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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.

Africa South of the Sahara↗

On the role of blue shadows in the visual behaviour of tsetse flies.

Tsetse flies (Glossina spp.), the vector for African trypanosomiasis, are highly attracted by blue and black surfaces. This phototactic behaviour has long been exploited to trap tsetse flies as one measure in the control of African trypanosomiasis. However, why blue and black are so attractive for tsetse flies is still unknown. We propose that the combination of blue and black is attractive for many Glossina species because when searching for a shady resting place to pass the day, the flies are probably guided by the blueness and darkness of daytime shadows. In contrast to people's experience that daytime shadows are colourless, actually on a sunny day all shadows are tinted bluish by the scattered blue skylight.

Animals↗

Tissue tropism, transmission and expression of foreign genes in vivo in midgut symbionts of tsetse flies.

Tsetse flies (Diptera: Glossinidae) harbour three different symbiotic organisms in addition to the pathogenic African trypanosomes they transmit. The two gut-associated symbionts (primary, P; secondary, S) are enteric and are nutritionally required, whereas the third microorganism Wolbachia (family Rickettsiaceae) affects the reproductive biology of the insects it infects. The bacteriome-associated P-symbiont (Wigglesworthia glossinidia) displays a concordant phylogeny with its host tsetse species, whereas midgut S-symbionts characterized from distant tsetse have identical 16S rDNA sequences and therefore may either represent recent independent acquisitions or horizontal transfer between species. The S-symbionts have been cultured in vitro and a genetic transformation system has been developed. Here we report on their density and tissue tropism in different species (G. m. morsitans, G. p. palpalis, G. austeni and G. brevipalpis) and on their maternal route of transmission to tsetse progeny. Using a bacterium-specific PCR-assay, the S-symbionts were found primarily in the midgut, haemolymph, milk gland and in G. palpalis also in salivary glands of teneral flies. In older flies these infections were found to spread to other tissues including muscle, testes and fat body. The S-symbionts were transformed to express the marker gene product, Green Fluorescent Protein (GFP) in vitro. When the recombinant symbionts were introduced into the haemoceal of fertile female flies via intrathoracic microinjection, they were detected in the intrauterine progeny, indicating that haemolymph may provide a possible route for their transmission. The implications of these results for symbiont-host interactions and for transgenic strategies in tsetse are discussed.

Animals↗

Inhibition of the DNA amplification of trypanosomes present in tsetse flies midguts: implications for the identification of trypanosome species in wild tsetse flies.

The present study was carried out in order to investigate if there was really a failure of PCR in identifying parasitologically positive tsetse flies in the field. Tsetse flies (Glossina palpalis gambiensis and Glossina morsitans morsitans) were therefore experimentally infected with two different species of Trypanosoma (Trypanosoma brucei gambiense or Trypanosoma congolense). A total of 152 tsetse flies were dissected, and organs of each fly (midgut, proboscis or salivary glands) were examined. The positive organs were then analysed using PCR. Results showed that, regardless of the trypanosome species, PCR failed to amplify 40% of the parasitologically positive midguts. This failure, which does not occur with diluted samples, is likely to be caused by an inhibition of the amplification reaction. This finding has important implications for the detection and the identification of trypanosome species in wild tsetse flies.

Animals↗

Ultrastructural localization of unique neurosecretory granules in the corpora cardiaca of the stable fly, Stomoxys calcitrans, and the tsetse fly, Glossina morsitans.

Ultrastructural analysis of the corpora cardiaca of the stable fly, Stomoxys calcitrans, and the tsetse fly, Glossina morsitans, revealed the presence of elementary neurosecretory granules (ENG) unique to the intrinsic neurosecretory cells (INC) of these species. In addition to electron-dense spheres, the INC of the corpus species. In addition to electron-dense spheres, the INC of the corpus cardiacum of the stable fly contain electrondense angular granules, either square or rectangular in shape, while the INC of the tsetse fly contain electron-dense spindle-shaped ENG. The distinctive granules of these INC can be traced within nerves to their sites of storage and release, eliminating the need for labeling with artificial probes. Although the INC of the corpus cardiacum of most species have been found to be fuchsinophilic, neither the INC of the stable fly nor the tsetse fly are aldehyde-fuchsinophilic. These peptigenic cells offer neuroendocrinologists a unique opportunity to study the physiology and biochemistry of neurosecretory cells.

