The tsetse fly Glossina palpalis palpalis is composed of several genetically differentiated small populations in the sleeping sickness focus of Bonon, Côte d'Ivoire.

Glossina palpalis is the main vector of human African trypanosomosis (HAT, or sleeping sickness) that dramatically affects human health in sub-Saharan Africa. Because of the implications of genetic structuring of vector populations for the design and efficacy of control campaigns, G. palpalis palpalis in the most active focus of sleeping sickness in Côte d'Ivoire was studied to determine whether this taxon is genetically structured. High and statistically significant levels of within population heterozygote deficiencies were found at each of the five microsatellite loci in two temporally separated samples. Neither null alleles, short allele dominance, nor trap locations could fully explain these deviations from random mating, but a clustering within each of the two samples into different genetic sub-populations (Wahlund effect) was strongly suggested. These different genetic groups, which could display differences in infection rates and trypanosome identity, were composed of small numbers of individuals that were captured together, leading to the observed Wahlund effect. Implications of this population structure on tsetse control are discussed.

Identification of midgut proteins that are differentially expressed in trypanosome-susceptible and normal tsetse flies (Glossina morsitans morsitans).

Molecules in the midgut of tsetse flies (Diptera: Glossinidiae) are thought to play important roles in the life cycle of African trypanosomes by influencing initial parasite establishment and subsequent differentiation events that ultimately lead to maturation of mammal-infective trypanosomes. The molecular composition of the tsetse midgut is, therefore, of critical importance to disease transmission by these medically important vectors. In this study we compared protein expression profiles of midguts of the salmon mutant and wild type Glossina morsitans morsitans Westwood that display marked differences in their susceptibility to infection by African trypanosomes. Isotope coded affinity tag (ICAT) technology was used to identify 207 proteins including 17 that were up regulated and nine that were down regulated in the salmon mutants. Several of the up regulated molecules were previously described as tsetse midgut or salivary gland proteins. Of particular interest was the up regulation in the salmon flies of tsetse midgut EP protein, a recently described molecule with lectin-like activity that was also found to be induced in tsetse by bacterial challenge. The up regulation of the EP protein in midguts of salmon mutants was confirmed by two-dimensional gel electrophoresis and tandem mass spectrometry.

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.

Isolation and characterization of hypervariable sequences from tsetse fly genome.

A Glossina brevipalpis Newstead genomic library, constructed using a Charomid 9-36 vector, was used to isolate putative clones that hybridize to polymorphic regions of the tsetse genome. Five types of probes, that reveal individual DNA polymorphic in humans and higher animal species, were used to screen 300 tsetse Charomid clones; 15% of the clones hybridized with at least one probe. Twenty four recombinants were further characterized by Southern blotting hybridization using DNA isolated from individual tsetse fly from Glossina morsitans centralis Machado. Two classes of DNA profiles were obtained upon hybridization with the recombinant inserts. The first, termed the "multilocus profile" displayed a complex DNA pattern of multiple components whilst the second, referred to as the "single locus profile" revealed one or two bands in each individual. Of the 24 recombinant inserts tested, 13 were multilocus probes and the remainder were single locus probes, each of which hybridized to a single location when G. m. centralis DNA had been cleaved with EcoRI. These single locus probes revealed a low level of genetic variability among individual flies from an inbred colony. The hybridization profiles using multilocus and single locus probes were also obtained on DNA from individual Glossina palpalis palpalis Robineau-Desvoidy and Glossina palpalis gambienis Vanderplank and some of the G. brevipalpis recombinant clones also detected multilocus profiles in honey bees and man.

Identification of major soluble salivary gland proteins in teneral Glossina morsitans morsitans.

