Cytogenetic and biometric observations on members of the Anopheles gambiae complex in Mozambique

Four species of the Anopheles gambiae complex were identified in Mozambique (East Africa) by chromosomal analysis. They were An. merus, An. gambiae s.s., An. arabiensis and An. quadriannulatus. An. merus was observed in coastal zones as well as in inner areas where the rivers are tidal and brackish and/or the soil is salty. An. gambiae s.s. is present in the central-northern regions (north of Save river) from the coast to the western mountains. On the coast it is often sympatric with An. merus. It is apparently absent south of Save river. An. arabiensis was observed in samples from the north-western hilly and mountainous areas, sympatrically with An. gambiae s.s., as well as south of Save river where often it is sympatric with An. merus. Only one specimen of An. quadriannulatus was observed. It was from a small sample collected feeding on bovid in a southern locality (Bela Vista-Maputo area). No inversion polymorphism was observed in the 446 An. merus identified. A quite low degree of inversion polymorphism was shown by both An. gambiae s.s. and An. arabiensis, involving 2Rb, 2La and 2Rb inversion systems respectively. By morphological analysis of cytogenetically identified samples of three species of the complex, number of sensilla coeloconica and palpal ratio were confirmed to be useful to distinguish An. merus from An. gambiae s.s./An. arabiensis. The overlapping areas between brackish and freshwater species become smaller when both characters are considered together, 1.5% and 3.5% being the probabilities of confusing An. merus with An. gambiae s.s. and An. arabiensis respectively.     

Multiple origins of cytologically identical chromosome inversions in the Anopheles gambiae complex.

For more than 60 years, evolutionary cytogeneticists have been using naturally occurring chromosomal inversions to infer phylogenetic histories, especially in insects with polytene chromosomes. The validity of this method is predicated on the assumption that inversions arise only once in the history of a lineage, so that sharing a particular inversion implies shared common ancestry. This assumption of monophyly has been generally validated by independent data. We present the first clear evidence that naturally occurring inversions, identical at the level of light microscopic examination of polytene chromosomes, may not always be monophyletic. The evidence comes from DNA sequence analyses of regions within or very near the breakpoints of an inversion called the 2La that is found in the Anopheles gambiae complex. Two species, A. merus and A. arabiensis, which are fixed for the "same" inversion, do not cluster with each other in a phylogenetic analysis of the DNA sequences within the 2La. Rather, A. merus 2La is most closely related to strains of A. gambiae homozygous for the 2L+. A. gambiae and A. merus are sister taxa, the immediate ancestor was evidently homozygous 2L+, and A. merus became fixed for an inversion cytologically identical to that in A. arabiensis. A. gambiae is polymorphic for 2La/2L+, and the 2La in this species is nearly identical at the DNA level to that in A. arabiensis, consistent with the growing evidence that introgression has or is occurring between these two most important vectors of malaria in the world. The parallel evolution of the "same" inversion may be promoted by the presence of selectively important genes within the breakpoints.     

Distribution of the species of the Anopheles gambiae complex and first evidence of Anopheles merus as a malaria vector in Madagascar

BACKGROUND: Members of the Anopheles gambiae complex are amongst the best malaria vectors in the world, but their vectorial capacities vary between species and populations. A large-scale sampling of An. gambiae sensu lato was carried out in various bioclimatic domains of Madagascar. Local abundance of an unexpected member of this complex raised questions regarding its role in malaria transmission. METHODS: Sampling took place at 38 sites and 2,067 females were collected. Species assessment was performed using a PCR targeting a sequence in the IGS of the rDNA. Analysis focused on the relative prevalence of the species per site, bioclimatic domain and altitude. Infectivity of Anopheles merus was assessed using an ELISA to detect the presence of malarial circumsporozoite protein in the head-thorax. RESULTS: Three species were identified: An. gambiae, Anopheles arabiensis and An. merus. The distribution of each species is mainly a function of bioclimatic domains and, to a lesser extent, altitude. An. arabiensis is present in all bioclimatic domains with highest prevalence in sub-humid, dry and sub-arid domains. An. gambiae has its highest prevalence in the humid domain, is in the minority in dry areas, rare in sub-humid and absent in sub-arid domains. An. merus is restricted to the coastal fringe in the south and west; it was in the majority in one southern village. The majority of sites were sympatric for at least two of the species (21/38) and two sites harboured all three species.The role of An. merus as malaria vector was confirmed in the case of two human-biting females, which were ELISA-positive for Plasmodium falciparum. CONCLUSION: Despite the huge environmental (mainly man-made) changes in Madagascar, the distribution of An. gambiae and An. arabiensis appears unchanged for the past 35 years. The distribution of An. merus is wider than was previously known, and its effectiveness as a malaria vector has been shown for the first time; this species is now on the list of Malagasy malaria vectors.     

hAT element population genetics in Anopheles gambiae s.l. in Mozambique

Herves is a functional Class II transposable element in Anopheles gambiae belonging to the hAT superfamily of elements. Class II transposable elements are used as gene vectors in this species and are also being considered as genetic drive agents for spreading desirable genes through natural populations as part of an effort to control malaria transmission. In this study, Herves was investigated in populations of Anopheles gambiae s.s., Anopheles arabiensis and Anopheles merus in Mozambique over a period of 2 years. The copy number of Herves within these three species was approximately 5 copies per diploid genome and did not differ among species or between years. Based on the insertion-site occupancy-frequency distribution and existing models of transposable element dynamics, Herves appears to be transpositionally active currently or, at least recently, in all species tested. Ninety-five percent of the individuals within the populations of the three species tested contained intact elements with complete Herves transposase genes and this is consistent with the idea that these elements are currently active.     

