Emergence of behavioural avoidance strategies of malaria vectors in areas of high LLIN coverage in Tanzania.

Despite significant reductions in malaria transmission across Africa since 2000, progress is stalling. This has been attributed to the development of insecticide resistance and behavioural adaptations in malaria vectors. Whilst insecticide resistance has been widely investigated, there is poorer understanding of the emergence, dynamics and impact of mosquito behavioural adaptations. We conducted a longitudinal investigation of malaria vector host choice over 3 years and resting behaviour over 4 years following a mass long-lasting insecticidal nets (LLINs) distribution in Tanzania. By pairing observations of mosquito ecology with environmental monitoring, we quantified longitudinal shifts in host-choice and resting behaviour that are consistent with adaptation to evade LLINs. The density of An. funestus s.l., declined significantly through time. In tandem, An. arabiensis and An. funestus s.l. exhibited an increased rate of outdoor relative to indoor resting; with An. arabiensis reducing the proportion of blood meals taken from humans in favour of cattle. By accounting for environmental variation, this study detected clear evidence of intra-specific shifts in mosquito behaviour that could be obscured in shorter-term or temporally-coarse surveys. This highlights the importance of mosquito behavioural adaptations to vector control, and the value of longer-term behavioural studies.

A set of broadly applicable microsatellite markers for analyzing the structure of Culex pipiens (Diptera: Culicidae) populations.

Microsatellite markers were isolated and developed from Culex pipiens quinquefasciatus Say (Diptera: Culicidae) sampled in Johannesburg, South Africa, to identify those that are broadly useful for analyzing Cx. pipiens complex populations between continents. Suitable loci should be 1) inherited in a codominant Mendelian manner, 2) polymorphic, 3) selectively neutral, 4) randomly associated, 5) without null alleles, and 6) applicable across broad regions and between diverse biotypes. Loci in Cx. p. quinquefasciatus from Johannesburg ranged from two to 17 alleles per locus and expected heterozygosities (H(e)) were 0.02-0.87. Loci in Cx. p. pipiens L. from Johannesburg had five to 19 alleles per locus and H(e) values ranging from 0.57 to 0.93, whereas those from George, South Africa, had five to 17 alleles per locus and H(e) values ranging from 0.54 to 0.88. Loci in North American mosquitoes were more variable. Cx. p. quinquefasciatus from South Carolina had five to 19 alleles per locus and H(e) values ranging from 0.64 to 0.90, whereas Cx. p. pipiens from Massachusetts had six to 28 alleles per locus and with H(e) values ranging from 0.65 to 0.94. All loci were associated randomly. Overall, four of nine of these new loci satisfied all six criteria for broad utility for analyzing the genetic structure of Cx. pipiens populations.

Investigating associations between biting time in the malaria vector Anopheles arabiensis Patton and single nucleotide polymorphisms in circadian clock genes: support for sub-structure among An. arabiensis in the Kilombero valley of Tanzania.

BACKGROUND: There is growing evidence that the widespread use of Long-Lasting Insecticidal Nets (LLINs) is prompting malaria vectors to shift their biting towards times and places where people are not protected, such as earlier in the evening and/or outdoors. It is uncertain whether these behavioural shifts are due to phenotypic plasticity and/or ecological changes within vector communities that favour more exophilic species, or involve genetic factors within vector species to limit their contact with LLINs. Possibly variation in the time and location of mosquito biting has a genetic basis, but as yet this phenomenon has received little investigation. Here we used a candidate gene approach to investigate whether polymorphisms in selected circadian clock genes could explain variation in the time and location of feeding (indoors versus outside) within a natural population of the major African malaria vector Anopheles arabiensis. METHODS: Host-seeking An. arabiensis were collected from two villages (Lupiro and Sagamaganga) in Tanzania by Human Landing Catch (HLC) technique. Mosquitoes were classified into phenotypes of "early" (7 pm-10 pm) or "late" biting (4 am -7 am), and host-seeking indoors or outdoors. In these samples we genotyped 34 coding SNPs in 8 clock genes (PER, TIM, CLK, CYC, PDP1, VRI, CRY1, and CRY2), and tested for associations between these SNPs and biting phenotypes. SNPs in 8 mitochondrial genes (ATP6, ATP8, COX1, COX2, COX3, ND3, ND5 and CYTB) were also genotyped to test population subdivision within An. arabiensis. RESULTS: The candidate clock genes exhibited polymorphism within An. arabiensis, but it was unrelated to variation in the timing and location of their biting activity. However, there was evidence of strong genetic structure within An. arabiensis populations in association with the TIM, which was unrelated to geographic distance. Substructure within An. arabiensis was also detected using mitochondrial markers. CONCLUSIONS: The variable timing and location of biting in An. arabiensis could not be linked to candidate clock genes that are known to influence behaviour in other Diptera. This finding does not rule out the possibility of a genetic basis to biting behaviour in this malaria vector, but suggests these are complex phenotypes that require more intensive ecological, neuronal and genomic analyses to understand.