Salivary glands of tsetse flies (Diptera: Glossinidiae) contain molecules that are involved in preventing blood clotting during feeding as well as molecules thought to be intimately associated with trypanosome development and maturation. Here we present a protein microchemical analysis of the major soluble proteins of the salivary glands of Glossina morsitans morsitans, an important vector of African trypanosomes. Differential solubilization of salivary proteins was followed by reverse-phase, high-performance liquid chromatography (HPLC) and analysis of fractions by 1-D gel electrophoresis to reveal four major proteins. Each protein was subjected to amino acid microanalysis and N-terminal microsequencing. A protein chemical approach using high-resolution 2-D gel electrophoresis and mass spectrometry was also used to identify the salivary proteins. Matrix-assisted, laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass mapping and sequencing, respectively. Sequence information and peptide mass maps queried against the NCBI non-redundant database confirmed the identity of the first protein as tsetse salivary gland growth factor-1 (TSGF-1). Two proteins with no known function were identified as tsetse salivary gland protein 1 (Tsal 1) and tsetse salivary gland protein 2 (Tsal 2). The fourth protein was identified as Tsetse antigen-5 (TAg-5), which is a member of a large family of anti-haemostatic proteins. The results show that these four proteins are the most abundant soluble gene products present in salivary glands of teneral G. m. morsitans. We discuss the possible functions of these major proteins in cyclical transmission of African trypanosomes.

The major protein in the midgut of teneral Glossina morsitans morsitans is a molecular chaperone from the endosymbiotic bacterium Wigglesworthia glossinidia.

Molecules in the midgut of the tsetse fly (Diptera: Glossinidiae) are thought to play an important role in the life cycle of African trypanosomes by influencing their initial establishment in the midgut and subsequent differentiation events that ultimately affect parasite transmission. It is thus important to determine the molecular composition of the tsetse midgut to aid in understanding disease transmission by these medically important insect vectors. Here, we report that the most abundant protein in the midguts of teneral (unfed) Glossina morsitans morsitans is a 60 kDa molecular chaperone of bacterial origin. Two species of symbiotic bacteria reside in the tsetse midgut, Sodalis glossinidius and Wigglesworthia glossinidia. To determine the exact origin of the 60 kDa molecule, a protein microchemical approach involving two-dimensional (2-D) gel electrophoresis and mass spectrometry was used. Peptide mass maps were compared to virtual peptide maps predicted for S. glossinidius and W. glossinidia 60 kDa chaperone sequences. Four signature peptides were identified, revealing that the source of the chaperone was W. glossinidia. Comparative 2-D gel electrophoresis and immunoblotting further revealed that this protein was localized to the bacteriome and not the distal portion of the tsetse midgut. The possible function of this highly abundant endosymbiont chaperone in the tsetse midgut is discussed.

Genetics of two populations of Glossina morsitans centralis (Diptera: Glossinidae) from Zambia.

Glossina morsitans centralis Machado was collected from the main fly belt west of Mumbwa Zambia and from the apparently isolated 'Keembe pocket' and 11 gene-enzyme systems were examined by polyacrylamide gel electrophoresis. There were no significant differences in allele frequencies among flies collected entering a vehicle, from fly-rounds, or from F3 traps in the main fly belt. Mean heterozygosity per locus is slightly higher in flies from the main fly belt than it is in flies from the 'Keembe pocket'. Allele frequencies at loci for xanthine oxidase (Xo), aldehyde oxidase (Ao) and a thoracic esterase (Est-2) were significantly different in the two populations and it is concluded that there is little gene flow between them.

Genetic polymorphism in three species of tsetse flies (Diptera: Glossinidae) in Upper Volta.

Natural populations of Glossina morsitans submorsitans Newstead, Glossina palpalis gambiensis Vanderplank and Glossina tachinoides Westwood occurring within 150 km of Bobo-Dioulasso, Upper Volta were examined using polyacrylamide gel electrophoresis. No variation was found in the banding pattern for arginine phosphokinase (EC 2.7.3.3). G. p. gambiensis and G. tachinoides had three alleles for each of the thoracic enzymes octanol dehydrogenase (EC 1.1.1.73), malic dehydrogenase (EC 1.1.1.37) and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8) and for midgut alkaline phosphatase (EC 3.1.3.1). The same situation was found with G. m. submorsitans except that only two alleles for alpha-glycerophosphate dehydrogenase and one for alkaline phosphatase were found. In each species, and for each of the enzymes, one allele was present at a frequency of 95% or greater. There was little or no variation between populations. Two laboratory colonies of G. p. gambiensis had less genetic variation than wild populations.

Influence of the salmon mutant of Glossina morsitans morsitans on the susceptibility to infection with Trypanosoma congolense.

