Tsetse fly evolution, genetics and the trypanosomiases - A review.

This reviews work published since 2007. Relative efforts devoted to the agents of African trypanosomiasis and their tsetse fly vectors are given by the numbers of PubMed accessions. In the last 10 years PubMed citations number 3457 for Trypanosoma brucei and 769 for Glossina. The development of simple sequence repeats and single nucleotide polymorphisms afford much higher resolution of Glossina and Trypanosoma population structures than heretofore. Even greater resolution is offered by partial and whole genome sequencing. Reproduction in T. brucei sensu lato is principally clonal although genetic recombination in tsetse salivary glands has been demonstrated in T. b. brucei and T. b. rhodesiense but not in T. b. gambiense. In the past decade most genetic attention was given to the chief human African trypanosomiasis vectors in subgenus Nemorhina e.g., Glossina f. fuscipes, G. p. palpalis, and G. p. gambiense. The chief interest in Nemorhina population genetics seemed to be finding vector populations sufficiently isolated to enable efficient and long-lasting suppression. To this end estimates were made of gene flow, derived from FST and its analogues, and Ne, the size of a hypothetical population equivalent to that under study. Genetic drift was greater, gene flow and Ne typically lesser in savannah inhabiting tsetse (subgenus Glossina) than in riverine forms (Nemorhina). Population stabilities were examined by sequential sampling and genotypic analysis of nuclear and mitochondrial genomes in both groups and found to be stable. Gene frequencies estimated in sequential samplings differed by drift and allowed estimates of effective population numbers that were greater for Nemorhina spp than Glossina spp. Prospects are examined of genetic methods of vector control. The tsetse long generation time (c. 50 d) is a major contraindication to any suggested genetic method of tsetse population manipulation. Ecological and modelling research convincingly show that conventional methods of targeted insecticide applications and traps/targets can achieve cost-effective reduction in tsetse densities.

Tsetse flies: genetics, evolution, and role as vectors.

Tsetse flies (Diptera: Glossinidae) are an ancient taxon of one genus, Glossina, and limited species diversity. All are exclusively haematophagous and confined to sub-Saharan Africa. The Glossina are the principal vectors of African trypanosomes Trypanosoma sp. (Kinetoplastida: Trypanosomatidae) and as such, are of great medical and economic importance. Clearly tsetse flies and trypanosomes are coadapted and evolutionary interactions between them are manifest. Numerous clonally reproducing strains of Trypanosoma sp. exist and their genetic diversities and spatial distributions are inadequately known. Here I review the breeding structures of the principle trypanosome vectors, G. morsitans s.l., G. pallidipes, G. palpalis s.l. and G. fuscipes fuscipes. All show highly structured populations among which there is surprisingly little detectable gene flow. Rather less is known of the breeding structure of T. brucei sensu lato vis à vis their vector tsetse flies but many genetically differentiated strains exist in nature. Genetic recombination in Trypanosoma via meiosis has recently been demonstrated in the laboratory thereby furnishing a mechanism of strain differentiation in addition to that of simple mutation. Spatially and genetically representative sampling of both trypanosome species and strains and their Glossina vectors is a major barrier to a comprehensive understanding of their mutual relationships.

Structure of some East African Glossina fuscipes fuscipes populations.

Glossina fuscipes fuscipes Newstead 1910 (Diptera: Glossinidae) is the primary vector of human sleeping sickness in Kenya and Uganda. This is the first report on its population structure. A total of 688 nucleotides of mitochondrial ribosomal 16S2 and cytochrome oxidase I genes were sequenced. Twenty-one variants were scored in 79 flies from three geographically diverse natural populations. Four haplotypes were shared among populations, eight were private and nine were singletons. The mean haplotype and nucleotide diversities were 0.84 and 0.009, respectively. All populations were genetically differentiated and were at demographic equilibrium. In addition, a longstanding laboratory culture originating from the Central African Republic (CAR-lab) in 1986 (or before) was examined. Haplotype and nucleotide diversities in this culture were 0.95 and 0.012, respectively. None of its 27 haplotypes were shared with the East African populations. A first approximation of relative effective population sizes was Uganda > CAR-lab > Kenya. It was concluded that the structure of G. f. fuscipes populations in East Africa is localized.

Phylogeography of stable fly (Diptera: Muscidae) estimated by diversity at ribosomal 16S and cytochrome oxidase I mitochondrial genes.

