Genetic diversity and population structure of Glossina morsitans morsitans in the active foci of human African trypanosomiasis in Zambia and Malawi.

The tsetse fly, Glossina morsitans morsitans, is a significant problem in Zambia and Malawi. It is the vector for the human infective parasite Trypanosoma brucei rhodesiense, which causes human African trypanosomiasis, and various Trypanosoma species, which cause African animal trypanosomiasis. Understanding the genetic diversity and population structure of G. m. morsitans is the basis of elucidating the connectivity of the tsetse fly populations, information that is essential in implementing successful tsetse fly control activities. This study conducted a population genetic study using partial mitochondrial cytochrome oxidase gene 1 (CO1) and 10 microsatellite loci to investigate the genetic diversity and population structure of G. m. morsitans captured in the major HAT foci in Zambia and Malawi. We have included 108 and 99 G. m. morsitans samples for CO1 and microsatellite analyses respectively. Our results suggest the presence of two different genetic clusters of G. m. morsitans, existing East and West of the escarpment of the Great Rift Valley. We have also revealed genetic similarity between the G. m. morsitans in Kasungu National Park and those in the Luangwa river basin in Zambia, indicating that this population should also be included in this historical tsetse belt. Although further investigation is necessary to illustrate the whole picture in East and Southern Africa, this study has extended our knowledge of the population structure of G. m. morsitans in Southern Africa.

Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with K(D) values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

Sphingomyelinase activity in mother's milk is essential for juvenile development: a case from lactating tsetse flies.

Sphingosine is a structural component of sphingolipids. The metabolism of phosphoethanolamine ceramide (sphingomyelin) by sphingomyelinase (SMase), followed by the breakdown of ceramide by ceramidase (CDase) yields sphingosine. Female tsetse fly is viviparous and generates a single progeny within her uterus during each gonotrophic cycle. The mother provides her offspring with nutrients required for development solely via intrauterine lactation. Quantitative PCR showed that acid smase1 (asmase1) increases in mother's milk gland during lactation. aSMase1 was detected in the milk gland and larval gut, indicating this protein is generated during lactation and consumed by the larva. The higher levels of SMase activity in larval gut contents indicate that this enzyme is activated by the low gut pH. In addition, cdase is expressed at high levels in the larval gut. Breakdown of the resulting ceramide is likely accomplished by the larval gut-secreted CDase, which allows absorption of sphingosine. We used the tsetse system to understand the critical role(s) of SMase and CDase during pregnancy and lactation and their downstream effects on adult progeny fitness. Reduction of asmase1 by short interfering RNA negatively impacted pregnancy and progeny performance, resulting in a 4-5-day extension in pregnancy, 10%-15% reduction in pupal mass, lower pupal hatch rates, impaired heat tolerance, reduced symbiont levels, and reduced fecundity of adult progeny. This study suggests that the SMase activity associated with tsetse lactation and larval digestion is similar in function to that of mammalian lactation and represents a critical process for juvenile development, with important effects on the health of progeny during their adulthood.

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.

Molecular characterization of a tsetse fly midgut proteolytic lectin that mediates differentiation of African trypanosomes.

Differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms is an important first step in the establishment of an infection within the tsetse fly. This complex process is mediated by a wide variety of factors, including those associated with the vector itself, the trypanosomes and the bloodmeal. As part of an on-going project in our laboratory, we recently isolated and characterized a bloodmeal-induced molecule with both lectin and trypsin activities from midguts of the tsetse fly, Glossina longipennis [Osir, E.O., Abubakar, L., Imbuga, M.O., 1995. Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly, Glossina longipennis. Parasitol. Res. 81, 276-281]. The protein (lectin-trypsin complex) was found to be capable of stimulating differentiation of bloodstream trypanosomes in vitro. Using polyclonal antibodies to the complex, we screened a G. fuscipes fuscipes cDNA midgut expression library and identified a putative proteolytic lectin gene. The cDNA encodes a putative mature polypeptide with 274 amino acids (designated Glossina proteolytic lectin, Gpl). The deduced amino acid sequence includes a hydrophobic signal peptide and a highly conserved N-terminal sequence motif. The typical features of serine protease trypsin family of proteins found in the sequence include the His/Asp/Ser active site triad with the conserved residues surrounding it, three pairs of cysteine residues for disulfide bridges and an aspartate residue at the specificity pocket. Expression of the gene in a bacterial expression system yielded a protein (M(r) approximately 32,500). The recombinant protein (Gpl) bound d(+) glucosamine and agglutinated bloodstream-form trypanosomes and rabbit red blood cells. In addition, the protein was found to be capable of inducing transformation of bloodstream-form trypanosomes into procyclic forms in vitro. Antibodies raised against the recombinant protein showed cross-reactivity with the alpha subunit of the lectin-trypsin complex. These results support our earlier hypothesis that this molecule is involved in the establishment of trypanosome infections in tsetse flies.

Antioxidant gene expression in the blood-feeding fly Glossina morsitans morsitans.

We report the characterization of 11 antioxidant genes from the tsetse fly Glossina m. morsitans. Through similarity searches which detected homology we suggest that these genes consist of two superoxide dismutases (one with a putative signal peptide), three thioredoxin peroxidases (one with a putative signal peptide), three peroxiredoxins, one further signal peptide-containing peroxidase with its closest similarity to a glutathione peroxidase, one catalase and one thioredoxin reductase. We describe the changes occurring in the expression levels of these genes during fly development, in different adult tissues, in the adult midgut through the digestive cycle and following trypanosome infection. Overall, nine of the 11 genes studied showed responses to changes in physiological circumstance, with the peroxiredoxin group showing the smallest variations throughout.

Adult midgut expressed sequence tags from the tsetse fly Glossina morsitans morsitans and expression analysis of putative immune response genes.

BACKGROUND: Tsetse flies transmit African trypanosomiasis leading to half a million cases annually. Trypanosomiasis in animals (nagana) remains a massive brake on African agricultural development. While trypanosome biology is widely studied, knowledge of tsetse flies is very limited, particularly at the molecular level. This is a serious impediment to investigations of tsetse-trypanosome interactions. We have undertaken an expressed sequence tag (EST) project on the adult tsetse midgut, the major organ system for establishment and early development of trypanosomes. RESULTS: A total of 21,427 ESTs were produced from the midgut of adult Glossina morsitans morsitans and grouped into 8,876 clusters or singletons potentially representing unique genes. Putative functions were ascribed to 4,035 of these by homology. Of these, a remarkable 3,884 had their most significant matches in the Drosophila protein database. We selected 68 genes with putative immune-related functions, macroarrayed them and determined their expression profiles following bacterial or trypanosome challenge. In both infections many genes are downregulated, suggesting a malaise response in the midgut. Trypanosome and bacterial challenge result in upregulation of different genes, suggesting that different recognition pathways are involved in the two responses. The most notable block of genes upregulated in response to trypanosome challenge are a series of Toll and Imd genes and a series of genes involved in oxidative stress responses. CONCLUSIONS: The project increases the number of known Glossina genes by two orders of magnitude. Identification of putative immunity genes and their preliminary characterization provides a resource for the experimental dissection of tsetse-trypanosome interactions.

Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans.