Four phenotypes of a sex-linked, maternally influenced semi-lethal eye color mutant of Glossina morsitans morsitans Westwood were fed on Trypanosoma congolense Broden infected guinea pigs. Infection rates were evaluated 25 days later by means of dissection. Procyclic as well as mature infections were significantly more common among females with salmon-colored eyes (sal/sal) than among heterozygous (+/sal, phenotypically wild-type) females. A tendency was found for more mature infections among sal/Y males than among wild-type males. Similarly, females tended to be more infected than males with both procyclic and mature infections. These results indicate that the genotype of the fly, exemplified by the allele salmon, might influence the development of T. congolense in G.m. morsitans. A possible explanation for this phenomenon is discussed.

Hybridization asymmetries in tsetse (Diptera: Glossinidae): role of maternally inherited factors and the tsetse genome.

Among the morsitans-group of tsetse there are several pairs of taxa in which there is a marked hybridization asymmetry (HA), i.e., one cross produces significantly more offspring than does the reciprocal cross. To investigate the relative contribution of maternally inherited factors (MIF) and chromosomal factors to HA, three hybrid lines were established in which flies have MIF from one taxon and chromosome from another. HA was then compared among crosses of the parental taxa and crosses of each parental taxon with the appropriate hybrid line. The results indicate that HA in reciprocal crosses of Glossina morsitans morsitans Westwood and Glossina swynnertoni Austin and in reciprocal crosses of G. m. morsitans and Glossina morsitans centralis Machado are caused by chromosomal factors, not MIF. Reciprocal crosses of G. m. centralis and G. swynnertoni do not display HA, and none developed as a result of a novel combination of MIF and tsetse chromosomes.

Effect of maternal age on offspring quality in tsetse (Diptera: Glossinidae).

The effects of maternal age on offspring quality were studied in 1 line of Glossina palpalis palpalis Robineau-Desvoidy, 1 line of G. p. gambiensis Vanderplank, and 3 lines of G. morsitans morsitans Westwood by measuring offspring adult size and the duration of puparial period. G. p. gambiensis males also were examined for effects of maternal age on fluctuating asymmetry of wing veins. The puparial period was shorter in offspring of old females (late offspring) than in offspring of young females (early offspring). The difference was small but was greater for male than for female offspring. Early male offspring were larger than late males. Wing vein fluctuating asymmetry was slightly greater in early than in late offspring in G. p. gambiensis. The differences between early and late offspring were very small, and we conclude that old females produce offspring of marginally lower quality than those produced by young females and that these differences are not biologically significant.

Genetic variation in a Tanzanian population of Glossina swynnertoni (Diptera: Glossinidae).

Adult Glossina swynnertoni Austen that emerged from puparia collected during 1989 and 1991 near Makuyuni, Tanzania, were examined by polyacrylamide gel electrophoresis. Fourteen of 17 enzymes were monomorphic. Midgut alkaline phosphatase (ALKPH), phosphoglucomutase (PGM), and glucose-6-phosphate isomerase (PGI) from the head and thorax were polymorphic. Banding patterns indicated that the locus for PGM was on the X chromosome and loci for ALKPH and PGI were autosomal. For the 17 loci studied, the mean heterozygosity per locus was 6.1 +/- 3.7% in the 1989 sample and 5.7 +/- 3.7% in the 1991 sample. The effective number of alleles per locus was 1.11 and 1.10 in these samples. This level of genetic variation was low compared with other populations of tsetse flies and indicated that the sample may have been drawn from a small inbred population or one that recently had gone through a genetic bottleneck.

Genetic variation in two field populations and a laboratory colony of Glossina pallidipes (Diptera: Glossinidae).

Glossina pallidipes Austen from Lambwe and Nguruman in Kenya and a laboratory colony, originating from flies collected at Lambwe, were compared for 12 enzyme-gene systems using polyacrylamide gel electrophoresis. In the Nguruman, Lambwe, and colony flies, mean heterozygosities were 9.1, 15.3, and 16.5%, and polymorphism was observed in 3, 4, and 5 loci, respectively. Significant differences in number of gene products were observed between Nguruman and Lambwe flies at three loci, between Nguruman and colony flies at four loci, and between Lambwe and colony flies at two loci. Evidence is presented indicating that the locus for phosphoglucomutase is on the X chromosome, whereas loci for octanol dehydrogenase, malate dehydrogenase, phosphoglucose isomerase, and a thoracic esterase (Esterase-1) are autosomal.