Michael J. Lehane

Interactions between tsetse and trypanosomes with implications for the control of trypanosomiasis

Tsetse flies (Diptera: Glossinidae) are vectors of several species of pathogenic trypanosomes in tropical Africa. Human African trypanosomiasis (HAT) is a zoonosis caused by Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West and Central Africa. About 100000 new cases are reported per year, with many more probably remaining undetected. Sixty million people living in 36 countries are at risk of infection. Recently, T. b. gambiense trypanosomiasis has emerged as a major public health problem in Central Africa, especially in the Democratic Republic of Congo, Angola and southern Sudan where civil war has hampered control efforts. African trypanosomes also cause nagana in livestock. T. vivax and T. congolense are major pathogens of cattle and other ruminants, while T. simiae causes high mortality in domestic pigs; T. brucei affects all livestock, with particularly severe effects in equines and dogs. Central to the control of these diseases is control of the tsetse vector, which should be very effective since trypanosomes rely on this single insect for transmission. However, the area infested by tsetse has increased in the past century. Recent advances in molecular technologies and their application to insects have revolutionized the field of vector biology, and there is hope that such new approaches may form the basis for future tsetse control strategies. This article reviews the known biology of trypanosome development in the fly in the context of the physiology of the digestive system and interactions of the immune defences and symbiotic flora.

Gene vector and transposable element behavior in mosquitoes

SUMMARY The development of efficient germ-line transformation technologies for mosquitoes has increased the ability of entomologists to find, isolate and analyze genes. The utility of the currently available systems will be determined by a number of factors including the behavior of the gene vectors during the initial integration event and their behavior after chromosomal integration. Post-integration behavior will determine whether the transposable elements being employed currently as primary gene vectors will be useful as gene-tagging and enhancer-trapping agents. The post-integration behavior of existing insect vectors has not been extensively examined. Mos1 is useful as a primary germ-line transformation vector in insects but is inefficiently remobilized in Drosophila melanogaster and Aedes aegypti. Hermes transforms D. melanogaster efficiently and can be remobilized in this species. This element is also useful for creating transgenic A. aegypti, but its mode of integration in mosquitoes results in the insertion of flanking plasmid DNA. Hermes can be remobilized in the soma of A. aegypti and transposes using a common cut-and-paste mechanism; however, the element does not remobilize in the germ line. piggyBac can be used to create transgenic mosquitoes and occasionally integrates using a mechanism other than a simple cut-and-paste mechanism. Preliminary data suggest that remobilization is infrequent. Minos also functions in mosquitoes and, like the other gene vectors, appears to remobilize inefficiently following integration. These results have implications for future gene vector development efforts and applications.

The use of specific and generic primers to identify trypanosome infections of wild tsetse flies in Tanzania by PCR

The accurate identification of trypanosome species and subspecies remains a challenging task in the epidemiology of human and animal trypanosomiasis in tropical Africa. Currently, there are specific PCR tests to identify about 10 different species, subspecies or subgroups of African tsetse-transmitted trypanosomes. These PCR tests have been used here to identify trypanosomes in four species of tsetse (Glossina brevipalpis, G. pallidipes, G. swynnertoni, G. morsitans morsitans) from two areas of Tanzania. PCR using species-specific primers was performed on 1041 dissection-positive proboscides, giving an overall positive identification in 254 (24%). Of these, 61 proboscides (24%) contained two or more trypanosomes. The trypanosome with the greatest overall prevalence at both field sites was Trypanosoma simiae Tsavo, which was identified in a total of 118 infected tsetse proboscides (46%). At Pangani, T. godfreyi was found in G. pallidipes but not in G. brevipalpis, suggesting that these flies might have different susceptibility to this trypanosome or might have fed on a different range of hosts. A high proportion (about 75%) of trypanosome infections remained unidentified. To investigate the identity of these unidentified samples, we used primers complementary to the conserved regions of trypanosomal small subunit ribosomal RNA (ssu rRNA) genes to amplify variable segments of the gene. Amplified DNA fragments were cloned, sequenced and compared with ssu rRNA genes on database of known trypanosome species. In this way, we have tentatively identified two new trypanosomes: a trypanosome related to Trypanosoma vivax and a trypanosome related to T. godfreyi. The T. godfreyi-related trypanosome occurred frequently in the Tanzanian field samples and appears to be widespread. Molecular identification of these two new trypanosomes should now facilitate their isolation and full biological characterisation.

Regulation of digestive enzyme levels in insects

Abstract Because of confusion over the use of the term secretagogue we propose that the direct interaction of an element of the meal with digestive enzyme-producing cells, resulting in increased rates of enzyme synthesis or secretion, should be referred to as a prandial mechanism. Most studies suggest that paracrine and/or prandial mechanisms are the main factors controlling digestive enzyme synthesis and secretion in insects. Distinguishing between the two mechanisms is a significant challenge. In many insects, soluble proteins are potent stimulants of proteinase synthesis and secretion probably through the prandial/paracrine pathways. The details of the mechanisms involved are unknown. Hormones (other than paracrine) probably play a role in modulating the levels of digestive enzymes rather than in their absolute control. Feeding can effect the control of digestive enzyme synthesis at either the transcriptional or translational level.

Post-integration behavior of a Mos1 mariner gene vector in Aedes aegypti.

