Population genetic structure of the malaria vector Anopheles minimus A in Vietnam

Anopheles minimus A, a major malaria vector in Southeast Asia, is the main target of vector control in this area. The impact of these control measures can be influenced by the population structure of the target species. In rural areas, An. minimus breeds along the banks of small clear-water streams, yet in the suburbs of Hanoi, northern Vietnam, there is an An. minimus population whose immature stages develop in water tanks. This study uses allozyme data (1) to assess the population structure of An. minimus A and (2) to evaluate the taxonomic status of the urban An. minimus population from Hanoi. The population from the suburbs of Hanoi was identified as An. minimus A. Although significant genetic differentiation was observed between rural and urban An. minimus A populations, they have not differentiated substantially by genetic drift. Limited macrogeographical differentiation was observed between two rural populations at distances of more than 1000 km. Consequently, geographical distance is not the primary factor in differentiating An. minimus A populations having the typical breeding ecology. The estimated effective population size is consistent with the moderate macrogeographical differentiation. Furthermore, no genetic structuring was observed between adult mosquitoes having different behaviour. The macrogeographical population structure indicates that genes may spread over large areas, whereas the presence of an 'urban' An. minimus A population shows the ability of this species to adapt to anthropogenic environmental changes.     

Barrier spraying to control the malaria vector Anopheles albimanus: laboratory and field evaluation in the Dominican Republic

Abstract. To develop a new strategy for control of exophilic/exophagic malaria vectors which rest on peridomestic foliage, Anopheles albimanus was used for laboratory bioassays of mosquito adulticides applied to various vegetation types. Of the three classes of insecticide tested, the pyrethroid (permethrin at 112 g/ha) showed greater foliar persistence than either the carbamate (bendiocarb at 340g/ha) or the organophosphorus compound (malathion at 277g/ha). Field evaluation of barrier spraying against An.albimanus was evaluated in rural villages of southwest Dominican Republic during 1989. The pyrethroid deltamethrin was sprayed aerially for ultra-low volume application at treatment rates of 17-19ga.i./ha in a radius of 500m around two villages. An.albimanus were monitored by light-traps and human bait collections at both treated villages, compared with two similar untreated villages, up to 9 nights post-treatment. Densities of female An.albimanus were significantly reduced in the sprayed villages for at least 8 or 9 nights. Further evaluation of barrier spraying is recommended to determine optimal pyrethroid formulations and applications rates, their impact on non-target fauna and efficacy against malaria transmission.     

Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi.

Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.     

Population genetic structure and demographic history of the fat greenling Hexagrammos otakii

Fat greenling (Hexagrammos otakii) is an important commercial fish in the Northwestern Pacific, being distributed along the coastal waters of the East/Japan Sea and the Yellow Sea. To investigate population genetic structure and demographic history of this species, one hundred and fifty five individuals were collected from five localities in the distribution range of the species and sequence variations in the mitochondrial genes COI, COIII-ND3-ND4L, and cytochrome b were examined. For all the genes in every sampling location, the nucleotide diversities were very low (0.001 ～ 0.005) although the haplotype diversities were relatively high, 0.55 ～ 0.81 for COI, 0.79 ～ 0.84 for COIII-ND3-ND4L, and 0.95 ～ 0.97 for cytochrome b. Hierarchical analysis of molecular variance (AMOVA), the conventional population statistic FST, and exact test of population differentiation revealed no significant genetic structuring among the samples, indicating that fat greening is panmictic throughout the range of its distribution. Neutrality tests such as Tajima’s D and Fu’s FS statistics and mismatch distribution analyses suggested that fat greening has undergone the demographic history of population expansion during the late Pleistocene period approximately 91,000 ～ 327,000 years ago. The star-burst patterns of haplotype networks and low nucleotide diversities also indicated recent population expansion. These results help establish the fisheries management strategy for fat greenling in the Northwestern Pacific.     

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.     