A chitinase cDNA, GChit1 was isolated from Glossina morsitans morsitans and shown to be specifically expressed in fat body tissue. GChit1 is encoded by a 1.6 kb mRNA with a putative open reading frame (ORF) of 460 amino acids (predicted pI=7.5, m.w.=51kDa) that contains a signal peptide domain and two potential N-linked glycosylation sites. The ORF exhibits homology to various chitinases characterized from insects. It has the conserved catalytic site residues and the cysteine-rich 3'-end domain associated with chitin binding although the serine/threonine rich domain is apparently missing. Southern blot data indicate that GChit1 is present as a single-copy locus in the Glossina genome. Northern analysis indicates that transcripts for GChit1 can be detected only from the fat body of adult flies. Similarly, chitinase activity could be detected in fat body but not in the gut or salivary gland tissues. The full-length cDNA was expressed in vitro in Drosophila S2 cells and the molecule was produced in a soluble form. Polyclonal antibodies raised against recGChit1 could recognize a protein of about 50 kDa in adult fat body extracts. In addition to fat body, chitinase protein was detected by Western analysis from the milk gland tissue of pregnant females as well as from the intrauterine larval and pupal developmental stages. No chitinase specific mRNA transcripts could be observed, however from larvae and pupae. The intrauterine larva of tsetse may receive the protein from its mother via the milk gland route. The molecular characteristics of GChit and its product and the potential role of this chitinase in tsetse biology are discussed.

Molecular characterization of three gut genes from Glossina morsitans morsitans: cathepsin B, zinc-metalloprotease and zinc-carboxypeptidase.

Insect gut enzymes are involved in digestion of dietary proteins. Additionally, these enzymes have been implicated in the process of pathogen establishment in several insects including the tsetse fly (Diptera:Glossinidae), which is the vector for African trypanosomes. Both the male and female tsetse can transmit trypanosomes and are strict blood feeders during all stages of their development. Here, we describe the molecular characterization of three gut genes: cathepsin B (GmCatB), zinc-metalloprotease (GmZmp) and zinc-carboxypeptidase (GmZcp). The cDNA for GmCatB encodes a protein for 340 amino acids with a predicted molecular mass of 38.2 kDa, while the 854 bp GmZmp cDNA encodes a protein of 254 amino acids with a molecular mass of 29 kDa. The GmZcp cDNA is 1319 bp in length and has a 354 amino acids open reading frame for coding a 40 kDa protein. All three cDNAs have signal peptide sequences associated with their N-terminal domains and structure analysis indicates that GmCatB and GmZmp are expressed as zymogens with pro-domains proteolytically removed for activity. The activation domain associated with the carboxypeptidase sequences is lacking in GmZcp. While GmCatB transcription is constitutive, teneral flies express very low levels of transcripts for GmZmp and GmZcp prior to the first bloodmeal. Transcription of all genes is induced and remains high throughout the digestion cycle within a few hours following the first bloodmeal ingestion. Both GmCatB and GmZcp are parasite responsive, with the expression of both genes being higher in trypanosome infected flies.

Molecular characterization of two serine proteases expressed in gut tissue of the African trypanosome vector, Glossina morsitans morsitans.

Serine proteases are major insect gut enzymes involved in digestion of dietary proteins, and in addition they have been implicated in the process of pathogen establishment in several vector insects. The medically important vector, tsetse fly (Diptera:Glossinidiae), is involved in the transmission of African trypanosomes, which cause devastating diseases in animals and humans. Both the male and female tsetse can transmit trypanosomes and both are strict bloodfeeders throughout all stages of their development. Here, we describe the characterization of two putative serine protease-encoding genes, Glossina serine protease-1 (Gsp1) and Glossina serine protease-2 (Gsp2) from gut tissue. Both putative cDNA products represent prepro peptides with hydrophobic signal peptide sequences associated with their 5'-end terminus. The Gsp1 cDNA encodes a putative mature protein of 245 amino acids with a molecular mass of 26 428 Da, while the predicted size of the 228 amino acid mature peptide encoded by Gsp2 cDNA is 24 573 Da. Both deduced peptides contain the Asp/His/Ser catalytic triad and the conserved residues surrounding it which are characteristic of serine proteases. In addition, both proteins have the six-conserved cysteine residues to form the three-cysteine bonds typically present in invertebrate serine proteases. Based on the presence of substrate specific residues, the Gsp1 gene encodes a chymotrypsin-like protease while Gsp2 gene encodes for a protein with trypsin-like activity. Both proteins are encoded by few loci in tsetse genome, being present in one or two copies only. The mRNA expression levels for the genes do not vary extensively throughout the digestive cycle, and high levels of mRNAs can be readily detected in the gut tissue of newly emerged flies. The levels of trypsin and chymotrypsin activities in the gut lumen increase following blood feeding and change significantly in the gut cells throughout the digestion cycle. Hence, the regulation of expression for trypsin and chymotrypsin occurs at the post-transcriptional level in tsetse. Both the coding sequences and patterns of expression of Gsp1 and Gsp2 genes are similar to the serine proteases that have been reported from the bloodfeeding insect Stomoxys calcitrans.

