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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Genetic differentiation of the malaria vector Anopheles gambiae across Nigeria suggests that selection limits gene flow.

Gene flow was investigated in Anopheles gambiae from eight localities that span the ecological zones of Nigeria (arid savanna zones in the north gradually turn into humid forest zones in the south). Genetic differentiation was measured over 10 microsatellite loci and, to determine any effects of selection, five loci were located within chromosome inversions and the other five were outside inversions. Over all loci, the largest estimates of differentiation were in comparisons between localities in the savanna vs. forest zones (range FST 0.024-0.087, Nm 2.6-10.1; RST 0.014-0.100, Nm 2.2-16.4). However, three loci located within inversions on chromosome II, whose frequencies varied clinically from north to south, were responsible for virtually all of the differentiation. When the three loci were removed, genetic distances across the remaining seven loci were markedly reduced even between localities in the forest and savanna zones (range FST 0.001-0.019, Nm 12.7-226.1) or no longer significant (P > 0.05) in the case of RST. Although tests of isolation by distance gave seemingly equivocal results, geographical distance does not appear to limit gene flow. These observations suggest that gene flow is extensive across the country but that selection on genes located within some inversions on chromosome II counters the homogenizing effects of gene flow.

Molecular and morphological phylogenetic analysis of an insular radiation in Pacific black flies (Simulium).

Ecological adaptation within islands may have figured prominently in the insular radiation of black flies (subgenus Inseliellum) in the Society Islands, French Polynesia. To aid in understanding the sequence of ecological shifts in this group, we have constructed a phylogeny by using morphology, the cytochrome oxidase I (COI) gene, and the small ribosomal subunit (12S) gene. The strong influence of COI on the combined analysis tree was evident from its contribution to the partitioned Bremer support (62%). The net effect of including 12S was to reduce overall tree support. Different character sets resolved different portions of the combined analysis tree, with COI resolving recent lineages, 12S resolving basal relationships, and morphology supporting the monophyly of taxa having smaller larval feeding fans (oviceps group). The Partition Homogeneity and Kashino-Hasegawa tests indicated significant incongruence between morphological and mitochondrial data. The Templeton test revealed that morphology and the combined (COI + 12S) mitochondrial data were incongruent. This conflict stems primarily from disagreement over the monophyly of taxa having much smaller larval feeding fans. Either convergence in a subset of morphological characters, low phylogenetic signal among mitochondrial sequences, or lineage-sorting causing the mitochondrial data to track an incorrect evolutionary history may be responsible for these results.

The distribution of two major malaria vectors, Anopheles gambiae and Anopheles arabiensis, in Nigeria.

The distribution of Anopheles gambiae and An. arabiensis across the ecological zones of Nigeria (arid savanna in the north gradually turns into humid forest in the south) was investigated. Results of the present study were compared to the distributions determined from samples of indoor-resting females reported by an earlier study over 20 years ago. Larvae were sampled in the rainy seasons of 1997 and 1999 from 24 localities, 10 of which were sampled in both years. Specimens were identified by the polymerase chain reaction method. Results showed that species composition changed significantly among the 10 localities in both years (chi2=13.62, P = 0.0002), but this change was significant in only four of the 10 localities. The identity of the prevalent (more abundant) species changed between 1997 and 1999 in only three of 10 localities. An. arabiensis was prevalent in several localities in the southern Guinea savanna, an area where it was virtually absent over 20 years ago. The data suggest that An. arabiensis has extend its range, although differences in sampling technique (larval sampling versus adult collection) can not be ruled out as a possible explanation.

Population genetic structure of the malaria mosquito Anopheles arabiensis across Nigeria suggests range expansion.

Ten microsatellite loci, four located within and six outside chromosome inversions, were employed to study the genetic structure of Anopheles arabiensis across the ecological zones of Nigeria (arid savannah in the north gradually turns into humid forest in the south). Regardless of location within or outside inversions, genetic variability at all loci was characterized by a reduction in both the number of alleles per locus and heterozygosity from savannah to forest. Across all loci, all but one allele in the forest also occurred in the savannah, whereas at least 78 alleles in the savannah were missing in the forest. Genetic differentiation increased with geographical distance; consequently, genetic distances between zones exceeded those within zones. The largest genetic distances were between localities at the extremes of the transect (range F(ST) = 0.196-0.258 and R(ST) = 0.183-0.468) and were as large as those between A. arabiensis and Anopheles gambiae s.s. Gene flow across the country was very low, so that Nm between the extremes of the transect was < 1. These data suggest that A. arabiensis has extended its range from the savannah into the forest during which it experienced a reduction in effective population size due to sequential founder effects. Gene flow post range expansion appears too restricted by geographical distance to homogenize the gene pool of A. arabiensis across Nigeria.

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.