Gene flow among populations of the malaria vector, Anopheles gambiae, in Mali, West Africa.

The population structure of the Anopheles gambiae complex is unusual, with several sibling species often occupying a single area and, in one of these species, An. gambiae sensu stricto, as many as three "chromosomal forms" occurring together. The chromosomal forms are thought to be intermediate between populations and species, distinguishable by patterns of chromosome gene arrangements. The extent of reproductive isolation among these forms has been debated. To better characterize this structure we measured effective population size, N(e), and migration rates, m, or their product by both direct and indirect means. Gene flow among villages within each chromosomal form was found to be large (N(e)m > 40), was intermediate between chromosomal forms (N(e)m approximately 3-30), and was low between species (N(e)m approximately 0.17-1.3). A recently developed means for distinguishing among certain of the forms using PCR indicated rates of gene flow consistent with those observed using the other genetic markers.

Effect of seminal fluids in mating between M and S forms of Anopheles gambiae.

Previous studies have shown that sympatric populations of M and S molecular forms of Anopheles gambiae sensu stricto exhibit strong assortative mating. In the few documented cases of cross-mating between M and S forms, females that mated with amale of the alternative form were often also mated with a male of their own form. A potential explanation for the association between cross-mating and double mating could be that male accessory gland or sperm proteins that are responsible for inducing refractoriness to further mating by females have diverged between the M and S forms. This mechanism of postmating reproductive isolation would have important implications for our understanding of the speciation processes in the An. gambiae complex. We tested for this mechanism, by comparing the likelihood of mating, feeding, and laying eggs, as well as the fertility of females presented with males of their own form or the alternate form in the laboratory. We also compared the likelihood of remating in cross-mated and assortatively-mated females, and we analyzed their progeny to unravel patterns of sperm precedence. We found that cross-mated females differed from assortatively-mated females only in terms of egg-hatching rate and larval survival but that these effects could be attributed to hybrid vigor rather than differential response to seminal products. Cross-mating between forms was not associated with remating behavior. These results indicate that the sex proteins responsible for inhibiting further insemination and triggering the gonotrophic cycle in females have not diverged between these M and S populations. We discuss alternative explanations for the patterns of cross-mating and multiple mating observed in the field.

Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences.

Sand flies in the Lutzomyia longipalpis species complex include the primary vector of Leishmania chagasi, the etiologic agent of visceral leishmaniasis in the Neotropics. Twelve L. longipalpis populations from South and Central America were compared using the cytochrome c oxidase I (COI) gene from the mitochondrial genome. The haplotype profiles for each population revealed that the majority of sequence variation was inter-population (98%) rather than intra-population, suggesting that sequence polymorphisms at the COI locus should provide excellent characters for the study of phylogenetic relationships among populations. Phylogenetic reconstruction using distance (neighbor-joining) and maximum parsimony analysis revealed the existence of four clades among the L. longipalpis populations studied: (1) Laran, (2) Brazilian, (3) cis-Andean and (4) trans-Andean. We suggest that these clades represent species. A biogeographical interpretation of the molecular phylogeny suggests that the process of speciation in the L. longipalpis complex began in the Pliocene, from a sub-Andean-Amazonian gene pool resulting from the Andean orogeny (formation of the East Andean Cordillera). The four clades probably diverged as a result of vicariance events that occurred throughout the late Pliocene and Pleistocene. We propose and discuss several historical scenarios, based on the biogeography and historical geology of Central and South America.

Lutzomyia longipalpis is a species complex: genetic divergence and interspecific hybrid sterility among three populations.