The blood-feeding cosmopolitan stable fly, Stomoxys calcitrans L. (Diptera: Muscidae), is thought to disperse rapidly and widely, and earlier studies of allozyme variation were consistent with high vagility in this species. The geographic origins of New World populations are unknown. Diversity at mitochondrial loci r16S and cytochrome oxidase I was examined in 277 stable flies from 11 countries, including five zoogeographical regions. Of 809 nucleotides, 174 were polymorphic and 133 were parsimony informative. Seventy-six haplotypes were found in frequencies consistent with the Wright-Fisher infinite allele model. None were shared among four or more zoogeographical regions. The null hypothesis of mutation neutrality was not rejected, thereby validating the observed distribution. Fifty-nine haplotypes were singular, eight were private and confined to the Old World, and three of 76 haplotypes were shared between the Old and New World. Only 19 haplotypes were found in the New World, 14 of which were singletons. Haplotype and nucleotide diversities were heterogeneous among countries and regions. The most diversity was observed in sub-Saharan Africa. Regional differentiation indices were C(RT) = 0.26 and N(RT) = 0.31, indicating populations were highly structured macrogeographically. Palearctic and New World flies were the least differentiated from each other. There were strong genetic similarities among populations in the Nearctic, Neotropical, and Palearctic regions, and it is most likely that New World populations were derived from the Palearctic after 1492 CE, in the colonial era.

Patterns of genetic diversity and differentiation in the tsetse fly Glossina morsitans morsitans Westwood populations in East and southern Africa.

Genetic diversity and differentiation within and among nine G. morsitans morsitans populations from East and southern Africa was assessed by examining variation at seven microsatellite loci and a mitochondrial locus, cytochrome oxidase (COI). Mean COI diversity within populations was 0.63+/-0.33 and 0.81 taken over all populations. Diversities averaged over microsatellite loci were high (mean number of alleles/locus>or=7.4; mean HE>or=65%) in all populations. Diversities averaged across populations were greater in East Africa (mean number of alleles=22+/-2.6; mean he=0.773+/-0.033) than in southern Africa (mean number of alleles=18.7+/-4.0; mean he=0.713+/-0.072). Differentiation among all populations was highly significant (RST=0.25, FST=0.132). Nei's Gij statistics were 0.09 and 0.19 within regions for microsatellites and mitochondria, respectively; between regions, Gij was 0.14 for microsatellites and 0.23 for mitochondria. GST among populations was 0.23 for microsatellite loci and 0.40 for mitochondria. The F, G and R statistics indicate highly restricted gene flow among G. m. morsitans populations separated over geographic scales of 12-917 km.

New polymorphic microsatellites in Glossina pallidipes (Diptera: Glossinidae) and their cross-amplification in other tsetse fly taxa.

We report the development and characterization of three new microsatellite markers in the tsetse fly, Glossina pallidipes (Diptera: Glossinidae). Fifty-eight alleles were scored in 192 individuals representing six natural populations. Allelic diversity ranged from 9 to 28 alleles per locus (mean 19.3 +/- 5.5). Averaged across loci, observed heterozygosity was 0.581 +/- 0.209, and expected heterozygosity was 0.619 +/- 0.181. Cross-species amplifications of the G. pallidipes loci in other tsetse fly taxa are reported.

Performance of the Nzi and other traps for biting flies in North America.

The performance of Nzi traps for tabanids (Tabanus similis Macquart, T. quinquevittatus Wiedemann, Chrysops aberrans Philip, C. univittatus Macquart, C. cincticornis Walker, Hybomitra lasiophthalma (Macquart)), stable flies (Stomoxys calcitrans Linnaeus) (Diptera: Muscidae) and mosquitoes (Aedes) (Diptera: Culicidae) was investigated at various sites in Canada (Ontario, Alberta) and USA (Iowa, Florida, Louisiana). Traps made from selected fabrics, insect nettings and hand-dyed blue cotton were compared to the African design to provide practical recommendations for temperate environments. Comparisons of substituted materials showed that trap performance was optimal only when traps were made from appropriate fabrics in the colours produced by either copper phthalocyanine (phthalogen blue), or its sulphonated forms (turquoise). Fabrics dyed with other blue chromophores were not as effective (anthraquinone, disazo, formazan, indanthrone, triphenodioxazine). An appropriate texture as well as an appropriate colour was critical for optimal performance. Smooth, shiny synthetic fabrics (polyester, nylon) and polyester blends reduced catches. Low catches occurred even for nominal phthalogen blue, but slightly-shiny, polyester fabrics in widespread use for tsetse. The most suitable retail fabric in place of phthalogen blue cotton was Sunbrella Pacific Blue acrylic awning/marine fabric. It was both attractive and durable, and had a matching colour-fast black. Nzi traps caught grossly similar numbers of biting flies as canopy, Vavoua, and Alsynite cylinder traps, but with differences in relative performance among species or locations.

