Genetic relationships between subspecies of the tsetse fly Glossina morsitans inferred from variation in mitochondrial DNA sequences

A 750 base pair segment of DNA from the tsetse fly Glossina morsitans morsitans was isolated by means of molecular cloning. It was shown by DNA hybridization to have substantial sequence homology with a defined region of the mitochondrial genomes of several Drosophila species. When used as a probe against DNA prepared from single tsetse flies, the cloned sequence revealed local restriction site variation between members of the G. morsitans subspecies complex. This feature was used to demonstrate maternal inheritance of the sequence in progeny of hybrid crosses and to assemble comparative restriction maps for a 3-kilobase segment of each mitochondrial genome. The data obtained from these exercises point to a higher genetic identity between G. m. morsitans and G. m. centralis than between either form and G. m. submorsitans.     

Transition stages of molecular drive in multiple-copy DNA families in Drosophila

Multigene and non-genic DNA families are in a state of turnover and hence are continually being replaced throughout a population by new variant repeats. To quantify such molecular processes, in the absence of selection, it is necessary to find and compare stages of transistion during the homogenization of at least two non-genic families evolving in parallel in a closely related group of species. Detailed sequence analysis of patterns of variation, at each nucleotide position considered independently, amongst repeats of two tandem DNA families from seven related Drosophila species, reveals all stages of transition during the spread of randomly produced variant repeats. Variant repeats are found at different stages of homogenization and fixation in a population, irrespective of the loci, chromosomes or individuals from which they were cloned. Differences between the families in the relatively small number of variants at each transition stage and the greater number of fully homogenized and fixed variants between species of greater divergence indicate that the process of spread (molecular drive) is rapid relative to the mutation rate and occurs at seemingly different constant rates for each family. Occasional gene conversions, in addition to unequal exchanges, have contributed to family turnover. The significance of these results to the evolution of functional multigene families and divergence and conservation of sequences is discussed.     

Sequence of PRAT Satellite DNA ``Frozen' in Some Coleopteran Species

The intriguing diversity of highly abundant satellite repeats found even among closely related species can result from processes leading to dramatic changes in copy number of a particular sequence in the genome and not from rapid accumulation of mutations. To test this hypothesis, we investigated the distribution of the PRAT satellite DNA family, a highly abundant major satellite in the coleopteran species Palorus ratzeburgii, in eight species belonging to the related genera (Tribolium, Tenebrio, Latheticus), the subfamily (Pimeliinae), and the family (Chrysomelidae). Dot blot analysis and PCR assay followed by Southern hybridization revealed that the PRAT satellite, in the form of low-copy number repeats, was present in all tested species. The PRAT satellite detected in the species Pimelia elevata has been sequenced, and compared with previously cloned PRAT monomers from Palorus ratzeburgii and Palorus subdepressus. Although the two Palorus species diverged at least 7 Myr ago, and the subfamily Pimeliinae separated from the genus Palorus 50–60 Myr ago, all PRAT clones exhibit high mutual homology, with average variability relative to the common consensus sequence of 1.3%. The presence of ancestral mutations found in PRAT clones from all three species as well as the absence of species diagnostic mutations illustrate extremely slow sequence evolution. This unexpectedly high conservation of PRAT satellite DNA sequence might be induced by a small bias of turnover mechanisms favoring the ancestral sequence in the process of molecular drive.     

Long and short repeats of sea urchin DNA and their evolution

Repeated sequences cloned from the DNA of the sea urchin S. purpuratus were used as probes to measure the lengths of individual families of repeats. Some probes reassociated much more rapidly with preparations of long repeats than with short repeats while others reassociated more rapidly with short repeats than with long repeats. In this way two of five cloned repeats were shown to represent families with a great majority of sequences in the long class. One represented a family with similar numbers of long and short class members. Two were members of predominantly short class families. — The cloned repeats representing long class families, formed more precise duplexes than those representing short class families. Thermal stability measurements using S. purpuratus or S. franciscanus driver DNA showed that precise repetitive sequences have as great an interspecies sequence difference as the less precise repeats. Thus the precision of many families may result from recent multiplication rather than from selective pressure on the DNA sequences. Measurements of evolutionary frequency change show a clear correlation between the frequency change and the size of families of repeats in S. purpuratus. Comparison with S. franciscanus indicates that many of the large size families in S. purpuratus are those that have grown in size since these two species diverged.     