Inversion Monophyly in African Anopheline Malaria Vectors

The African Anopheles gambiae complex of six sibling species has many polymorphic and fixed paracentric inversions detectable in polytene chromosomes. These have been used to infer phylogenetic relationships as classically done with Drosophila. Two species, A. gambiae and A. merus, were thought to be sister taxa based on a shared X inversion designated X(ag). Recent DNA data have conflicted with this phylogenetic inference as they have supported a sister taxa relationship of A. gambiae and A. arabiensis. A possible explanation is that the X(ag) is not monophyletic. Here we present data from a gene (soluble guanylate cyclase) within the X(ag) that strongly supports the monophyly of the X(ag). We conjecture that introgression may be occurring between the widely sympatric species A. gambiae and A. arabiensis and that the previous DNA phylogenies have been detecting the introgression. Evidently, introgression is not uniform across the genome, and species-specific regions, like the X-chromosome inversions, do not introgress probably due to selective elimination in hybrids and backcrosses.     

P elements are found in the genomes of nematoceran insects of the genus Anopheles

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.     

Change in composition of the Anopheles gambiae complex and its possible implications for the transmission of malaria and lymphatic filariasis in north-eastern Tanzania

ABSTRACT: BACKGROUND: A dramatic decline in the incidence of malaria due to Plasmodium falciparum infection in coastal East Africa has recently been reported to be paralleled (or even preceded) by an equally dramatic decline in malaria vector density, despite absence of organized vector control. As part of investigations into possible causes for the change in vector population density, the present study analysed the Anopheles gambiae s.l. sibling species composition in north-eastern Tanzania. METHODS: The study was in two parts. The first compared current species complex composition in freshly caught An. gambiae s.l. complex from three villages to the composition reported from previous studies carried out 2-4 decades ago in the same villages. The second took advantage of a sample of archived dried An. gambiae s.l. complex specimens collected regularly from a fourth study village since 2005. Both fresh and archived dried specimens were identified to sibling species of the An. gambiae s.l. complex by PCR. The same specimens were moreover examined for Plasmodium falciparum and Wuchereria bancrofti infection by PCR. RESULTS: As in earlier studies, An. gambiae s.s., Anopheles merus and Anopheles arabiensis were identified as sibling species found in the area. However, both study parts indicated a marked change in sibling species composition over time. From being by far the most abundant in the past An. gambiae s.s. was now the most rare, whereas An. arabiensis had changed from being the most rare to the most common. P. falciparum infection was rarely detected in the examined specimens (and only in An. arabiensis) whereas W. bancrofti infection was prevalent and detected in all three sibling species. CONCLUSION: The study indicates that a major shift in An. gambiae s.l. sibling species composition has taken place in the study area in recent years. Combined with the earlier reported decline in overall malaria vector density, the study suggests that this decline has been most marked for An. gambiae s.s., and least for An. arabiensis, leading to current predominance of the latter. Due to differences in biology and vectorial capacity of the An. gambiae s.l. complex the change in sibling species composition will have important implications for the epidemiology and control of malaria and lymphatic filariasis in the study area.     

Use of the polymerase chain reaction to identify mosquito species of the Anopheles gambiae complex

A nonradiometric method has been developed for distinguishing between the sibling species Anopheles gambiae Giles and An. arabiensis Patton, two important Afrotropical vectors of malaria. DNA fragments of species diagnostic length are amplified by polymerase chain reaction (PCR) from a small amount of unknown DNA and three different PCR primers. All three PCR primers are based on ribosomal DNA (rDNA) sequences. A universal plus-strand primer (A0) is derived from a conserved region at the 3' end of the 28S rDNA coding region. Two species-specific minus-strand primers (Aa0.5 and Ag1.3) are derived from sequences in the intergenic spacers. The Ag1.3 sequence is approximately 1.3 kb downstream of A0; the Aa0.5 sequence is about 0.5 kb downstream of A0. When mosquito DNA is amplified in the presence of all three primers, a 1.3 kb fragment is produced if An. gambiae DNA is used as template, and a 0.5 kb fragment is produced if An. arabiensis DNA is used. Amplification of DNA from An.gambiae/An. arabiensis hybrids produces both the 1.3 kb and the 0.5 kb fragments. Neither diagnostic fragment is produced when DNA from other species in the An. gambiae complex is used as template.     

MAX, a novel retrotransposon of the BEL-Pao family, is nested within the Bari1 cluster at the heterochromatic h39 region of chromosome 2 in Drosophila melanogaster

A homogeneous array of 80 tandem repeats of the Bari1 transposon is located in the pericentromeric h39 region of chromosome 2 of Drosophila melanogaster. Here, we report that the Bari1 cluster is interrupted by an 8556-bp insertion. DNA sequencing and database searches identified this insertion as a previously unannotated retrotransposon that we have named MAX. MAX possesses two ORFs; ORF1 putatively encodes a polyprotein comprising GAG and RT domains, while ORF2 could encode a 288-amino acid protein of unknown function. Alignment with the RT domains of known LTR retrotransposons shows that MAX belongs to the BEL-Pao family, which remarkable for its widespread presence in different taxa, including lower chordates. We have analyzed the distribution of MAX elements within representative species of the Sophophora subgroup and found that they are restricted to the species of the melanogaster complex, where they are heavily represented in the heterochromatin of all autosomes and on the Y chromosome.Communicated by G. P. Georgiev