Glossina swynnertoni (Diptera: Glossinidae): effective population size and breeding structure estimated by mitochondrial diversity.

Nucleotide diversity was examined at mitochondrial COI and r16S2 loci in eight Glossina swynnertoni Austen collections from northern Tanzania and from a culture maintained by the International Atomic Energy Agency. Eighteen composite haplotypes were observed among 149 flies, two of which were common to all samples and 10 were private. Mean haplotype diversity was 0.59 and nucleotide diversity was 0.0013. There were excess singular haplotypes and mutation-drift disequilibrium suggesting that populations had experienced an earlier bottleneck and subsequent expansion. Factorial correspondence analysis showed that haplotype frequencies varied much more temporally (G ST=0.18) than spatially (G ST=0.04). The estimate of effective population size N e in Tarangire was a harmonic mean approximately 50 reproductive flies averaged over approximately 47 generations. The mean rate of gene flow was estimated to be approximately 5+/-1 reproducing females per generation but inflated because of mutation-drift disequilibrium arising from likely earlier bottlenecks.

Shared microsatellite loci in Glossina morsitans sensu lato (Diptera: Glossinidae).

Estimation of allelic frequencies at three microsatellite loci among 20 populations of Glossina morsitans morsitans Westwood, Glossina morsitans submorsitans Newstead, and Glossina morsitans centralis Machado indicated only two of 99 alleles were shared between three subspecies and 18 between any two subspecies; 81 alleles were unshared. The conserved flanking regions of each locus were completely shared. Genetic differentiation among subspecies, based on allele size, was RST = 0.87, close to the theoretic maximum value. All evidence suggests longstanding and complete reproductive isolation in nature among the sibling species. They should be elevated to specific rank.

Microgeographical breeding structure of the tsetse fly, Glossina pallidipes in south-western Kenya.

The origins of extant Glossina pallidipes Austen (Diptera: Glossinidae) populations in the ecologically well-studied Lambwe and Nguruman valleys in Kenya are controversial because populations have recovered after seemingly effective attempts to achieve high levels of control. The microgeographical breeding structure of the tsetse fly, G. pallidipes, was investigated by analysing spatial and temporal variation at eight microsatellite loci to test hypotheses about endemism and immigration. Samples were obtained at seasonal intervals from trap sites separated by 200 m to 14 km and arranged into blocks. G. pallidipes populations nearest to Lambwe and Nguruman also were sampled. Spatial analysis indicated that genetic differentiation by genetic drift was much less among trapping sites within Lambwe and Nguruman (F(ST) < or = 0.049) than between them (F(ST) = 0.232). F(ST) between Serengeti and Nguruman was 0.16 and F(ST) between Kodera Forest and Lambwe was 0.15. The genetic variance in G. pallidipes explained by dry and wet seasons (0.33%) was about one-fifth the variance among collection dates (1.6%), thereby indicating reasonable temporal stability of genetic variation. Gene frequencies in Kodera and Serengeti differed greatly from Lambwe and Nguruman, thereby falsifying the hypothesis that Lambwe and Nguruman were repopulated by immigrants. Harmonic mean effective (= breeding) population sizes were 180 in Lambwe and 551 in Nguruman. The genetic data suggest that G. pallidipes in Lambwe and Nguruman have been endemic for long intervals.

Macrogeographic population structure of the tsetse fly, Glossina pallidipes (Diptera: Glossinidae).

Tsetse flies are confined to sub-Saharan Africa where they occupy discontinuous habitats. In anticipation of area-wide control programmes, estimates of gene flow among tsetse populations are necessary. Genetic diversities were partitioned at eight microsatellite loci and five mitochondrial loci in 21 Glossina pallidipes Austin populations. At microsatellite loci, Nei's unbiased gene diversity averaged over loci was 0.659 and the total number of alleles was 214, only four of which were shared among all populations. The mean number of alleles per locus was 26.8. Random mating was observed within but not among populations (fixation index FST=0.18) and 81% of the genetic variance was within populations. Thirty-nine mitochondrial variants were detected. Mitochondrial diversities in populations varied from 0 to 0.85 and averaged 0.42, and FST=0.51. High levels of genetic differentiation were characteristic, extending even to subpopulations separated by tens and hundreds of kilometres, and indicating low rates of gene flow.