The post-integration behavior of insect gene vectors will determine the types of applications for which they can be used. Transposon mutagenesis, enhancer trapping, and the use of transposable elements as genetic drive systems in insects requires transposable elements with high rates of remobilization in the presence of transposase. We investigated the post-integration behavior of the Mos1 mariner element in transgenic Aedes aegypti by examining both germ-line and somatic transpositions of a non-autonomous element in the presence of Mos1 transposase. Somatic transpositions were occasionally detected while germ-line transposition was only rarely observed. Only a single germ-line transposition event was recovered after screening 14,000 progeny. The observed patterns of transposition suggest that Mos1 movement takes place between the S phase and anaphase. The data reported here indicate that Mos1 will be a useful vector in Ae. aegypti for applications requiring a very high degree of vector stability but will have limited use in the construction of genetic drive, enhancer trap, or transposon tagging systems in this species.

Transformation of Stomoxys calcitrans with a Hermes gene vector

The ability of the Hermes transposable element to function as a germ line transformation vector was tested in the stable fly, Stomoxys calcitrans. Plasmid‐based transposable element mobility assays indicated moderate mobility of Hermes in this species. Germline transformants were created using a Hermes element containing the enhanced green fluorescent protein (EGFP) under the regulatory control of the promoter from Actin5C gene of Drosophila melanogaster. Approximately 4% of the fifty‐five adults that developed from the 1903 G0 embryos injected with the vector produced transgenic progeny. In the four transgenic lines established, the EGFP expression pattern was distinctly nonuniform and levels of expression were low. Promoters other than the one from the Actin5C gene of D.melanogaster should be considered for widespread, constitutive expression. All transgenic lines contained multiple (2–4) integrated Hermes elements. Hermes integration events occurred through a canonical cut‐and‐paste mechanism.

Protein stimulation of trypsin secretion from the opaque zone midgut cells of Stomoxys calcitrans

Abstract The epithelial layer of the opaque zone of the midgut of Stomoxys calcitrans contains putative endocrine cells as well as cells synthesizing and secreting digestive enzymes. Soluble protein stimulates secretion of trypsin from in vitro preparations of the opaque zone in a time and dose-dependent manner. The half-maximal biological response (ED50) values for the proteins tested lie between 1 × 10−6and 1 × 10−7M. Trypsin secretion is not stimulated by amino acids or poly- l amino acids and the response to albumin is diminished by 50% following boiling. There is no apparent relationship between the type of protein, molecular weight or pI point and the ability to promote trypsin secretion—all proteins tested here promoting secretion to similar degrees. There is no stimulation of secretion by brain tissue homogenate beyond that which would be expected because of its protein content.

Regulation of midgut defensin production in the blood-sucking insect Stomoxys calcitrans

The Stomoxys midgut defensin (Smd) family of genes are exclusively expressed in the anterior midgut of adult flies. Their putative function is protection of the stored bloodmeal from microbial attack. Smd genes are constitutively expressed, up‐regulated in response to a bloodmeal and further up‐regulated by immune stimulation per os but only in the presence of a bloodmeal not a sugar meal. Smd genes are down‐regulated in response to a systemic immune challenge. Smd gene constructs transfected into l(2)mbn cells undertake constitutive expression but are not up‐regulated by immune challenge. Electrophoretic mobility shift assays (EMSA) suggest the rel‐like sites in the proximal promoter region of Smd genes do not bind midgut factors and so are non‐functional.

Association of midgut defensin with a novel serine protease in the blood-sucking fly Stomoxys calcitrans

Using ELISA we provide direct evidence that the midgut defensins of the blood‐sucking fly Stomoxys calcitrans are secreted into the gut lumen. We show that midgut defensin peptide levels increase up to fortyfold in response to a blood meal but not to a sugar meal. The data suggests the midgut defensin genes are post‐transcriptionally regulated and that their function is protection of the stored blood meal from bacterial attack while it awaits digestion. Using recombinant defensins produced in Pichia pastoris we demonstrate that while in the gut cells the midgut defensins are bound in an SDS‐stable complex to proteins with an apparent molecular weight of > 26 kDa from which they are released when secreted into the gut lumen. This > 26 kDa protein (Ssp3) has been cloned and sequenced and is a member of the serine protease S1 family with homologies to multiple insect proteases and to vertebrate trypsins and elastases.

Studies on the synthesis and secretion of trypsin in the midgut of Stomoxys calcitrans

Abstract Iso-electric focusing of affinity-purified trypsin produced at least 15 bands which also stained positively in protease zymograms. Fresh homogenate containing unactivated trypsin produced bands which were missing from activated samples. Affinity purified, fully active trypsin separated by SDS-PAGE gave major bands with molecular weights of 22,900 and 31,600 daltons (Da). These bands were absent from unactivated homogenates which instead produced major protein bands at 24,700 and 33,500 Da. Tritium labelling experiments suggest there is a baseline level of asynchronous secretion from unstimulated opaque zone cells. While trypsin is stored as an inactive pro-enzyme it is secreted in a fully activated form. Inhibitor studies suggest the burst in trypsin synthesis immediately following the blood meal is regulated at the translational, rather than the transcriptional, level. Inhibitor studies show secretory granules in opaque zone cells are acidified by a strongly ATP-dependent proton pump.