Isolation of polymorphic microsatellite markers from the malaria vector Anopheles darlingi

High molecular weight DNA was extracted from the primary Neotropical malaria vector, Anopheles darlingi from Capanema, Pará, Brazil, to create a small insert genomic library, and then a phagemid library. Enriched sublibraries were constructed from the phagemid library using a microsatellite oligo primed second strand synthesis protocol. The resulting 242 760 individual clones were screened. The mean clone size of the positive clones was 302 bp. Flanking primers were designed for each suitable microsatellite sequence. Eight polymorphic loci were optimized and characterized. The allele size ranges are based on 253 samples of A. darlingi from eastern Amazonian and central Brazil.     

Isolation and characterization of trinucleotide microsatellites in African malaria mosquito Anopheles funestus

We developed microsatellite markers for an important African malaria mosquito Anopheles funestus Giles. The microsatellite‐enriched genomic library was constructed and screened with single‐strand oligonucleotides [(CCT)₁₇, (AAT)₁₇, (CAG)₁₇ and (GA)₂₅] as probes. Among the 47 pairs of polymerase chain reaction primers screened, 31 produced successful and consistent amplification. Although only a few A. funestus individuals from one geographical location were used to screen microsatellite marker polymorphism, 27 markers were found polymorphic and four markers monomorphic. Most polymorphic markers are trinucleotide markers. Isolation of polymorphic microsatellite markers provide useful tools for A. funestus population genetic studies and genome mapping.     

Isolation and characterization of polymorphic microsatellite markers from the mosquito Anopheles moucheti,malaria vector in Africa

Anopheles moucheti is a major human malaria vector in Equatorial Africa. The screening of an Anopheles moucheti genomic microsatellite library allowed us to select 36 sequences with AC/GT dinucleotide tandem repeats. Primer pairs were designed to amplify the loci and 25 out of 36 gave a repeatable and scorable amplification. In total, 17 loci were selected for their high degree of polymorphism (the number of alleles per locus ranged from four to 16, and observed heterozygosity from 0.43 to 0.87) and suspicion of absence of null alleles, using 30 wild females from South‐Cameroon. No linkage disequilibrium was found between the loci.     

Genetic isolation within the malaria mosquito Anopheles melas

Anopheles melas is a brackish water–breeding member of the Anopheles gambiae complex that is distributed along the coast of West Africa and is a major malaria vector within its range. Because little is known about the population structure of this species, we analysed 15 microsatellite markers and 1161 bp of mtDNA in 11 A. melas populations collected throughout its range. Compared with its sibling species A. gambiae, A. melas populations have a high level of genetic differentiation between them, representing its patchy distribution due to its fragmented larval habitat that is associated with mangroves and salt marsh grass. Populations clustered into three distinct groups representing Western Africa, Southern Africa and Bioko Island populations that appear to be mostly isolated. Fixed differences in the mtDNA are present between all three clusters, and a Bayesian clustering analysis of the microsatellite data found no evidence for migration from mainland to Bioko Island populations, and little migration was evident between the Southern to the Western cluster. Surprisingly, mtDNA divergence between the three A. melas clusters is on par with levels of divergence between other species of the A. gambiae complex, and no support for monophyly was observed in a maximum‐likelihood phylogenetic analysis. Finally, an approximate Bayesian analysis of microsatellite data indicates that Bioko Island A. melas populations were connected to the mainland populations in the past, but became isolated, presumably when sea levels rose after the last glaciation period (≥10 000–11 000 bp ). This study has exposed species‐level genetic divergence within A. melas and also has implications for control of this malaria vector.     

Chromosomal evidence of incipient speciation in the Afrotropical malaria mosquito Anopheles funestus

The analysis of chromosomal polymorphism of paracentric inversions in anopheline mosquitoes has often been instrumental to the discovery of sibling species complexes and intraspecific genetic heterogeneities associated with incipient speciation processes. To investigate the population structure of Anopheles funestus Giles (Diptera: Culicidae), one of the three most important vectors of human malaria in sub-Saharan Africa, a three-year survey of chromosomal polymorphism was carried out on 4,638 karyotyped females collected indoors and outdoors from two villages of central Burkina Faso. Large and temporally stable departures from Hardy-Weinberg equilibrium due to significant deficits of heterokaryotypes were found irrespective of the place of capture, and of the spatial and temporal units chosen for the analysis. Significant linkage disequilibrium was observed among inversion systems on independently assorting chromosomal arms, indicating the existence of assortative mating phenomena. Results were consistent with the existence of two chromosomal forms characterized by contrasting degrees of inversion polymorphism maintained by limitations to gene flow. This hypothesis was supported by the reestablishment of Hardy-Weinberg and linkage equilibria when individual specimens were assigned to each chromosomal form according to two different algorithms. This pattern of chromosomal variability is suggestive of an incipient speciation process in An. funestus populations from Burkina Faso.     