Geographic differentiation in the house fly estimated by microsatellite and mitochondrial variation.

Gene flow over very large geographic scales has been investigated in few species. Examples include Drosophila melanogaster, Drosophila subobscura, Drosophila simulans, and the Mediterranean fruit fly (Ceratitis capitata). The cosmopolitan house fly, a highly vagile, fecund, colonizing species offers an additional exemplar. Genotypes at seven microsatellite loci were scored in 14 widely separated natural house fly populations from the Nearctic, neotropics, Afrotropics, Palearctic, and Asia. Allelic diversities and heterozygosities differed significantly among populations. Averaged over all populations, Weir and Cockerham's theta = 0.13 and RST = 0.20. Pairwise genetic distance measures were uncorrelated with geographic distance. Microsatellite frequencies were compared with mitochondrial data from 13 of the same populations in which theta = 0.35 and Nei's GST = 0.72. Mitochondrial variation indicated up to threefold greater indices of genetic differentiation than the microsatellites. We were unable to draw any biogeographical inferences from these results or from tree or network topologies constructed from the genetic data. It is likely that high microsatellite diversities, mutation rates, and homoplasy greatly compromised their usefulness in estimating gene flow. House fly colonization dynamics include a large number of primary and secondary colonizations coupled with substantial genetic drift, but no detectable bottlenecks.

Population genetics of wood-feeding cockroaches in the genus Cryptocercus.

Members of the genus Cryptocercus are xylophagous, wingless, subsocial cockroaches that inhabit decaying logs in temperate forests. Given their winglessness, subsocial living, and the patchy distribution of food resources (decomposing logs), it is likely that Cryptocercus populations are substructured. Allozyme variation at eight polymorphic loci was assayed for 10 subpopulations of Cryptocercus darwini and 13 subpopulations of Cryptocercus wrighti, both of which are distributed in the Appalachian Mountains. The mean F(IS) was 0.13 and F(ST) was about 0.25 for both C. darwini and C. wrighti. The relatedness among individuals of a subpopulation of both species was not significantly different from that expected among full sibs. In terms of how genetic variation is partitioned, C. darwini and C. wrighti differed from each other substantially. Most of the genetic variation occurred among subpopulations of C. wrighti in the same region and among subpopulations of C. darwini in different regions. We discuss the factors that may have contributed to the observed similarities and differences in the breeding structure of the two species.

North American face flies Old World origins: mitochondrial evidence.

Analysis of a 513 base sequence of mitochondrial cytochrome oxidase I gene in Musca autumnalis De Geer (Diptera: Muscidae) from the U.S.A., England, Russia and Kazakhstan confirms that North American flies originated in Western Europe. Flies from the U.S.A., England and southern Russia shared most of their mitochondrial diversities, but face flies from Kazakhstan were substantially dissimilar, suggesting highly restricted gene flow and a species complex within the Palearctic.

Spatial diversity in mitochondrial cytochrome c oxidase in house flies.

DNA sequence analysis at mitochondrial gene COI was surveyed in 293 house flies, Musca domestica Linneaus (Diptera: Muscidae), in 29 populations from North, Central and South America, Europe, Asia, Africa, and the Western Pacific. Nei's gene diversity index (H(S)) was 0.47, the chance that two randomly chosen flies have different COI haplotypes. Haplotype diversity was greater in the Old World (H(S) = 0.58) than the New World (H(S) = 0.31). The hierarchical partition of the total diversity indicated substantial differentiation at all levels (G(ST) = 0.30), and highly structured populations. All pairwise estimates of gene flow between zoogeographical regions were less than 0.70 reproducing females per generation. The results are compared to those of a similar study based on the single-strand conformation polymorphism method. Probable colonization scenarios for house flies into the New World are discussed and it is concluded that house flies are a recent addition to the fauna of the Western Hemisphere.

Tsetse genetics: contributions to biology, systematics, and control of tsetse flies.

Tsetse flies (Diptera: Glossinidae) constitute a small, ancient taxon of exclusively hematophagous insects that reproduce slowly and viviparously. Because tsetse flies are the only vectors of pathogenic African trypanosomes, they are a potent and constant threat to humans and livestock over much of sub-Saharan Africa. Despite their low fecundity, tsetse flies demonstrate great resilience, which makes population suppression expensive, transient, and beyond the capacities of private and public sectors to accomplish, except over small areas. Nevertheless, control measures that include genetic methods are under consideration at national and supranational levels. There is a pressing need for sufficient laboratory cultures of tsetse flies and financial support to carry out genetic research. Here we review tsetse genetics from organismal and population points of view and identify some research needs.