Univariate analysis of tsetse habitat in the common fly belt of Southern Africa using climate and remotely sensed vegetation data

Abstract Tsetse are vectors of trypanosomes that cause diseases both in humans and livestock. Traditional tsetse surveys, using sampling methods such as Epsilon traps and black screen fly rounds, are often logistically difficult, costly and time-consuming. The distribution of tsetse, as revealed by such survey methods, is strongly influenced by environmental conditions, such as climate and vegetation cover, which may be readily mapped using satellite data. These data may be used to make predictions of the probable distribution of tsetse in unsurveyed areas by determining the environmental characteristics of areas of tsetse presence and absence in surveyed areas. The same methods may also be used to characterize differences between tsetse species and subspecies. In this paper we analyse the distribution of Glossina morsitans centralis, Glossina morsitans morsitans and Glossina pallidipes in southern Africa with respect to single environmental variables. For G.m.centralis the best predictions were made using the average NDVI (75% correct predictions; range > 0.37) and the average of the maximum temperature (70% correct predictions; 27.0–29.2°C). For G.m.morsitans the best prediction was given by the maximum of the minimum temperature (84% correct predictions; range > 18.8°C), and for G.pallidipes , also by the maximum of the minimum temperature (86% correct predictions; range > 19.6 °C). The following paper compares a range of multivariate techniques for making predictions about the distribution of these species in the same region.     

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 variablity, measured by comparison of DNAfp profiles of tsetse flies from 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 characteristics 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.     

New Culture Medium for Maintenance of Tsetse Tissues and Growth of Trypanosomatids*

SYNOPSIS. A new culture medium (SM), based on the amino-acid composition of tsetse hemolymph and containing fetal bovine serum, was designed for the maintenance of tsetse organs and the cultivation of various trypanosomatids. For optimum growth 20% (v/v) serum was required. The medium supported prolonged peristalsis of the alimentary tract and salivary glands of pre-emerged Glossina morsitans morsitans. In established cultures, derived from bloodstream forms of pleomorphic Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense strains, inocula of ～ 10⁶ procyclics/ml yielded 4–5 × 10⁷ organisms/ml after 4 or 5 days of incubation at 28 C. Bloodstream forms of a cloned monomorphic T. b. brucei strain were also able to transform into procyclics, which, however, multiplied at a lower rate, with maximum yields of ～ 2 × 10⁷ after 5 days. Cultures of Trypanosoma congolense and of a nearly monomorphic Trypanosoma brucei gambiense strains could be established in SM medium only in the presence of tsetse alimentary tract. The procyclic trypomastigotes of these species, adapted to SM medium and able to grow in it without Glossina organs, gave maximum populations of ～ 4.5 × 10⁷ cells/ml. Promastigotes of Leishmania donovani, cultivated routinely in a diphasic Table's medium, multiplied actively upon being transferred into SM medium, producing yields of ～ 4 × 10⁷ cells/ml.     

Comparison of the susceptibility of different Glossina species to simple and mixed infections with Trypanosoma (Nannomonas) congolense savannah and riverine forest types