Animals↗

[Improving the salivation technic in the tsetse fly for the detection of infective metatripanosomes: study of the effect of biologic and non-biologic factors in the probing behavior of the tsetse fly].

The probing and salivation behaviour on a warm slide of three tsetse fly species or subspecies (glossina morsitans morsitans, Glossina palpalis gambiensis, Glossina tachinoides) was examined with respect to various parameters (species, sex, age, starvation period, trypanosome infection, quality of support). Each fly was given the opportunity to probe the warm slide (38 degrees C) for 5 minutes (we mean by probing an attempt to touch the glass slide by the proboscis in a biting position). G.m morsitans is by far the most efficient at probing (70.50%) when compared with G. tachinoides (50.50%) and G. palpalis gambiensis (45.80%). Globally, males (61.30%) are more active than females (52%) and those of the morsitans group are more active than those of the palpalis group. Teneral flies probe more easily than non-teneral flies, with an increased advantage in G. m. morsitans. The starvation period increases the probing behaviour, but at 48 h. G. m. morsitans probed as much as G. palpalis gambiensis and G. tachinoides at 72 h. The males of G. m. morsitans and G. palpalis gambiensis are more precocious than females, but the inverse is observed in G. tachinoides. Infection by T. congolense (EATRO 325 strain) does not affect the probing behaviour of males of all 3 species but seems to lower that of females in the palpalis group. Addition of a drop of PSG or blood improves the probing behaviour of infected G. m. morsitans females (the only ones tested). The results are discussed in relation to biological data and knowledge of the receptor systems of tsetse flies.

Animals↗

[Comparison of the longevity of tsetse flies (Glossina morsitans morsitans Westwood, 1850) infected with trypanosomes (Trypanosoma nannomonas congolense Broden, 1904) and uninfected tsetse flies].

This preliminary note results from a comparative study on susceptibility to pyrethroid insecticides of tsetse flies infected and not with trypanosomes. Trypanosoma infection increases the susceptibility to insecticides as previous study showed it (Golder et al., 1982, 1984). Moreover, infected control flies showed a significant lower longevity than uninfected ones.

Animals↗

Spiroplasma endosymbiont reduction of host lipid synthesis and Stomoxyn-like peptide contribute to trypanosome resistance in the tsetse fly Glossina fuscipes.

