula (Dyar and Knab) (6.2 bites/person/night). These two species were also the most common, present in 99.1 and 74.9%, respectively, of the sites. Anopheles (Nyssorhynchus) aquasalis (Curry) was encountered mostly in the indigenous Kuna Yala Comarca along the eastern Atlantic coast, where malaria case history and average human biting rate (9.3 bites/person/night) suggest a local role in malaria transmission. An. albimanus, An. punctimacula, and Anopheles (Anopheles) vestitipennis (Dyar and Knab) were more abundant during the rainy season (May-December), whereas An. aquasalis was more abundant in the dry season (January-April). Other vector species collected in this study were Anopheles (Kerteszia) neivai (Howard, Dyar, and Knab) and Anopheles (Anopheles) pseudopunctipennis s.l. (Theobald). High diversity of Anopheles species and six confirmed malaria vectors in endemic areas of Panama emphasize the need for more detailed studies to better understand malaria transmission dynamics.

Molecular differentiation in natural populations of Anopheles oswaldoi sensu lato (Diptera: Culicidae) from the Brazilian Amazon, using sequences of the COI gene from mitochondrial DNA.

Anopheles (Nyssorhynchus) oswaldoi (Peryassú, 1922) s. l., which has been incriminated as a potential human malaria vector in Western Brazilian Amazon, may constitute a cryptic species complex. However, the most recent study with isozymes indicated high similarity among samples from the States of Acre, Amazonas and Rondônia in the Brazilian Amazon. In the present study, 45 individuals were sequenced from Sena Madureira (State of Acre), Coari (State of Amazonas), São Miguel (State of Rondônia), and Moju (State of Pará), using the cytochrome oxidase I gene from mitochondrial DNA. Twenty-five haplotypes were identified in the four localities, and no haplotype was shared among them. The lowest haplotype number was detected in the Coari sample. The dendrogram based on maximum parsimony analysis yielded four groups: I) haplotypes 1, 2, 3, 4, and 5 from Sena Madureira and haplotypes 17 and 18 from São Miguel; II) haplotypes 13 to 16 and 19 to 22 from São Miguel; III) haplotypes 23 to 25 from Moju, and IV) haplotypes 6 to 9 from Sena Madureira and haplotypes 10 to 12 from Coari. The genetic distance (uncorrected p) obtained among the four groups ranged from 0.08 to 5.3%, whereas the highest values (4.97 to 5.3%) were found between groups I (Sena Madureira) and III (Moju). Based on male genitalia identification, it was suggested that group I may be A. oswaldoi s. s. whereas group IV may be A. konderi. Groups II and III could constitute other lineages or species within A. oswaldoi s. l., whose taxonomic status remains to be clarified. These results suggest that additional studies are necessary using samples of A. oswaldoi s. l. from a larger geographic area.

Intragenomic heterogeneity of internal transcribed spacer rDNA in neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae).

Intragenomic heterogeneity of the internal transcribed spacer (ITS) array was investigated in Anopheles aquasalis Curry mosquitoes from two geographic locations in each of Brazil and Venezuela, and one in Suriname. Polymerase chain reaction-amplified copies of the ITS were cloned and sequenced. The length of the entire array ranged from 782 to 990 bp, with most variation due to microsatellite insertions in ITS1. We detected 40 different ITSL sequences and 15 different ITS2 sequences of the 71 to 72 clones examined. The sequence divergence within localities ranged from 0.002 to 0.043 for ITS1 and from 0 to 0.006 for ITS2. Point mutations were common to both spacer regions, but dinucleotide microsatellite repeats were restricted to ITS1. Sequences from neither ITS1 nor ITS2 had a diagnostic distribution or were informative in distinguishing these populations, providing additional support for the status of An. aquasalis as a single species.

Cryptic Species in the Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) Complex: Incongruence Between Random Amplified Polymorphic DNA-Polymerase Chain Reaction Identification and Analysis of Mitochondrial DNA COI Gene Sequences.