A family of genes with growth factor and adenosine deaminase similarity are preferentially expressed in the salivary glands of Glossina m. morsitans.

A cDNA library constructed from salivary glands of tsetse fly, Glossina morsitans morsitans (Diptera: Glossinidae), was differentially screened, and two related full-length cDNAs were molecularly characterized: tsetse salivary growth factor, TSGF-1 and TSGF-2. The cDNAs encode for open reading frames (ORFs) of 494 and 506aa, respectively, and display an overall 45% amino acid identity and 61% similarity to one another. Both genes are preferentially expressed in the salivary glands of male and female adult flies. In addition to salivary glands, both transcripts can be detected from the gut tissue. Only transcripts specific for TSGF-2 are detected in ovary and testes tissues of adults as well as in puparia, while neither gene is expressed during the larval developmental stages. The N-terminal region of both putative proteins contains a hydrophobic sequence with secretory signal peptide characteristics, and analysis of proteins in saliva by Western blot indicates that both are secreted. Western blot analysis indicates that TSGF-1 is synthesized at significantly higher levels than TSGF-2. The deduced protein sequences of both cDNAs display extensive similarities to two other proteins: insect derived growth factor (IDGF) characterized from Sarcophaga peregrina with growth-factor activity, and atrial gland specific antigen (AGSA or MDSF) characterized from Apylasia californica. In addition to growth factor similarity, all four related proteins share the evolutionarily conserved amino acid residues associated with the enzymatic deamination of adenosine, which is shown here to be present in salivary gland extracts of tsetse. While both genes are present and expressed in G. m. morsitans and G. p. palpalis, only TSGF-1 is present in G. austeni. We present the molecular characteristics of the cDNAs, their genomic arrangement and their regulation of expression in different fly tissues and species. We discuss the potential role of these proteins in hemostatis and in African trypanosome transmission by different species of tsetse.

Isozymic comparison of five laboratory lines of tsetse flies belonging to the two subspecies of Glossina palpalis (Diptera: Glossinidae).

Three laboratory colonies of Glossina palpalis palpalis and two of G. p. gambiensis have been characterized by means of 14 polymorphic enzyme loci. The presence/absence of some alleles for three enzymes (octanol dehydrogenase, phosphoglucomutase and aldehyde oxidase) distinguished the two subspecies. Other differences in allozymes could not be used to discriminate between subspecies but could be used to distinguish populations within each of the subspecies. The genetic differences between populations of a given subspecies are briefly discussed.

Tsetse-trypanosome interactions: rites of passage.

Trypanosomes that cause sleeping sickness (Trypanosoma brucei rhodesiense and T. b. gambiense) are entirely dependent on tsetse for their transmission between hosts, but the flies are not easily infected. This situation has not arisen by chance - the tsetse has evolved an efficient defence system against trypanosome invasion. In this review, Susan Welburn and Ian Maudlin chart the progress of trypanosomes through the fly and identify some of the hazards faced by both parasite and fly that affect vector competence of tsetse.

Glossina morsitans morsitans and Glossina palpalis palpalis: dosage compensation raises questions about the Milligan model for control of trypanosome development.

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 in Glossina m. morsitans and hexokinase and phosphoglucomutase in Glossina 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 of Trypanozoon maturation in tsetse are discussed in light of these findings and previously published reports on the lack of fitness effects of mature Trypanozoon infections in tsetse and on published results on antitrypanosomal factors in male and female tsetse flies.

A new method for identifying blood meals of human origin in tsetse flies.