Tsetse flies (Glossina spp.) vector African trypanosomes that cause devastating diseases in humans and domestic animals. Within the Glossina genus, species in the Palpalis subgroup exhibit greater resistance to trypanosome infections compared to those in the Morsitans subgroup. Varying microbiota composition and species-specific genetic traits can significantly influence the efficiency of parasite transmission. Notably, infections with the endosymbiotic bacterium Spiroplasma have been documented in several Palpalis subgroup species, including Glossina fuscipes fuscipes (Gff). While Spiroplasma infections in Gff are known to hinder trypanosome transmission, the underlying mechanisms remain unknown. To investigate Spiroplasma-mediated factors affecting Gff vector competence, we conducted high-throughput RNA sequencing of the gut tissue along with functional assays. Our findings reveal elevated oxidative stress in the gut environment in the presence of Spiroplasma, evidenced by increased expression of nitric oxide synthase, which catalyzes the production of trypanocidal nitric oxide. Additionally, we observed impaired lipid biosynthesis leading to a reduction of this important class of nutrients essential for parasite and host physiologies. In contrast, trypanosome infections in Gff's midgut significantly upregulated various immunity-related genes, including a small peptide, Stomoxyn-like, homologous to Stomoxyn first discovered in the stable fly, Stomoxys calcitrans. We observed that the Stomoxyn-like locus is exclusive to the genomes of Palpalis subgroup tsetse species. GffStomoxyn is constitutively expressed in the cardia (proventriculus) and synthetic GffStomoxyn exhibits potent activity against Escherichia coli and bloodstream form of Trypanosoma brucei parasites, while showing no effect against insect stage procyclic forms or tsetse's commensal endosymbiont Sodalis in vitro. Reducing GffStomoxyn levels significantly increased trypanosome infection prevalence, indicating its potential trypanocidal role in vivo. Collectively, our results suggest that the enhanced resistance to trypanosomes observed in Spiroplasma-infected Gff may be due to the reduced lipid availability necessary for parasite metabolic maintenance. Furthermore, GffStomoxyn could play a crucial role in the initial immune response(s) against mammalian parasites early in the infection process in the gut and prevent gut colonization. We discuss the molecular characteristics of GffStomoxyn, its spatial and temporal expression regulation and its microbicidal activity against Trypanosome parasites. Our findings reinforce the nutritional influences of microbiota on host physiology and host-pathogen dynamics.

Animals↗

GIS and multiple-criteria evaluation for the optimisation of tsetse fly eradication programmes.

Tsetse flies are the vectors of trypanosomes, the causal agent of trypanosomiasis, a widespread disease of livestock and people in Africa. Control of tsetse may open vast areas of land to livestock-keeping, with the associated benefits of developing mixed crop-livestock production systems. However, as well as possible positive impacts there are also risks: bush clearing would accelerate and cattle numbers would rise, leading to a reduction of vegetation cover, and an increase in runoff and erosion; there may also be increased pressure on conserved areas and reductions in biodiversity. The objective of this study is to show how remotely sensed and other environmental data can be combined in a decision support system to help inform tsetse control programmes in a manner that could be used to limit possible detrimental effects of tsetse control. For Zambia, a methodology is developed that combines a tree-based decision-support approach with the use of Multiple-Criteria Evaluation (MCE), within a Geographical Information System (GIS), in order to target areas for tsetse control. The results show clear differentiation of priority areas under a series of hypothetical scenarios, and some areas (e.g. northwest of Petauke in the Eastern Province of Zambia) are consistently flagged as high priority for control. It is also demonstrated that priority areas do not comprise isolated tsetse populations, meaning that disease control using an integrated approach is likely to be more economically viable than local eradication.

Animals↗

Cyclic AMP is a likely mediator of ovulation in the tsetse fly.

Ovulation in tsetse flies is normally induced by mating, but virgins can be stimulated to ovulate with an injection of dibutyryl cyclic AMP, cholera toxin (a cyclic AMP generator), or aminophylline (a phosphodiesterase inhibitor). Thus, elevation of cyclic AMP is a likely link in the events leading to ovulation.

Aminophylline↗

Tissue distribution and prevalence of Wolbachia infections in tsetse flies, Glossina spp.

Tsetse flies Glossina spp. (Diptera: Glossinidae) harbor three different symbiotic microorganisms, one being an intracellular Rickettsia of the genus Wolbachia. This bacterium infects a wide range of arthropods, where it causes a variety of reproductive abnormalities, one of which is termed cytoplasmic incompatibility (CI) that, when expressed, results in embryonic death due to disruptions in fertilization events. We report here that in colonized flies, Wolbachia infections can be detected in 100% of sampled individuals, while infections vary significantly in field populations. Based on Wolbachia Surface Protein (wsp) gene sequence analysis, the infections associated with different fly species are all unique within the A group of the Wolbachia pipientis clade. In addition to being present in germ-line tissues, Wolbachia infections have been found in somatic tissues of several insects. Using a Wolbachia-specific PCR-based assay, the tissue tropism of infections in Glossina morsitans morsitans Westwood, Glossina brevipalpis Newstead and Glossina austeni Newstead were analysed. While infections in G. m. morsitans and G. brevipalpis were limited to reproductive tissues, in G. austeni, Wolbachia could be detected in various somatic tissues.