Satellite DNA from the Y chromosome of the malaria vector Anopheles gambiae

Satellite DNA is an enigmatic component of genomic DNA with unclear function that has been regarded as "junk." Yet, persistence of these tandem highly repetitive sequences in heterochromatic regions of most eukaryotic chromosomes attests to their importance in the genome. We explored the Anopheles gambiae genome for the presence of satellite repeats and identified 12 novel satellite DNA families. Certain families were found in close juxtaposition within the genome. Six satellites, falling into two evolutionarily linked groups, were investigated in detail. Four of them were experimentally confirmed to be linked to the Y chromosome, whereas their relatives occupy centromeric regions of either the X chromosome or the autosomes. A complex evolutionary pattern was revealed among the AgY477-like satellites, suggesting their rapid turnover in the A. gambiae complex and, potentially, recombination between sex chromosomes. The substitution pattern suggested rolling circle replication as an array expansion mechanism in the Y-linked 53-bp satellite families. Despite residing in different portions of the genome, the 53-bp satellites share the same monomer lengths, apparently maintained by molecular drive or structural constraints. Potential functional centromeric DNA structures, consisting of twofold dyad symmetries flanked by a common sequence motif, have been identified in both satellite groups.     

Isolation and characterization of histones from Anopheles albimanus Weidemann

1. Histones from Anopheles albimanus adults were prepared by a combination of techniques including chromatin isolation and selective extractions. 2. The anopheline histones were identified on acid urea gels by comparing their electrophoretic profile with that of calf thymus histones and histones isolated from other tissue. 3. Excellent separation of histones was obtained after the extractions by a single electrophoretic run. 4. In addition to the five major classes of histones found in eukaryotes, a sixth class was detected and tentatively identified as histone H5. 5. This is the first report of histone H5 and its function in insects.     

Population structure and population history of Anopheles dirus mosquitoes in Southeast Asia

Separating the confounding effects of long-term population history from gene flow can be difficult. Here, we address the question of what inferences about gene flow can be made from mitochondrial sequence data in three closely related species of mosquitoes, Anopheles dirus species A, C, and D, from southeast Asia. A total of 84 sequences of 923 bp of the mitochondrial cytochrome oxidase I gene were obtained from 14 populations in Thailand, Myanmar, and Bangladesh. The genealogy of sequences obtained from two populations of AN: dirus C indicates no contemporary gene flow between them. The F(ST) value of 0.421 therefore probably represents a recent common history, perhaps involving colonization events. Anopheles dirus A and D are parapatric, yet no differentiation was seen either within or between species. The starlike genealogy of their haplotypes, smooth unimodal mismatch distributions, and excess of low frequency mutations indicate population expansion in An. dirus A and D. This, rather than widespread gene flow, explains their low within-species F(ST) values (0.018 and 0.022). The greater genetic diversity of An. dirus D suggests that expansion occurred first in species D and subsequently in species A. The current geographical separation and low hybrid fitness of these species also argue against ongoing interspecific gene flow. They suggest instead either historical introgression of mtDNA from An. dirus D into species A followed by independent range expansions, or a selective sweep of mtDNA that originated in An. dirus D. While not excluding contemporary gene flow, historical population processes are sufficient to explain the data in An. dirus A and D. The genealogical relationships between haplotypes could not be used to make inferences of gene flow because of extensive homoplasy due to hypervariable sites and possibly also recombination. However, it is concluded that this approach, rather than the use of fixation indices, is required in the future to understand contemporary gene flow in these mosquitoes. The implications of these results for understanding gene flow in another important and comparable group of malaria vector mosquitoes in Africa, the An. gambiae complex, are also discussed.     

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F(ST)) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.     