Random amplified polymorphic DNA (RAPD) diagnostic bands are one tool used to differentiate cryptic mosquito species in the Anopheles albitarsis Complex. Monophyly of four species (A. albitarsis Lynch-Arribálzaga, A. albitarsis B, A. deaneorum Rosa-Freitas, and A. marajoara Galvão & Damasceno) currently identified with the RAPD technique was assessed using sequences of the cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) gene. Maximum parsimony, maximum likelihood, and Bayesian analyses support monophyly for A. albitarsis s.s., A. albitarsis B, and A. deaneorum. Anopheles marajoara, as identified by RAPD banding patterns, was either polyphyletic or paraphyletic in all phylogenetic analyses. The phylogenetic pattern and within-species genetic distances observed in A. marajoara suggest the existence of a previously unidentified species (species E) in northern Brazil and Venezuela. Diagnostic RAPD bands were unable to distinguish between A. marajoara and species E, probably because of the low number of correlated bands used to identify species and weaknesses of the RAPD technique, in particular, violations of the untested assumption of homology of comigrating bands. A. marajoara (even without species E) is paraphyletic with respect to A. deaneorum; if A. deaneorum is a separate species from A. marajoara, then A. marajoara may consist of two or more species in Amazonian Brazil. Based on mtDNA COI sequences, there are at least four phylogenetic species within the Albitarsis Complex: A. albitarsis s.s., A. albitarsis B, A. marajoara, and species E; the species status of A. deaneorum is ambiguous.

Population structure of the primary malaria vector in South America, Anopheles darlingi, using isozyme, random amplified polymorphic DNA, internal transcribed spacer 2, and morphologic markers.

A genetic and morphologic survey of Anopheles darlingi populations collected from seven countries in Central and South America was performed to clarify the taxonomic status of this major malaria vector species in the Americas. Population genetics was based on three techniques including isozyme, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), and internal transcribed spacer 2 (ITS2) markers. The results of the isozyme analysis indicated moderate differences in the allele frequencies of three putative loci (glutamate oxalaoacetate transaminase-1, isocitrate dehydrogenase-1, and phosphoglucomutase) of the 31 analyzed. No fixed electromorphic differences separated the populations of An. darlingi, which showed little genetic divergence (Nei distances = 0.976-0.995). Fragments produced by RAPD-PCR demonstrated evidence of geographic partitioning and showed that all populations were separated by small genetic distances as measured with the 1 - S distance matrix. The ITS2 sequences for all samples were identical except for four individuals from Belize that differed by a three-base deletion (CCC). The morphologic study demonstrated that the Euclidean distances ranged from 0.02 to 0.14, with the highest value observed between populations from Belize and Bolivia. Based on these analyses, all the An. darlingi populations examined demonstrated a genetic similarity that is consistent with the existence of a single species and suggest that gene flow is occurring throughout the species' geographic range.

The distribution of M and S molecular forms of Anopheles gambiae in Nigeria.

The distribution of M and S molecular forms of Anopheles gambiae sensu stricto across Nigeria was determined. The molecular form of 40 to 45 specimens per locality from 9 localities was determined using mostly the same specimens from our recent study of genetic differentiation of A. gambiae across Nigeria (Onyabe & Conn, 2001). These samples were previously genotyped at 10 microsatellite loci, 5 located within chromosome inversions and 5 outside inversions. Both molecular forms occurred throughout the country, with no apparent relationship to the ecological transition from dry savannah in the north to humid forest in southern Nigeria. In all localities, however, 1 form or the other occurred virtually exclusively. No hybrids between forms were found. Across all loci, F(ST) values were as high within molecular forms as between forms. Regardless of molecular form, F(ST) values calculated across loci within inversions were much higher (range 0.0016 to 0.1988) than those calculated across loci outside inversions (range -0.0035 to 0.0260). Genetic distance was not significantly correlated with geographical distance within either form (P> 0.05). These observations suggest that, in addition to partial reproductive barriers between molecular forms, selection is a major factor shaping genetic differentiation of A. gambiae across Nigeria.

Evaluation of the Amazon River delta as a barrier to gene flow for the regional malaria vector, Anopheles aquasalis (Diptera: Culicidae) in northeastern Brazil.