The sand fly Lutzomyia longipalpis is the vector of Leishmania donovani chagasi in Latin America. An analysis of genetic variability at 27 enzyme coding loci among three laboratory populations of Lu. longipalpis revealed substantial genetic polymorphism. Levels of genetic distance between all pairwise comparisons of colonies were very high, and consistent with those previously reported among separate species in the genus Lutzomyia. Between 7% and 22% of the loci studied were diagnostic for any two of the colony populations. Experimental hybridization between colonies resulted in the production of sexually sterile male progeny. Our results provide strong evidence that Lu. longipalpis exists in nature as a complex of at least three distinct species. The possible effects of colonization on the genetic makeup of laboratory populations is considered in extending our results to natural populations.

Microsatellite polymorphism in Anopheles maculatus, a malaria vector in Thailand.

Dinucleotide microsatellites were characterized from Anopheles maculatus, a species of mosquito that transmits malaria. A partial genomic library of An. maculatus, consisting of 3,960 kilobases (kb), was screened with either (GT)12 or (CT)12 probes. Approximately 1.5% of the recombinants contained sequences that hybridized to either (GT)12 or (CT)12 dinucleotide probes, suggesting that microsatellites are abundant in the genome of An. maculatus. Estimation of abundance of the two dinucleotide repeats revealed that (GT)n or (CA)n microsatellites occur on average every 68 kb and (CT)n or (GA)n repeats every 495 kb. Among 23 microsatellite loci sequenced, four loci were selected to synthesize primers to perform polymerase chain reaction scoring for genetic polymorphism in a population of An. maculatus. A high level of polymorphism was observed with all four microsatellite loci analyzed. The number of alleles detected at each locus ranged from eight to 12 and the heterozygosities ranged from 0.25 to 0.54. A total of 42 alleles were found among four microsatellite loci. The large number of alleles and polymorphic nature resolved from microsatellite loci make these markers valuable for the study of population genetic structure and gene flow. Knowledge of gene flow is required to develop vector control strategies using genetic manipulations of malaria vector populations.

Breeding structure of the sand fly Lutzomyia longipalpis (Lutz & Neiva) in Brazil.

Eleven populations of Lutzomyia longipalpis (Lutz & Neiva), the sand fly vector of Leishmania chagasi, from different areas of Brazil were analyzed for genetic variation at 16 enzyme loci. In this region, the prevalence of visceral leishmaniasis (VL) caused by L. chagasi is spotty and reproductive isolation among populations of Lu. longipalpis has been reported. It is thought that morphologically similar cryptic species with varying vectorial capacity may be responsible for the discontinuous distribution of VL. The aim was to study the genetic structure of populations within this region and to identify demes that may represent sibling species. Genotypic frequencies within populations were in close compliance to Hardy-Weinberg expectations, suggesting there are no sympatric species among these 11 populations. Levels of genetic distance between pairs of populations were very low (< 0.03), consistent with local populations within a single sand fly species. When genotypic frequency data for all populations were pooled, 9 of the 13 polymorphic loci deviated from Hardy-Weinberg expectations, indicating some degree of genetic substructuring. Estimates of effective migration rates (N(e)m) among all populations were low, 2.73, suggesting that gene flow is restricted among populations, which is probably the reason for the observed genetic substructuring.

Evidence through crossmating experiments of a species complex in Anopheles pseudopunctipennis sensu lato: a primary malaria vector of the American continent.

Crossmating experiments were conducted to determine if postmating reproductive barriers are involved in the maintenance of genetic divergence among populations of Anopheles pseudopunctipennis sensu lato, a primary malaria vector of the American continent. Reciprocal crosses were conducted between colony and wild strains from Mexico, Bolivia, and Peru. Hybridization experiments revealed unidirectional male/female hybrid sterility in crosses between Mexican females and South American males. The data presented provide the first evidence that genetic differences exist among geographic strains of An. pseudopunctipennis in neotropical America. There is a consistent pattern suggesting the presence of at least two allopatric sibling species. One species occurs in central Mexico, the other in the South American Andean Cordillera.

Characterization of Anopheles pseudopunctipennis sensu lato from three countries of neotropical America from variation in allozymes and ribosomal DNA.