Isolation and characterization of chromosomal proteins from the mosquito Anopheles albimanus Weidemann

1. Nuclei were isolated from adult anopheline mosquitoes and fractionated into nucleolar chromatin, nucleoplasmic chromatin and ribonucleoprotein particles by sucrose density gradients. 2. Histones and nonhistone proteins were selectively dissociated from chromatin by treatment with sodium chloride, urea and guanidine HC1. 3. A special class of nonhistone proteins (tight proteins) were extracted from chromatin with Na4P2O7. 4. The electrophoretic properties of the histones, nonhistone proteins and ribonucleoprotein particles were examined by isoelectric focusing and SDS multiphase polyacrylamide gel electrophoresis. 5. By contrast to the histones, the nonhistone proteins displayed considerable heterogeneity. 6. Possible functional implications of the chromosomal proteins are discussed.     

Mitochondrial DNA variation in the malaria vector Anopheles minimus across China,Thailand and Vietnam: evolutionary hypothesis,population structure and population history

The effects of Pleistocene environmental fluctuations on the distribution and diversity of organisms in Southeast Asia are much less well known than in Europe and North America. In these regions, the combination of palaeoenvironmental reconstruction and inferences about population history from genetic data has been very powerful. In Southeast Asia, mosquitoes are good candidates for the genetic approach, with the added benefit that understanding the relative contributions of historical and current processes to population structure can inform management of vector species. Genetic variation among populations of Anopheles minimus was examined using 144 mtDNA COII sequences from 23 sites in China, Thailand and Vietnam. Haplotype diversity was high, with two distinct lineages that have a sequence divergence of over 2% and exhibit different geographical distributions. We compare alternative hypotheses concerning the origin of this pattern. The observed data deviate from the expectations based on a single-panmictic population with or without growth, or a stable but spatially structured population. However, they can be readily accommodated by a model of past fragmentation into eastern and western refugia, followed by growth and range expansion. This is consistent with the palaeoenvironmental reconstructions currently available for the region.     

Esterases in the malaria vector mosquito, Anopheles culicifacies. Genetic and linkage analyses of an alpha and beta polymorphism

The genetic and linkage analyses of an alpha and beta esterase polymorphism in Anopheles culicifacies are presented. A survey of laboratory strains uncovered four electrophoretic variants for the alpha esterase and two for the beta esterase. Genetic analyses indicated that the variants of the alpha esterase are under the control of codominant alleles of a single locus and that this locus is linked to the locus controlling the expression of the codominant alleles of the beta esterases. The esterases are not linked either to sex (chromosome 1) or to maroon eye (chromosome 2) but to the chromosome 3 markers, dieldrin resistance and phosphoglucomutase. The gene sequence is Est-alpha--Dl--Pgm--Est-beta dnd the recombination frequencies are as follows: Est-alpha--Dl = 2.9 percent; Dl--Pgm = 5.8 percent; Pgm--Est-beta = 6.4 percent; Est-alpha--Est-beta = 15.1 percent, Est-alpha--Pgm = 8.7 percent and Dl--Est-beta = 12.2 percent.     

Low-volume application by mist-blower compared with conventional compression sprayer treatment of houses with residual pyrethroid to control the malaria vector Anopheles albimanus in Mexico

Abstract. Village-scale trials were carried out in southern Mexico to compare the efficacy of indoor-spraying of the pyrethroid insecticide lambda-cyhalothrin applied either as low-volume (LV) aqueous emulsion or as wettable-powder (WP) aqueous suspension for residual control of the principal coastal malaria vector Anopheles albimanus. Three indoor spray rounds were conducted at 3-month intervals using back-pack mist-blowers to apply lambda-cyhalothrin 12.5 mg a.i./m² by LV, whereas the WP was applied by conventional compression sprayer at a mean rate of 26.5 mg a.i./m².
Both treatments caused mosquito mortality indoors and outdoors (collected inside house curtains) as a result of contact with treated surfaces before and after feeding, but had no significant impact on overall population density of An. albimanus resting indoors or assessed by human bait collections. Contact bioassays showed that WP and LV treatments with lambda-cyhalothrin were effective for 12–20 weeks (>75% mortality) without causing excito-repellency.
Compared to the WP treatment (8 houses/man/day), LV treatment (25 houses/man/day) was more than 3 times quicker per house, potentially saving 68% of labour costs. This is offset, however, by the much lower unit price of a compression sprayer (e.g. Hudson 'X-pert' at US120) than a mist-blower (e.g. 'Super Jolly' at US350), and higher running costs for LV applications. It was calculated, therefore, that LV becomes more economical than WP after 18.8 treatments/100 houses/10 men at equivalent rates of application, or after 7.6 spray rounds with half-rate LV applications.     