Isolation and characterization of polymorphic microsatellite markers of Anopheles sinensis,a malaria vector mosquito in the East Asia region

Eleven polymorphic dinucleotide (GA and CA) microsatellites were isolated and characterized in the mosquito Anopheles sinensis; this species is distributed over the East Asia region and is a primary vector of malaria, particularly in Korea. The number of alleles per locus ranged from 4 to 13. The observed heterozygosity (H_O) and expected heterozygosity (H_E) varied from 0.30 to 0.89 and from 0.59 to 0.90, respectively. These microsatellites could be useful in studying the evolution of the widely distributed A. sinensis in diverse environments.     

Temperature-related duration of aquatic stages of the Afrotropical malaria vector mosquito Anopheles gambiae in the laboratory

Vector abundance is an important factor governing disease risk and is often employed when modelling disease transmission. The longevity of the aquatic stages of mosquitoes (Diptera: Culicidae) dictates the rate of production of adults and hence the intensity of disease transmission. We examined how temperature influences the survival of larval stages (larvae and pupae) of Anopheles gambiae Giles sensu stricto and subsequent adult production of this most efficient malaria vector. Groups of 30 mosquitoes were reared at constant temperatures (from 10 to 40 degrees C) from the first instar and observed until death or metamorphosis of the last individual. Larvae developed into adults at temperatures ranging from 16 to 34 degrees C. Larval survival was shortest (< 7 days) at 10-12 degrees C and 38-40 degrees C, and longest (> 30 days) at 14-20 degrees C. Within the temperature range at which adults were produced, larval mortality was highest at the upper range 30-32 degrees C, with death (rather than adult emergence) representing over 70% of the terminal events. The optimal survival temperatures were lower than the temperatures at which development was quickest, suggesting a critical relationship between temperature and the life cycle of the insect. These data provide fundamental information about An. gambiae s.s. adult productivity at different temperatures, which may facilitate the construction of process-based models of malaria risk in Africa and the development of early warning systems for epidemics.     

Isolation and characterization of polymorphic microsatellite markers from Asian malaria mosquito Anopheles sinensis (Diptera: Culicidae)

Microsatellite-containing region were isolated and characterized in Anopheles sinensis, a primary vector of malaria parasites in Asia. An enrichment protocol yielded 252 microsatellite sequences. We designed primers to amplify 20 unique microsatellites, 14 of which amplify cleanly and were polymorphic. A survey of 24 individuals showed that 12 loci are highly variable with the number of alleles ranging from two to 11, and expected heterozygosity ranging from 0.116 to 0.903. These markers will be useful for population genetic studies and genome mapping in A. sinensis.     

Anopheles nili as the main vector of human malaria in villages of southern Cameroon

In villages near Sanaga river, in the forest zone of south Cameroon, Anopheles nili Theobald is the main species of mosquito regularly found biting man inside houses. Its densities are related to the flow level of the river. It is also the main malaria vector in terms of intensity and seasonal duration of transmission. The yearly malaria inoculation rate due to An. nili alone is 104 infective bites/man, varying between 3 infective bites/man in October and 20 in March. Anopheles gambiae Giles is a less important vector in the area but reached its peak in the dry season, when the Sanaga river is at its lowest level. These observations underline the influence of local ecology on malaria transmission and the need for entomological studies in each situation.     

Anion inhibition studies of a beta carbonic anhydrase from the malaria mosquito Anopheles gambiae

An anion inhibition study of the β-class carbonic anhydrase, AgaCA, from the malaria mosquito Anopheles gambiae is reported. A series of simple as well as complex inorganic anions, and small molecules known to interact with CAs were included in the study. Bromide, iodide, bisulphite, perchlorate, perrhenate, perruthenate, and peroxydisulphate were ineffective AgaCA inhibitors, with K_Is?>?200?mM. Fluoride, chloride, cyanate, thiocyanate, cyanide, bicarbonate, carbonate, nitrite, nitrate, sulphate, stannate, selenate, tellurate, diphosphate, divanadate, tetraborate, selenocyanide, and trithiocarbonate showed K_Is in the range of 1.80–9.46?mM, whereas N,N-diethyldithiocarbamate was a submillimolar AgaCA inhibitor (K_I of 0.65?mM). The most effective AgaCA inhibitors were sulphamide, sulphamic acid, phenylboronic acid and phenylarsonic acid, with inhibition constants in the range of 21–84?µM. The control of insect vectors responsible of the transmission of many protozoan diseases is rather difficult nowadays, and finding agents which can interfere with these processes, as the enzyme inhibitors investigated here, may arrest the spread of these diseases worldwide.     