Abstract Teneral Glossina morsitans mositans, G.m.submorsitans, G.palpalis gambiensis and G.tachinoides were allowed to feed on rabbits infected with Trypanosoma congolense savannah type or on mice infected with T.congolense riverine-forest type. The four tsetse species and subspecies were also infected simultaneously in vitro on the blood of mice infected with the two clones of T.congolense via a silicone membrane. The infected tsetse were maintained on rabbits and from the day 25 after the infective feed, the surviving tsetse were dissected in order to determine the infection rates.
Results showed higher mature infection rates in morsitans-gwup tsetse flies than in palpalis-group tsetse flies when infected with the savannah type of T.congolense. In contrast, infection rates with the riverine-forest type of T.congolense were lower, and fewer flies showed full development cycle. The intrinsec vectorial capacity of G.m.submorsitans for the two T.congolense types was the highest, whereas the intrinsic vectorial capacity of G.p.gambiensis for the Savannah type and G.m.morsitans for the riverine-forest type were the lowest. Among all tsetse which were infected simultaneously with the two types of T.congolense , the polymerase chain reaction detected only five flies which had both trypanosome taxa in the midgut and the proboscis. All the other infections were attributable to the savannah type.
The differences in the gut of different Glossina species and subspecies allowing these two sub-groups of T.congolense to survive better and undergo the complete developmental cycle more readily in some species than other are discussed.     

Glossina morsitans morsitansandGlossina palpalis palpalis:Dosage Compensation Raises Questions about the Milligan Model for Control of Trypanosome Development

Gooding, R. H., and McIntyre, G. S. 1998.Glossina morsitans morsitansandGlossina palpalis palpalis: Dosage compensation raises questions about the Milligan model for control of trypanosome development.Experimental Parasitology90, 244–249. Evidence that dosage compensation occurs in tsetse flies was obtained by comparing the activities of X chromosome-linked enzymes, arginine phosphokinase and glucose-6-phosphate dehydrogenase inGlossina m. morsitansand hexokinase and phosphoglucomutase inGlossina p. palpalis, with the activity of an autosome-linked enzyme, malate dehydrogenase, in each species. The shortcomings of the X chromosome model for the control ofTrypanozoonmaturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of matureTrypanozooninfections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.     

Polytene chromosome maps in four species of tsetse flies Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans (Diptera: Glossinidae): a comparative analysis

Photographic polytene chromosome maps from pupal trichogen cells of four tsetse species, Glossina austeni, G. pallidipes, G. morsitans morsitans and G. m. submorsitans were constructed and compared. The homology of chromosomal elements between the species was achieved by comparing banding patterns. The telomeric and subtelomeric chromosome regions were found to be identical in all species. The pericentromeric regions were found to be similar in the X chromosome and the left arm of L1 chromosome (L1L) but different in L2 chromosome and the right arm of L1 chromosome (L1R). The L2 chromosome differs by a pericentric inversion that is fixed in the three species, G. pallidipes, G. morsitans morsitans and G. m. submorsitans. Moreover, the two morsitans subspecies appeared to be homosequential and differ only by two paracentric inversions on XL and L2L arm. Although a degree of similarity was observed across the homologous chromosomes in the four species, the relative position of specific chromosome regions was different due to chromosome inversions established during their phylogeny. However, there are regions that show no apparent homology between the species, an observation that may be attributed to the considerable intra—chromosomal rearrangements that have occurred following the species divergence. The results of this comparative analysis support the current phylogenetic relationships of the genus Glossina.     

Relationships between protease activity, host blood and infection rates in Glossina morsitans sspp. infected with Trypanosoma congolense, T.brucei and T.simiae

Abstract. Midgut protease activity in Glossina morsitans centralis and G. m. morsitans , at 48h post bloodmeal averaged 1.8IU of trypsin-like activity. These two tsetse subspecies differ in their susceptibility to trypanosome infection. Except for low levels in flies fed on waterbuck blood (0.7IU), activity did not differ in flies fed a variety of host bloods (goat, pig, cow, buffalo, eland) and trypanosome species ( Trypanosoma congolense, T.brucei, T.simiae ). Protease activity was also not correlated with infection rates, despite large differences in infection rates among experiments. Nevertheless, addition of 0.06M D(+)-glucosamine to parasitaemic blood resulted in a three-fold reduction in protease activity, coincident with a large increase in infection rate. This effect did not occur when parasites or D(+)-glucosamine were added alone to the bloodmeal, suggesting that the effect was due to metabolism of D(+)-glucosamine by parasites.     