Enzyme electrophoresis and restriction fragment length polymorphism (RFLP) analysis of Anopheles pseudopunctipennis sensu lato from nine isolated populations in neotropical America confirmed previous observations that it constitutes a species complex. Electrophoretic studies showed fixed differences at two enzyme loci, glycerol dehydrogenase (Gcd) and phosphoglucomutase (Pgm), suggesting limited or no gene flow between populations from Mexico and South America. In addition, analysis of genetic distance showed two distinctive clusters, one from Mexico and the other from South America, separated at a Nei's distance level of 0.13, a value consistent in magnitude with that of other anopheline sibling species. The RFLP analysis revealed the presence of a ribosomal DNA fragment in Mexican strains that was absent in strains from South America. Two species have been identified through these studies, one provisionally named An. pseudopunctipennis A, a species from central Mexico, and the other An. pseudopunctipennis B, for the species found in the interAndean valleys and Andean slopes in regions of Peru and Bolivia. This research provides information required to elucidate the status of the different species of the An. pseudopunctipennis complex as vectors of malaria in the Americas.

Occurrence and evolutionary significance of a California encephalitis-like virus in Aedes squamiger (Diptera: Culicidae).

More than 12,000 Aedes increpitus Dyar and 4,600 Aedes squamiger (Coquillett) were tested for the presence of arboviruses to test the hypothesis that there is a coevolutionary relationship between Aedes (Ochlerotatus) mosquitoes and California serogroup viruses. Five strains of a California encephalitis-like virus were isolated from adults reared from larvae of Ae. squamiger collected in January 1989 from a coastal salt marsh at Morro Bay, San Luis Obispo County, California. Viruses were isolated in Vero cell cultures and serotyped by cross-neutralization tests. These isolates represent the first arboviruses isolated from this species. On the basis of morphology, Aedes squamiger has been included in the Aedes stimulans group of the subgenus Ochlerotatus. Other species within the Ae. stimulans group are vectors of California (CAL) serogroup viruses elsewhere in North America. Analysis of isozyme variability supports the inclusion of Ae. squamiger in the Ae. stimulans group and suggests that coastal populations of Ae. increpitus are the closest California relatives of Ae. squamiger. Recovery of virus from Ae. squamiger reinforces the relationship between CAL serogroup viruses and Aedes (Ocherlotatus) mosquitoes. However, the failure to isolate virus from large samples of Ae. increpitus from coastal and low elevation inland habitats suggests a complex evolutionary history involving both vertical and horizontal transmission mechanisms.

Microsatellite DNA polymorphism and heterozygosity among field and laboratory populations of Anopheles gambiae ss (Diptera: Culicidae).

We compared microsatellite polymorphism at nine loci located on chromosome 3 among two colonies and a field population of Anopheles gambiae sensu stricto Giles mosquitoes. Numbers of microsatellite alleles observed at each locus and mean heterozygosities were drastically reduced among laboratory colonies. Genetic analysis of the field population used in this study revealed an unprecedented frequency of rare alleles (<0.05). In contrast, colony samples revealed large numbers of alleles with frequencies >0.50. Partitioning of field data to assess the impact of rare alleles, null alleles, and sample size on estimates of mean heterozygosity revealed the plasticity of this measurement and suggests that heterozygosity may be reliably estimated from relatively small collections using microsatellites.

Genetic variability among populations of the sand fly Lutzomyia (Lutzomyia) longipalpis (Diptera: Psychodidae) from Central America.

Eleven Central American populations of Lutzomyia longipalpis (Lutz & Neiva) were analyzed for genetic variation at 16 enzyme loci. The aim was to study the genetic structure among populations within this region and to identify demes that may represent different sibling species. Genotypic frequencies within populations agreed with Hardy-Weinberg expectations, indicating that there were no sympatric sibling species among these 11 populations. Levels of genetic distance between pairs of populations were very low (< 0.02). Some substructing was evident, because after genotypes of all populations mere pooled, 7 of the 16 enzyme loci deviated from Hardy-Weinberg expectations. Estimates of effective migration rates among populations (Nm) were low (3.7), indicating that gene flow was restricted. These data explained observed genetic substructuring when all genotypes were pooled.

Comparison of host-feeding patterns between Anopheles quadrimaculatus sibling species A and B.