Animals↗

Interspecific transfer of bacterial endosymbionts between tsetse fly species: infection establishment and effect on host fitness.

Tsetse flies (Glossina spp.) can harbor up to three distinct species of endosymbiotic bacteria that exhibit unique modes of transmission and evolutionary histories with their host. Two mutualist enterics, Wigglesworthia and Sodalis, are transmitted maternally to tsetse flies' intrauterine larvae. The third symbiont, from the genus Wolbachia, parasitizes developing oocytes. In this study, we determined that Sodalis isolates from several tsetse fly species are virtually identical based on a phylogenetic analysis of their ftsZ gene sequences. Furthermore, restriction fragment-length polymorphism analysis revealed little variation in the genomes of Sodalis isolates from tsetse fly species within different subgenera (Glossina fuscipes fuscipes and Glossina morsitans morsitans). We also examined the impact on host fitness of transinfecting G. fuscipes fuscipes and G. morsitans morsitans flies with reciprocal Sodalis strains. Tsetse flies cleared of their native Sodalis symbionts were successfully repopulated with the Sodalis species isolated from a different tsetse fly species. These transinfected flies effectively transmitted the novel symbionts to their offspring and experienced no detrimental fitness effects compared to their wild-type counterparts, as measured by longevity and fecundity. Quantitative PCR analysis revealed that transinfected flies maintained their Sodalis populations at densities comparable to those in flies harboring native symbionts. Our ability to transinfect tsetse flies is indicative of Sodalis ' recent evolutionary history with its tsetse fly host and demonstrates that this procedure may be used as a means of streamlining future paratransgenesis experiments.

Animals↗

A new Sodalis lineage from bloodsucking fly Craterina melbae (Diptera, Hippoboscoidea) originated independently of the tsetse flies symbiont Sodalis glossinidius.

Symbiotic bacterium closely related to the secondary symbiont of tsetse flies, Sodalis glossinidius, has been described from the bloodsucking fly Craterina melbae. Phylogenetic analysis of two genes, 16S rRNA gene and component of type three secretion system, placed the bacterium closer to the Sitophilus-derived branch of Sodalis than to the tsetse symbionts. This indicates that the Craterina-derived lineage of Sodalis originated independent of the tsetse flies symbionts and documents the capability of Sodalis bacteria either to switch between different host groups or to establish the symbiosis by several independent events.

Amino Acid Sequence↗

The micro-organisms of tsetse flies.

Micro-organisms from tsetse fly mycetomes were maintained in culture, where they were more pleomorphic than in the mycetomes, but were in some cases very similar to those observed in ovaries by other authors. Agglutination tests on the cultured forms indicated in affinity to Rickettsia. They were sensitive to antibiotics introduced by feeding flies on hosts treated with Ampicillin; this reduced the longevity and fecundity of the tsetse flies and appeared to disturb normal digestion of bloodmeals.

Agglutination Tests↗

Genetic analysis by DNA fingerprinting in tsetse fly genomes.

Genomic DNA from tsetse flies (Diptera: Glossinidae: Glossina Wiedemann) was analyzed by hybridization using the whole M13 phage as a probe to reveal DNA fingerprinting (DNAfp) profiles. Intrapopulation variability, measured by comparison of DNAfp profiles of tsetse flies from a large colony of G. brevipalpis, showed a high degree of polymorphism similar to that found in other animal species. Different lines of G. m. morsitans, G. m. centralis, G. m. submorsitans, G. p. palpalis and G. p. gambiensis established from small colonies displayed less genetic variability than the G. brevipalpis population. The analysis of pedigree relationships within an inbred line of G. m. centralis conformed to a Mendelian inheritance pattern. In the pedigree presented no mutations were observed, one fragment was linked to the X chromosome, and three fragment sets were linked, but most fragments showed independent segregation. M13 revealed no characteristic DNAfp profile differences between the subgenus Glossina and the subgenus Nemorhina, but a conserved distribution pattern was found in the laboratory colonies within each subspecies. M13 also revealed line specific DNA fragments that may be useful as genetic markers to expand the present linkage map of G. m. morsitans.