The Neotropical malaria vector Anopheles aquasalis Curry is distributed predominantly along the Atlantic and Pacific Coasts because of its tolerance for breeding in salt water. We tested the hypothesis that the freshwater Amazon River acts as a barrier to gene flow in northeastern Brazil, by examining variation at a 588-nucleotide fragment of the mitochondrial cytochrome oxidase Igene from five populations. We identified 15 haplotypes of which 5 were shared both (1) between sample localities and (2) across the Amazon River Delta. Sequence divergence ranged from 0.0017-0.0272 (average = 0.0137). Estimates of genetic subdivision based on the presence of the Amazon Riverwere greatest within localities (phi = 0.029) and among regions (phi = 0.018), followed by among localities (phi = 0.011), but none were significant. Parsimony, neighbor-joining, and Nested Clade Analyses were used to estimate relationships among populations and infer evolutionary processes. Two phylogenetically distinct clusters of populations were moderately supported by parsimony. Neighbor-joining trees were poorly resolved, thus providing no geographical resolution and no support for the Amazon River as a barrier to migration. Phylogeographic structure as detected by the Nested Clade Analysis was consistent with restricted gene flow coupled with isolation by distance. Taken together, these analyses suggest that the localities within this region of northeastern Brazil constitute a single large population of An. aquasalis that spans the Amazon Delta.

Effects of local geographic barriers and latitude on population structure in Anopheles punctipennis (Diptera: Culicidae).

We sampled Anopheles punctipennis (Say) from 11 localities throughout Vermont to examine the effects of latitude and two local geographical boundaries, Lake Champlain and the Green Mountains, on the population genetic structure of this species. Thirty-five mitochondrial haplotypes were detected in 104 individuals using a variable region of the COI gene. When latitude was examined, we detected significant structure within localities and among localities within latitudinal regions. For geographic analysis, significant genetic structure was detected only within localities. Estimates of gene flow across geographic regions indicate that the Green Mountains, but not Lake Champlain, is a barrier to dispersal for this species. We found no correlation between genetic and geographic distances for An. punctipennis.

Morphological, molecular, and chromosomal discrimination of cryptic Anopheles (Nyssorhynchus) (Diptera: Culicidae) from South America.

Based on similarity of male genitalia, the malaria vector Anopheles trinkae Faran from the eastern Andean piedmont of Colombia, Ecuador, Peru, and Bolivia was determined by Peyton (1993) to be a junior synonym of An. dunhami Causey, then known from a single locality in Amazonian Brazil. Following an appraisal of molecular, chromosomal, and morphological characters, we conclude herein that the 2 taxa are specifically distinct and remove An. trinkae from synonymy with An. dunhami. Eggs of the 2 species are distinguished easily by the anterior crown, long floats, and closed deck that occur only in An. trinkae. The X chromosome of larval polytenes is divisible into R and L arms in An. dunhami, but not in An. trinkae. A phenogram based on banding pattern scores from 18 random amplified polymorphic DNA primers separated with 100% resolution An. dunhami, An. trinkae, Anopheles nuneztovari Gabaldón and Anopheles darlingi Root. In the ITS2 region of rDNA, 25% of base sites distinguished An. trinkae from An. dunhami and 21% from the related An. nuneztovari; males of these 3 species had accessory glands of significantly different sizes. Preliminary isoenzyme screening indicated that 3 of 11 loci were diagnostic for separating An. trinkae from An. dunhami. The results indicate that An. dunhami is related more closely to An. nuneztovari than to An. trinkae and illustrate the merits of a multidisciplinary approach to mosquito systematics.

Molecular population genetics of the primary neotropical malaria vector Anopheles darlingi using mtDNA.

Samples of the neotropical malaria vector Anopheles darlingi from Bolivia, Brazil, and Venezuela were analyzed to test for differences in mitochondrial haplotype frequencies. With the use of molecular variance components and F-statistics, significant genetic variability of An. darlingi was found apportioned primarily among populations within regions or within populations, with regions defined either as biomes (n = 5) or ecoregions (n = 2). The Mantel analysis resulted in a significant correlation [Prob (r) = 0.009] between genetic and geographic distances, evidence that these populations are genetically isolated by distance. Such isolation could reflect differences in phenotypes for factors affecting vector capacity.

Review of genetic diversity in malaria vectors (Culicidae: Anophelinae).