Bloodmeal sources for sympatric species A and B of Anopheles quadrimaculatus in an area of Mississippi were identified using an indirect enzyme-linked immunosorbent assay. Females had fed only on 6 species of mammals including dog/fox, pig, opossum, raccoon, cottontail rabbit and white-tailed deer. The latter species was the predominant host, representing 96.7 and 89.5% of the bloodmeals taken by species A and B, respectively. No marked difference in feeding patterns was found.

Hybridization of laboratory strains of sibling species A and B of Anopheles quadrimaculatus.

Adult mosquitoes of the Anopheles quadrimaculatus complex were collected from Montgomery County, AL (MON) and Alachua County, FL, (KBG) and laboratory stocks of species A and B were established through a selection procedure employing isofemale lines. Progeny from a cross of species B females to ORL males were usually semisterile females and sterile males. Progeny of the reciprocal cross were also semisterile females and sterile males, but the sex ratio was variable and ranged from normal to no males because of male mortality during the pupal stage. Conspecific crosses between strains from the two locations resulted in fertile offspring. Crosses between the sibling species from the two locations invariably gave semisterile females and sterile males (or lethal effects). This evidence confirmed previous data from hybridization and electrophoretic analyses of field populations indicating that Anopheles quadrimaculatus is a species complex.

Evidence for subdivision within the M molecular form of Anopheles gambiae.

The principal vector of malaria in sub-Saharan Africa, Anopheles gambiae is subdivided into two molecular forms M and S. Additionally, several chromosomal forms, characterized by the presence of various inversion polymorphisms, have been described. The molecular forms M and S each contain several chromosomal forms, including the Savanna, Mopti and Forest forms. The M and S molecular forms are now considered to be the reproductive units within A. gambiae and it has recently been argued that a low recombination rate in the centromeric region of the X chromosome has facilitated isolation between these forms. The status of the chromosomal forms remains unclear however. Therefore, we studied genetic differentiation between Savanna S, Forest S, Forest M and Mopti M populations using microsatellites. Genetic differentiation between Savanna S and Forest S populations is very low (F(ST) = 0.0053 +/- 0.0049), even across large distances. In comparison, the Mopti M and Forest M populations show a relatively high degree of genetic differentiation (F(ST) = 0.0406 +/- 0.0054) indicating that the M molecular form may not be a single entity, but could be subdivided into at least two distinct chromosomal forms. Previously it was proposed that inversions have played a role in the origin of species within the A. gambiae complex. We argue that a possible subdivision within the M molecular form could be understood through this process, with the acquisition of inversions leading to the expansion of the M molecular form into new habitat, dividing it into two distinct chromosomal forms.

An unusual distribution of the kdr gene among populations of Anopheles gambiae on the island of Bioko, Equatorial Guinea.

In West Africa, Anopheles gambiae exists in discrete subpopulations known as the M and S molecular forms. Although these forms occur in sympatry, pyrethroid knock-down resistance (kdr) is strongly associated with the S molecular form. On the island of Bioko, Equatorial Guinea we found high frequencies of the kdr mutation in M form individuals (55.8%) and a complete absence of kdr in the S form. We also report the absence of the kdr allele in M and S specimens from the harbour town of Tiko in Cameroon, representing the nearest continental population to Bioko. The kdr allele had previously been reported as absent in populations of An. gambiae on Bioko. Contrary to earlier reports, sequencing of intron-1 of this sodium channel gene revealed no fixed differences between M form resistant and susceptible individuals. The mutation may have recently arisen independently in the M form on Bioko due to recent and intensive pyrethroid application.

Genetic variation in the sand fly salivary protein, SP-15, a potential vaccine candidate against Leishmania major.

SP-15 is a sandfly salivary protein that provides strong protection against cutaneous leishmaniasis, caused by Leishmania major, and has been proposed as a potential vaccine against this disease. To investigate possible antigenic variation in this protein, we examined genetic polymorphism of SP-15 in 100 Phlebotomus papatasi sandflies, from a natural population from Sudan and four laboratory colonies from Egypt, Jordan, Israel and Saudi Arabia. We found that although many variants of SP-15 may be found in nature, differences among them are minimal (mean+/-SD pairwise differences=1.69+/-0.83% for forty nucleotide sequences and 3.06+/-1.13% for thirty amino acid sequence variants). Analysis of proportions of synonymous and non-synonymous substitutions indicated that SP-15 is not under diversifying selection. Our results suggest that a vaccine based on SP-15 protein should result in a uniform immune response.