Mitochondrial diversity analysis of Glossina palpalis gambiensis from Mali and Senegal.

West African riverine tsetse populations of Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae) were investigated for gene flow, inferred from mitochondrial diversity in samples of 69 flies from Senegal and 303 flies from three river drainages in Mali. Four polymorphic mitochondrial loci were scored. Mean haplotype diversities were 0.30 in Mali, 0 in Senegal and 0.18 over both Mali and Senegal. These diversities estimate the probabilities that two randomly chosen tsetse have different haplotypes. Substantial rates of gene flow were detected among flies sampled along tributaries belonging to the river basins of the Senegal, Niger, and Bani in Mali. There was virtually no gene flow between tsetse in Senegal and Mali. No seasonal effects on gene flow were detected. The implications of these preliminary findings for the implementation of area-wide integrated pest management (AW-IPM) programmes against riverine tsetse in West Africa are discussed.

Mitochondrial diversity of Musca domestica housefly populations in the Asian and western Pacific biogeographical regions.

Houseflies (Musca domestica L., Diptera: Muscidae) are cosmopolitan, colonizing, and eusynanthropic. Their distribution in the Malaysian archipelago provides an opportunity to study successive waves of colonization and extinction during the Pleistocene and Recent epochs. We scored single-strand conformation polymorphisms (SSCPs) at 16S2 and COII mitochondrial loci in 47 housefly samples from the Australian, Austro-Malayan, Indo-Malayan, Manchurian and Indo-Chinese subregions of Wallace's zoogeographical classification. We discuss the results in light of the Pleistocene vs. post-Pleistocene dispersal and faunal exchange in the Asia-Pacific area. Fourteen haplotypes were detected, of which 10 were confined to a single subregion. No haplotype was ubiquitous and only one was found in four subregions. Population diversity, HS, was greatest in the Indo-Malayan (0.36) and heterogeneous among subregions. The mean subregional diversity was 0.21 +/- 0.03, representing the probability that two randomly chosen flies, from any subregion, had different haplotypes. The hierarchical partition of diversity indicated restricted maternal gene flow among subregions (GRT = 0.60, Nm approximately 0.32). These results suggest long-standing genetic isolation of houseflies in the Malaysian archipelago and support the hypothesis that they dispersed widely during the Pleistocene. Haplotypes common among mainland populations but shared with island groups in low frequencies (<1%) indicate surprisingly little recent gene flow.

Mitochondrial diversity evaluated by the single strand conformation polymorphism method in African and North American house flies (Musca domestica L.).

Single strand conformation polymorphisms (SSCPs) provide a convenient and inexpensive method of surveying mitochondrial genetic variation in large samples. We investigated how much variation should be incorporated into such surveys by scoring SSCP variation at eight mitochondrial loci in each of four sub-Saharan African and four North American house fly (Diptera: Muscidae.) populations. Hierarchical analysis of diversity was performed on haplotype frequencies at each locus and on haplotype frequencies formed by combining haplotypes from two, three, four, five and eight loci. Composite haplotypes at two loci were as informative about population structure as those composed of a greater number of loci. Increasing the number of loci increased diversity estimates within, but not between, populations. Mean composite haplotype diversities (16S2 and COII) were 0.49 +/- 0.09 among the African populations and 0.32 +/- 0.08 among the North American populations. Only two of 16 haplotypes were shared between continents. Nei's genetic differentiation statistic between populations in continents GPC was 0.30 +/- 0.06 and mean genetic differentiation between continents GCT was 0.39 +/- 0.06. We conclude that there has been little detectable gene flow between North America and sub-Saharan Africa.

Mitochondrial diversity in new world house flies (Diptera: Muscidae).

Mitochondrial diversity in house flies was examined by using the single-strand conformation polymorphism method in house flies, Musca domestica L. sampled in six zoogeographical subregions in the New World. The number of haplotypes and haplotype diversities were homogeneous among subregions, but a strong spatial component was found in the distribution of particular haplotypes. Nei's differentiation index among subregions, GRT, was 0.53 and that among populations within subregions, GPR, was 0.31. Greater genetic differentiation was found among populations in the Nearctic than in the Neotropics. Haplotype frequency distributions in two of three Nearctic subregions deviated from that expected under the neutral infinite allele model, suggesting the existence of differential selection patterns.