We review previous studies on the genetic diversity of malaria vectors to highlight the major trends in population structure and demographic history. In doing so, we outline key information about molecular markers, sampling strategies and approaches to investigate the causes of genetic structure in Anopheles mosquitoes. Restricted gene flow due to isolation by distance and physical barriers to dispersal may explain the spatial pattern of current genetic diversity in some Anopheles species. Nonetheless, there is noteworthy disagreement among studies, perhaps due to variation in sampling methodologies, choice of molecular markers, and/or analytical approaches. More refined genealogical methods of population analysis allowing for the inclusion of the temporal component of genetic diversity facilitated the evaluation of the contribution of historical demographic processes to genetic structure. A common pattern of past unstable demography (i.e., historical fluctuation in the effective population size) by several Anopheles species, regardless of methodology (DNA markers), mosquito ecology (anthropophilic vs zoophilic), vector status (primary vs secondary) and geographical distribution, suggests that Pleistocene environmental changes were major drivers of divergence at population and species levels worldwide.

Population analysis using the nuclear white gene detects Pliocene/Pleistocene lineage divergence within Anopheles nuneztovari in South America.

Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae), a locally important malaria vector in some regions of South America, has been hypothesized to consist of at least two cryptic incipient species. We investigated its phylogeographic structure in several South American localities to determine the number of lineages and levels of divergence using the nuclear white gene, a marker that detected two recently diverged genotypes in the primary Neotropical malaria vector Anopheles darlingi Root. In An. nuneztovari, five distinct lineages (1-5) were elucidated: (1) populations from northeastern and central Amazonia; (2) populations from Venezuela east and west of the Andes; (3) populations from Colombia and Venezuela west of the Andes; (4) southeastern and western Amazonian Brazil populations, and (5) southeastern and western Amazonian Brazil and Bolivian populations. There was a large amount of genetic differentiation among these lineages. The deepest and earliest divergence was found between lineage 3 and lineages 1, 2 and 4, which probably accounts for the detection of lineage 3 in some earlier studies. The multiple lineages within Amazonia are partially congruent with previous mtDNA and ITS2 data, but were undetected in many earlier studies, probably because of their recent (Pleistocene) divergence and the differential mutation rates of the markers. The estimates for the five lineages, interpreted as recently evolved or incipient species, date to the Pleistocene and Pliocene. We hypothesize that the diversification in An. nuneztovari is the result of an interaction between the Miocene/Pliocene marine incursion and Pleistocene climatic changes leading to refugial isolation. The identification of cryptic lineages in An. nuneztovari could have a significant impact on local vector control measures.

The biogeography and population genetics of neotropical vector species.

Phylogenetic and population genetic data support the Pliocene or Pleistocene divergences of the co-distributed hematophagous insect vectors, the sand fly Lutzomyia longipalpis s.l., the mosquitoes Anopheles darlingi and A. albitarsis s.l., and the triatomines Rhodnius prolixus and R. robustus. We examined patterns of divergence and distribution in relation to three hypotheses of neotropical diversification: Miocene/Pliocene marine incursion, Pliocene/Pleistocene riverine barriers and Pleistocene refugia. Only R. prolixus has a pattern concordant with the refugia hypothesis, and R. robustus conforms to the marine incursion predictions. A. darlingi partially fits the refugia hypothesis. For L. longipalpis s.l. and A. albitarsis s.l., elements of both incursion and refugia hypotheses seem to fit, suggesting perhaps an interaction of factors determining their distribution patterns.

Genetic variation tracks ecological segregation in Pacific island black flies.

Geographic isolation is widely viewed as a key component of insular radiations on islands. However, strong ecological affinities may also reinforce isolation and promote genetic divergence. The black fly fauna in the Society Islands French Polynesia is notable for the number of closely related endemic species (31), and the morphological and habitat diversity of the larvae. Here, we measure ecological and morphological differences within and between two closely related species, Simulium oviceps and Simulium dussertorum and relate these differences to genetic distance. Phylogenetic analyses of a 920 bp fragment of the cytochrome oxidase I (COI) gene revealed a well-supported, ecologically divergent S. oviceps clade (larvae found in rivers instead of cascades) that shows little morphological differentiation. For both S. oviceps and S. dussertorum, genetic distance among populations is related to larval habitat, with cascade populations showing greater isolation from each other than river populations. Our data support the hypothesis that larval ecological shifts have played a role in the radiation of this black fly fauna.

