Anopheles melas is a brackish water mosquito found along the coast of West-Africa where it can be the dominant malaria vector locally. In order to facilitate genetic studies of this species and to examine the usefulness of microsatellite markers when used in a sibling species, 45 microsatellite loci originally developed for Anopheles gambiae were sequenced in An. melas. These loci were evaluated on their suitability as polymorphic markers based on repeat structure, length, and polymorphism in wild An. melas populations. Of the 45 loci, 18 were not considered promising markers in An. melas. A total of 48 out of 90 An. gambiae primers contained at least one mismatch with the An. melas annealing site. An. melas-specific primers were designed for 27 loci, and their variability was examined in two wild populations from Equatorial Guinea. Based on a low level of polymorphism, Hardy-Weinberg disequilibrium, or poor amplification, a further 12 loci were excluded. The remaining fifteen loci were screened in four additional wild populations from a wider geographic region including Equatorial Guinea, Cameroon, The Gambia, and Guinea Bissau. These loci showed an average heterozygosity ranging from 0.18 to 0.79, with 2.5 to 15 average alleles per locus, yielding 13 highly polymorphic markers and two loci with more limited variability in a wide geographic region. To examine the effects of cross species amplification, five of the original An. gambiae markers were also amplified in the An. melas populations. Null alleles were found for one of these An. gambiae markers. We discuss the pitfalls of using microsatellite loci even in a very closely related species, and conclude that in addition to the well-known 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. Fifteen An. melas-specific markers were subsequently amplified and analyzed in 11 wild An. melas populations from throughout the range of this species, including Bioko Island, Equatorial Guinea. We analyzed pair-wise population differentiation between all populations, and found that all but two comparisons were significant (p-val.<0.05), and populations clustered into three distinct groups representing Bioko Island, Central Africa, and West Africa populations. A Bayesian clustering analysis found little, if any, evidence for migration from mainland to Bioko Island populations, although there was evidence of migration from Bioko Island to the West population cluster, and from the Central to the West population cluster. Simulations of historical gene followed these same patterns and further support our predictions of unidirectional gene flow. Comparison of 1161 nucleotides amplified and sequenced from the ND4 and ND5 regions of the mtDNA showed that differentiation between An. melas population clusters is on par with levels of differentiation between member species of the An. gambiae complex, with low support for internal nodes in a maximum likelihood tree, which suggests that observed An. melas clusters are not monophyletic. From this we hypothesize that Bioko Island An. melas populations are derived from Tiko, Cameroon, and that these populations became isolated from one another when sea levels rose after the last glaciation period (?10,000-11,000 years ago), cutting off Bioko Island populations from the mainland and significantly reducing migration. Our conclusions have implications for vector control within the region, as Bioko Island is the subject of an intensive malaria control campaign, and the lack of migration from mainland West Africa to Bioko Island make it unlikely that eradicated populations of this malaria vector will be repopulated by mainland immigrants.

Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Understanding the chromosome evolution and mechanisms of speciation can shed light on developing novel ecological-friendly vector control techniques. Sibling species of the An. gambiae complex provide an excellent model system for these topics. To understand the mechanisms of speciation, we investigated the cellular basis and phenotypes of hybrid male sterility in species crosses of the An. gambiae complex. By performing inter-species crosses of An. coluzzii/An. gambiae and An. merus lab strains, we found an asymmetric pattern of hybrid male sterility existed in sons from reciprocal interspecies crosses. Compared with pure species, hybrid males from crosses of ♀An. merus  ♂An. gambiae/An. coluzzii were normal in the morphology of male reproductive tracts; however, the testes of which that process the reductional meiotic division failed to produce primary spermatocytes and were accompanied with unpaired and insufficiently condensed chromosomes. As a result, primary spermatocytes undergo a mitosis-like anaphase division, producing nonmotile and malfunctional diploid sperm with two tails. However, individuals can mate with females normally and form the mating plug to induce the female monogamy. In contrast, hybrid males from the opposite crosses manifest severely underdeveloped reproductive tracts and a premeiotic arrest of germline stem cells in the testis, accompanied by a strong suppression of premeiotic and meiotic genes. In addition, hybrid males from this cross suffered from a shorter copulation time and failed to form mating plugs to induce female monogamous behaviors, albeit the expression of male accessory gland specific genes were similar between hybrids and pure species. To figure out chromosome evolution in the An. gambiae complex, we studied the molecular organization of heterochromatin and investigated the spatial organizations of autosomal regions of polytene chromosomes in soma and germline cells. We found that molecular composition of pericentrometric autosome and sex chromosome repetitive DNA differs among sibling species of An. gambiae complex with highly similarity between An. coluzzii and An. arabiensis. In addition, heterochromatin blocks of chromosomes have distinct compositions of satellite DNA sequences. Next, in order to address the relationship between inter-chromosomal (Chr-Chr) contacts and chromosome-nuclear envelope (Chr-NE) attachments during the development of the organism, we conducted microscopic analyses of the 3D organization of polytene chromosome in An. gambiae, An. coluzzii, and An. merus. Our quantitative study on chromosome territories in larval salivary gland cells and adult ovarian nurse cells showed that, compared with autosomal arms, the X chromosome has a significantly smaller volume and occupies more compact territories. The number of Chr-Chr contacts and the percentage of Chr-NE attachment were conserved among the species within the same cell type. Our data also demonstrated that there is a significantly and consistently inverse relationship between the frequencies of Chr–NE and Chr–Chr attachments on autosomes of two cell types in all tested species. / Doctor of Philosophy / Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Despite being treatable and preventable, malaria is estimated to cause large numbers of deaths every year. Since 2015, the malaria elimination program has stalled largely due to increased insecticide resistance. Novel transgenic techniques have a huge potential in reducing malaria transmission more effectively. However, there are large concerns about the potential negative effects of releasing genetically modified mosquitoes, such as a possibility of accidental spread to non-target species with incomplete reproductive barriers and unpredicted ecological damage. Understanding the mechanisms of speciation about how reproductive isolation occurred and developed as well as chromosome evolution can not only empower the development of ecologically friendly vector control techniques but also improve our basic knowledge. To study mechanisms of speciation, we mated males and females from different closely related species in the Anopheles gambiae complex to investigate the fecundity of hybrid generations. Our study identified two different types of reproductive abnormalities leading to hybrid male sterility. Hybrid males from female An. merus and male An. gambiae or An. coluzzii have normal appearing testes and male accessary glands but the testes produce abnormal sperms, which cannot move and have two tails. Hybrid males from female An. gambiae or An. coluzzii and An. merus have severely underdeveloped testes and male accessary glands. The sperm producing process stops unusually very early in their tiny underdeveloped testes. We also investigated chromosome evolution in species of An. gambiae complex. We found that chromosomal parts containing repetitive DNA, the sequence in the genome not producing proteins, evolve rapidly in An. coluzzii, An. arabiensis, An. quadriannulatus, and An. merus. In contrast, chromosome territories of gene rich regions in giant polytene chromosomes from larval salivary gland cells and adult ovarian nurse cells of An. gambiae, An. coluzzii, and An. merus, were relatively conserved within the same cell type among different species. However, the chromosomal 3D distribution pattern is different among various cell types in these species.