Animals↗

Analysis of fat body transcriptome from the adult tsetse fly, Glossina morsitans morsitans.

Tsetse flies (Diptera: Glossinidia) are vectors of pathogenic African trypanosomes. To develop a foundation for tsetse physiology, a normalized expressed sequence tag (EST) library was constructed from fat body tissue of immune-stimulated Glossina morsitans morsitans. Analysis of 20,257 high-quality ESTs yielded 6372 unique genes comprised of 3059 tentative consensus (TC) sequences and 3313 singletons (available at http://aksoylab.yale.edu). We analysed the putative fat body transcriptome based on homology to other gene products with known functions available in the public domain. In particular, we describe the immune-related products, reproductive function related yolk proteins and milk-gland protein, iron metabolism regulating ferritins and transferrin, and tsetse's major energy source proline biosynthesis. Expression analysis of the three yolk proteins indicates that all are detected in females, while only the yolk protein with similarity to lipases, is expressed in males. Milk gland protein, apparently important for larval nutrition, however, is primarily synthesized by accessory milk gland tissue.

Adipose Tissue↗

Wigglesworthia gen. nov. and Wigglesworthia glossinidia sp. nov., taxa consisting of the mycetocyte-associated, primary endosymbionts of tsetse flies.

The primary endosymbionts (P-endosymbionts) of tsetse flies (Diptera: Glossinidae) are harbored inside specialized cells (mycetocytes) in the anterior region of the gut, and these specialized cells form a white, U-shaped organelle called mycetome. The P-endosymbionts of five tsetse fly species belonging to the Glossinidae have been characterized morphologically, and their 16S ribosomal DNA sequences have been determined for phylogenetic analysis. These organisms were found to belong to a distinct lineage related to the family Enterobacteriaceae in the gamma subdivision of Proteobacteria, which includes the secondary endosymbionts of various insects and Escherichia coli. These bacteria are also related to the P-endosymbionts of aphids, Buchnera aphidicola. Signature sequences in the 16S ribosomal DNA and genomic organizational differences which distinguish the tsetse fly P-endosymbionts from members of the Enterobacteriaceae and from the genus Buchnera are described in this paper. I propose that the P-endosymbionts of tsetse flies should be classified in a new genus, the genus Wigglesworthia, and a new species, Wigglesworthia glossinidia. The P-endosymbiont found in the mycetocytes of Glossina morsitans morsitans is designated the type strain of this species.

Animals↗

Microsatellite diversities and gene flow in the tsetse fly, Glossina morsitans s.l.

Tsetse flies occupy discontinuous habitats and gene flow among them needs to be investigated in anticipation of area-wide control programs. Genetic diversities were estimated at six microsatellite loci in seven Glossina morsitans submorsitans Newstead (Diptera: Glossinidae) populations and five microsatellite loci in six G. m. morsitans Westwood populations. Nei's unbiased diversities were 0.808 and 76 alleles in G. m. submorsitans and 0.727 and 55 alleles in G. m. morsitans. Diversities were less in three laboratory cultures. Matings were random within populations. Populations were highly differentiated genetically. Populations were strongly subdivided, as indicated by fixation indices (F(ST)) of 0.18 in G. m. morsitans and 0.17 in G. m. submorsitans. 35% of the genetic variance in G. m. submorsitans was attributed to differences between populations from The Gambia and Ethiopia. All available genetic evidence suggests that genetic drift is much greater than gene flow among G. morsitans s.l. populations.

Alleles↗