Molecular phylogenetics of tsetse flies (Diptera: Glossinidae) based on mitochondrial (COI, 16S, ND2) and nuclear ribosomal DNA sequences, with an emphasis on the palpalis group

Relationships of 13 species of the genus Glossina (tsetse flies) were inferred from mitochondrial (cytochrome oxidase 1, NADH dehydrogenase 2 and 16S) and nuclear (internal transcribed spacer 1 of rDNA) sequences. The resulting phylogeny confirms the monophyly of the morphologically defined fusca, morsitans and palpalis subgenera. Genetic distances between palpalis and morsitans subspecies suggest that their status needs revision. In particular, cytochrome oxidase 1 sequences showed large geographical differences within G. palpalis palpalis, suggesting the existence of cryptic species within this subspecies. The morphology of palpalis group female genital plates was examined, and individuals were found varying outside the ranges specified by the standard identification keys, making definitive morphological classification impossible. A diagnostic PCR to distinguish G. palpalis palpalis, G. tachinoides and G. palpalis gambiensis based on length differences of internal transcribed spacer 1 sequences is presented.     

Cloning and Expression of a Bacillus thuringiensis (L1-2) Gene Encoding a Crystal Protein Active Against Glossina morsitans morsitans and Chilo partellus

A local isolate of Bacillus thuringiensis, designated L1-2, that is toxic to Chilo partellus was found to be toxic to the adult tsetse fly, Glossina morsitans morsitans. The δ-endotoxin crystals derived from the isolate gave a major protein band with a molecular weight of M_r 130,000–140,000 on denaturing polyacrylamide gel electrophoresis. The sequence of the cloned gene was found to be similar to that of the B. thuringiensis subsp. kurstaki HD-73 cryIA(c) gene, having one amino acid difference at position 148 and four additional DNA differences. Received: 29 June 1996 / Accepted: 1 August 1996     

Preservation and High Sequence Conservation of Satellite DNAs Suggest Functional Constraints

Due to a high evolutionary turnover many satellite DNAs are restricted to a group of closely related species. Here we demonstrate that the satellite DNA family PSUB, abundant in the beetle Palorus subdepressus, is distributed in a low number of copies among diverse taxa of Coleoptera (Insecta), some of them separated for an evolutionary period of up to 60 Myr. Comparison of PSUB cloned from the species Tribolium brevicornis with the PSUB family previously characterized in Palorus subdepressus revealed high sequence conservation and absence of fixed species-specific mutations. The most polymorphic sites are those with ancestral mutations shared among clones of both species. Since the ancestral mutations contribute significantly to overall diversity, it could be proposed that a similar mutational profile already existed in an ancestral species. The pattern of variability along the satellite monomer is characterized by the presence of conserved and variable regions. The nonrandom pattern of variability as well as the absence of sequence divergence is also discerned for PRAT satellite DNA, cloned previously from two Palorus species and a distantly related Pimelia elevata. Since PRAT and PSUB are present in parallel in diverse taxa of Coleoptera, we propose that their long evolutionary preservation suggests a possible functional significance. This indication is additionally supported not only by the high evolutionary conservation of the sequences, but also by the presence of significantly conserved and variable regions along the monomers. [Reviewing Editor: Dr. Jerzy Jurka]     

Prevalence of trypanosomes,salivary gland hypertrophy virus and Wolbachia in wild populations of tsetse flies from West Africa

Background

Tsetse flies are vectors of African trypanosomes, protozoan parasites that cause sleeping sickness (or human African trypanosomosis) in humans and nagana (or animal African trypanosomosis) in livestock. In addition to trypanosomes, four symbiotic bacteria Wigglesworthia glossinidia, Sodalis glossinidius, Wolbachia, Spiroplasma and one pathogen, the salivary gland hypertrophy virus (SGHV), have been reported in different tsetse species. We evaluated the prevalence and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in four tsetse species (Glossina palpalis gambiensis, G. tachinoides, G. morsitans submorsitans, and G. medicorum) that were collected between 2008 and 2015 from 46 geographical locations in West Africa, i.e. Burkina Faso, Mali, Ghana, Guinea, and Senegal.