Evidence for late Pleistocene population expansion of the malarial mosquitoes, Anopheles arabiensis and Anopheles gambiae in Nigeria.

Anopheles gambiae Giles s.s. and Anopheles arabiensis Patton (Diptera: Culicidae) are major vectors of malaria in Nigeria. We used 1115 bp of the mitochondrial COI gene to assess their population genetic structures based on samples from across Nigeria (n = 199). The mtDNA neighbour-joining tree, based on F(ST) estimates, separated An. gambiae M and S forms, except that samples of An. gambiae M from Calabar clustered with all the An. gambiae S form. Anopheles arabiensis and An. gambiae could be combined into a single star-shaped, parsimonious haplotype network, and shared three haplotypes. Haplotype diversity values were high in An. arabiensis and An. gambiae S, and intermediate in An. gambiae M; all nucleotide diversities were relatively low. Taken together, patterns of haplotype diversity, the star-like genealogy of haplotypes, five of seven significant neutrality tests, and the violation of the isolation-by-distance model indicate population expansion in An. arabiensis and An. gambiae S, but the signal was weak in An. gambiae M. Selection is supported as an important factor shaping genetic structure in An. gambiae in Nigeria. There were two geographical subdivisions in An. arabiensis: one included all southern localities and all but two central localities; the other included all northern and two central localities. Re-analysing an earlier microsatellite dataset of An. arabiensis using a Bayesian method determined that there were two distinctive clusters, northern and southern, that were fairly congruent with the mtDNA subdivisions. There was a trend towards decreasing genetic diversity in An. arabiensis from the northern savannah to the southern rainforest that corroborated previous data from microsatellites and polytene chromosomes.

Amazonian malaria: asymptomatic human reservoirs, diagnostic challenges, environmentally driven changes in mosquito vector populations, and the mandate for sustainable control strategies

Across the Americas and the Caribbean, nearly 561,000 slide-confirmed malaria infections were reported officially in 2008. The nine Amazonian countries accounted for 89% of these infections; Brazil and Peru alone contributed 56% and 7% of them, respectively. Local populations of the relatively neglected parasite Plasmodium vivax, which currently accounts for 77% of the regional malaria burden, are extremely diverse genetically and geographically structured. At a time when malaria elimination is placed on the public health agenda of several endemic countries, it remains unclear why malaria proved so difficult to control in areas of relatively low levels of transmission such as the Amazon Basin. We hypothesize that asymptomatic parasite carriage and massive environmental changes that affect vector abundance and behavior are major contributors to malaria transmission in epidemiologically diverse areas across the Amazon Basin. Here we review available data supporting this hypothesis and discuss their implications for current and future malaria intervention policies in the region. Given that locally generated scientific evidence is urgently required to support malaria control interventions in Amazonia, we briefly describe the aims of our current field-oriented malaria research in rural villages and gold-mining enclaves in Peru and a recently opened agricultural settlement in Brazil.(AU)

Molecular population genetics of the malaria vector Anopheles darlingi in Central and South America.

To analyze the genetic relatedness and phylogeographic structure of Anopheles darlingi from 19 localities throughout Central and South America, we used a minimum spanning network, diversity measures, differentiation, neutrality tests, and mismatch distribution with mitochondrial cytochrome oxidase subunit I (COI) sequences. All the Central American haplotypes were separated by seven mutational steps from the South American haplotypes and the FST distance-based neighbor-joining tree showed a primary division between Central and South America, evidence for a putative gene pool division. More ancestral and diverse haplotypes were found in Amazonian and southern Brazil populations, suggesting that Central American populations may have originated in South America. The patterns of the mtDNA haplotype diversity and five of six tests for equilibrium implicate demographic expansion in the South American populations as the historical structure, but mismatch distribution depicts populations at mutation drift equilibrium (MDE). In South America, the departure from equilibrium was consistent with an expansion that occurred during the Pleistocene.