A new sensitive technique using the electrophoresis of superoxide dismutase to distinguish between tsetse blood meals of human and non-human origin is described. In Côte d'Ivoire, 602 blood meals were collected; 170 were from man (28.3%), 377 from animals (62.6%) and 55 were unidentified (9.1%) because no pattern was observed. When calculating the index of epidemiological risk, it is strongly correlated with the incidence of sleeping sickness cases.

Genetics of Glossina palpalis palpalis: designation of linkage groups and the mapping of eight biochemical and visible marker genes.

The loci for three enzymes (hexokinase, phosphoglucomutase, and testicular esterase) and two eye-color mutants (brick and tan) are mapped on the X chromosome of Glossina palpalis palpalis. The loci occur in the order brick Hex (tan/Pgm) Est-t, with a recombination frequency of approximately 78% between the outer two loci. The locus for octanol dehydrogenase is located in linkage group II and the loci for malate dehydrogenase and phosphoglucose isomerase are separated by a recombination frequency of about 42.5% in linkage group III. Intrachromosomal recombination occurs at a much lower frequency in males than in females. The distribution of five biochemical marker genes in the linkage groups of G. p. palpalis is markedly different from that found in other higher flies.

Relationships between host blood factors and proteases in Glossina morsitans subspecies infected with Trypanosoma congolense.

Host blood effects on Trypanosoma congolense establishment in Glossina morsitans morsitans and Glossina morsitans centralis were investigated using goat, rabbit, cow and rhinoceros blood. Meals containing goat erythrocytes facilitated infection in G.m.morsitans, whereas meals containing goat plasma facilitated infection in G.m.centralis. Goat blood effects were not observed in the presence of complementary rabbit blood components. N-acetyl-glucosamine (a midgut-lectin inhibitor) increased infection rates in some, but not all, blood manipulations. Cholesterol increased infection rates in G.m.centralis only. Both compounds together added to cow blood produced superinfection in G.m.centralis, but not in G.m.morsitans. Midgut protease levels did not differ 6 days post-infection in flies maintaining infections versus flies clearing infections. Protease levels were weakly correlated with patterns of infection, but only in G.m.morsitans. These results suggest that physiological mechanisms responsible for variation in infection rates are only superficially similar in these closely-related tsetse.

Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly Glossina longipennis.

A blood-meal-induced lectin (agglutinin) with proteolytic activity was isolated from midgut extracts of Glossina longipennis by a two-step procedure involving anion-exchange chromatography. It is a glycoprotein [native molecular weight (M(r) 61,000 +/- 3000 da) composed of two noncovalently-linked subunits designated alpha (M(r), approximately 27,000 da) and beta (M(r), approximately 33,000 da). The trypsin activity and the glycosyl residues were present on the alpha- and beta-subunits, respectively. The native protein was capable of agglutinating both bloodstream-form and procyclic trypanosomes as well as rabbit red blood cells. This activity was strongly inhibited by D-glucosamine and weakly inhibited by N-acetyl-D-glucosamine. Similarly, soybean trypsin inhibitor abrogated agglutination of bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination activity was sensitive to temperatures above 40 degrees C but was unaffected by chelators of metal ions. Antibodies raised against the protein were used in immunoblotting experiments to show the presence of a similar protein in several members of the Glossina species. However, no cross-reactivity was detected with midgut extracts prepared from sandflies, mosquitoes, or stable flies. It is proposed that this molecule might play an important role in differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms.

Cytogenetic and isozymic comparisons of two laboratory lines of Glossina palpalis gambiensis.

The genetics of two laboratory colonies of Glossina palpalis gambiensis were characterized by C-banding and isoenzyme studies. The colonies, derived from flies collected in the same locality, had different histories in the laboratory and different susceptibilities to trypanosome infection. Although the two lines were also found to differ in the frequencies of chromosome and isozyme variants, the variation was not enough to put their specific status in doubt; it was probably the result of genetic drift since the foundation of the colonies.