Results

The results indicated an overall low prevalence of SGHV and Wolbachia and a high prevalence of trypanosomes in the sampled wild tsetse populations. The prevalence of all three infections varied among tsetse species and sample origin. The highest trypanosome prevalence was found in Glossina tachinoides (61.1%) from Ghana and in Glossina palpalis gambiensis (43.7%) from Senegal. The trypanosome prevalence in the four species from Burkina Faso was lower, i.e. 39.6% in Glossina medicorum, 18.08%; in Glossina morsitans submorsitans, 16.8%; in Glossina tachinoides and 10.5% in Glossina palpalis gambiensis. The trypanosome prevalence in Glossina palpalis gambiensis was lowest in Mali (6.9%) and Guinea (2.2%). The prevalence of SGHV and Wolbachia was very low irrespective of location or tsetse species with an average of 1.7% for SGHV and 1.0% for Wolbachia. In some cases, mixed infections with different trypanosome species were detected. The highest prevalence of coinfection was Trypanosoma vivax and other Trypanosoma species (9.5%) followed by coinfection of T. congolense with other trypanosomes (7.5%). The prevalence of coinfection of T. vivax and T. congolense was (1.0%) and no mixed infection of trypanosomes, SGHV and Wolbachia was detected.

Conclusion

The results indicated a high rate of trypanosome infection in tsetse wild populations in West African countries but lower infection rate of both Wolbachia and SGHV. Double or triple mixed trypanosome infections were found. In addition, mixed trypanosome and SGHV infections existed however no mixed infections of trypanosome and/or SGHV with Wolbachia were found.

     

Sodalis glossinidius (Enterobacteriaceae) and Vectorial Competence of Glossina palpalis gambiensis and Glossina morsitans morsitans for Trypanosoma congolense Savannah Type

Sodalis glossinidius is an endosymbiont of Glossina palpalis gambiensis and Glossina morsitans morsitans, the vectors of Trypanosoma congolense. The presence of the symbiont was investigated by PCR in Trypanosoma congolense savannah type-infected and noninfected midguts of both fly species, and into the probosces of flies displaying either mature or immature infection, to investigate possible correlation with the vectorial competence of tsetse flies. Sodalis glossinidius was detected in all midguts, infected or not, from both Glossina species. It was also detected in probosces from Glossina palpalis gambiensis flies displaying mature or immature infection, but never in probosces from Glossina morsitans morsitans. These results suggest that, a) there might be no direct correlation between the presence of Sodalis glossinidius and the vectorial competence of Glossina, and b) the symbiont is probably not involved in Trypanosoma congolense savannah type maturation. It could however participate in the establishment process of the parasite.     

Olfactory and behavioural responses of tsetse flies, <Emphasis Type="Italic">Glossina</Emphasis> spp., to rumen metabolites

Herbivores provide tsetse flies with a blood meal, and both wild and domesticated ruminants dominate as hosts. As volatile metabolites from the rumen are regularly eructed with rumen gas, these products could serve tsetse flies during host searching. To test this, we first established that the odour of rumen fluid is attractive to hungry Glossina pallidipes in a wind tunnel. We then made antennogram recordings from three tsetse species (G. pallidipes morsitans group, G. fuscipes palpalis group and G. brevipalpis fusca group) coupled to gas chromatographic analysis of rumen fluid odour and of its acidic, mildly acidic and neutral fractions. This shows tsetse flies can detect terpenes, ketones, carboxylic acids, aliphatic aldehydes, sulphides, phenols and indoles from this biological substrate. A mixture of carboxylic acids at a ratio similar to that present in rumen fluid induced behavioural responses from G. pallidipes in the wind tunnel that were moderately better than the solvent control. The similarities in the sensory responses of the tsetse fly species to metabolites from ruminants demonstrated in this study testify to a contribution of habitat exploitation by these vertebrates in the Africa-wide distribution of tsetse.     

Tsetse EP Protein Protects the Fly Midgut from Trypanosome Establishment

African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis.     

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.     

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.