Stable and heritable gene silencing in the malaria vector Anopheles stephensi

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles. This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.     

A cluster of candidate odorant receptors from the malaria vector mosquito,Anopheles gambiae

Olfaction is critical to the host preference selection behavior of many disease-transmitting insects, including the mosquito Anopheles gambiae sensu stricto (hereafter A. gambiae), one of the major vectors for human malaria. In order to more fully understand the molecular biology of olfaction in this insect, we have previously identified several members member of a family of candidate odorant receptor proteins from A. gambiae (AgORs). Here we report the cloning and characterization of an additional AgOR gene, denoted as AgOr5, which shows significant similarity to putative odorant receptors in A. gambiae and Drosophila melanogaster and which is selectively expressed in olfactory organs. AgOr5 is tightly clustered within the A. gambiae genome to two other highly homologous candidate odorant receptors, suggesting that these genes are derived from a common ancestor. Analysis of the developmental expression within members of this AgOR gene cluster reveals considerable variation between these AgORs as compared to candidate odorant receptors from D. melanogaster.     

Identification and characterization of a new putative c-type lysozyme from malaria vector Anopheles stephensi

Lysozyme (E.C. 3.2.1.17) activity is reported from the malaria vector Anopheles stephensi. The activity was detected in the salivary gland and midgut using bacteriolytic radial diffusion assay. Spectrophotometric analysis indicated that higher level of lysozyme activity was maintained in both midgut and salivary gland tissues. The activity reached the highest level in 4-8 days old mosquitoes. Genomic PCR amplification revealed the presence of at least two putative lysozyme genes in the mosquito genome. Preliminary analysis of one of the 413 bp genomic fragments showed 56% identity to the lysozyme of mosquito A. gambiae. However, the nature and origin of the putative cloned lysozyme gene remains elusive.     

Genetic and demographic structure of Russian populations from the Tver' and Rostov regions

Based on the data from 569 questionnaires collected in Udomlya and Ostashkov raions of Tver oblast and 436 questionnaires collected in Tsimlyansk and Dubovsk raions of Rostov oblast, genetic demographic characteristics and Crow's index for urban and rural populations of these regions were calculated. The data obtained were compared with those for other Russian populations obtained earlier.     

Limited usefulness of microsatellite markers from the malaria vector Anopheles gambiae when applied to the closely related species Anopheles melas

Anopheles melas is a brackish water mosquito found in coastal West Africa where it is a dominant malaria vector locally. In order to facilitate genetic studies of this species, 45 microsatellite loci originally developed for Anopheles gambiae were sequenced in An. melas. Those that were suitable based on repeat number and flanking regions were examined in 2 natural populations from Equatorial Guinea. Only 15 loci were eventually deemed suitable as polymorphic markers in An. melas populations. These loci were screened in 4 populations from a wider geographic range. Heterozygosity estimates ranged from 0.18 to 0.79, and 2.5-15 average alleles were observed per locus, yielding 13 highly polymorphic markers and 2 loci with lower variability. To examine the usefulness of microsatellite markers when applied in a sibling species, the original An. gambiae specific markers were used to amplify 5 loci in An. melas. Null alleles were found for 1 An. gambiae marker. We discuss the pitfalls of using microsatellite loci across closely related species and conclude that in addition to the problem of null alleles associated with this practice, many loci may prove to be of very limited use as polymorphic markers even when used in a sibling species.