Oogenesis in the malaria mosquito Anopheles gambiae

Summary Oogenesis has been followed with the electron microscope in 2 strains of the malaria mosquito Anopheles gambiae, from the emergence of the adult (oocytes at leptonema) till shortly before the oocytes are ready for oviposition. After pachynema the chromosomes form a karyosphere and a fibrous capsule develops around it. Work on other mosquitoes suggests that the capsule may be related to the synaptonemal complexes. Both Anopheles strains contain at some time an extrachromosomal (not DNA-containing) body comparable to the karyosphere in size. Clusters of granules are present at the surface of the nucleolus and free in the nucleoplasm. Tentative results indicate that they may contain DNA. During oogenesis the nucleolus becomes very large, mainly because of proliferation of the nucleolonema. Towards the end of oocyte development the nucleus assumes the large canoe-shape also seen in Aedes and Culex. Nucleolonema traverse the entire nucleus, and modified granular clusters are found throughout.     

Polymorphic chromosomal inversions in Anopheles moucheti,a major malaria vector in Central Africa

Anopheles moucheti Evans (Diptera: Culicidae) is a major vector of malaria in forested areas of Central Africa. However, few genetic tools are available for this species. The present study represents the first attempt to characterize chromosomes in An. moucheti females collected in Cameroon. Ovarian nurse cells contained polytene chromosomes, which were suitable for standard cytogenetic applications. The presence of three polymorphic chromosomal inversions in An. moucheti was revealed. Two of these inversions were located on the 2R chromosome arm. The homology between the 2R chromosome arms of An. moucheti and Anopheles gambiae Giles was established by fluorescent in situ hybridization of six An. gambiae genic sequences. Mapping of the probes on chromosomes of An. moucheti detected substantial gene order reshuffling between the two species. The presence of polytene chromosomes and polymorphic inversions in An. moucheti provides a new basis for further population genetic, taxonomic and ecological studies of this neglected malaria vector.     

Characterization of microsatellite loci in Anopheles flavirostris,the principal malaria vector in the Philippines

Microsatellites were isolated and characterized from Anopheles flavirostris, the principal malaria vector in the Philippines. Fifty of the 150 positive clones sequenced contained mostly dinucleotide microsatellites and only 16 had trinucleotide repeats. We designed primers from the unique sequences flanking 18 microsatellite loci. Of these, 11 loci produced successful amplification and revealed high levels of polymorphism; 86 alleles were detected with allele number ranging from 2 to 16 at each locus. The high allelic variability will make these microsatellite loci very useful for taxonomic and population genetic studies.     

The Genome of Anopheles darlingi,the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.     

Interindividual variation in the attractiveness of human odours to the malaria mosquito Anopheles gambiae s. s

Differences between human individuals in their attractiveness to female mosquitoes have been reported repeatedly, but the underlying mechanisms are not well understood. Skin emanations from 27 human individuals, collected on glass marbles, were tested against ammonia in a dual-choice olfactometer to establish their degrees of attractiveness to anthropophilic Anopheles gambiae s.s. Giles (Diptera: Culicidae) mosquitoes. Ammonia was used as a standard odour source because of its proven attractiveness to An. gambiae s.s. Skin emanations from most volunteers attracted significantly more mosquitoes than ammonia. There were clear differences in the attractiveness of skin emanations from different volunteers relative to that of ammonia, as well as in the strength of the trap entry response. Consistent differences were observed when emanations from the three most and the three least attractive volunteers were tested pairwise. No gender or age effect was found for relative attractiveness or trap entry response. Emanations from volunteers with higher behavioural attractiveness elicited higher electroantennogram response amplitudes in two pairs, but in a third pair a higher electroantennogram response was found for the less attractive volunteer. These results confirm that odour contributes to the differences in attractiveness of humans to mosquitoes.     

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.