2. Tsetse biology

(a) Rearing of tsetse flies

(b) Taxonomy, anatomy, physiology, biochemistry

[See also 27: no. 12702.]

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 100 000 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 defenses and symbiotic flora.

A study of reproductive disorders was carried out through the dissection of 11 012 tsetse flies caught over a period of one year in different forested habitats of Glossina palpalis palpalis, at Daloa in Ivory Coast. The proportion of females with reproductive disorders was very low and estimated at 0.79 percent. Out of 87 tsetse flies with reproductive disorders, 93.10 percent were abortions, 5.77 percent were ovariole blockage and 1.13 percent were uterine pupariation. Reproductive disorders were recorded from all age groups: 0.78 percent in young parous flies (out of 6 398 tsetse flies examined) and 0.80 percent in old parous flies (out of 4 614 tsetse flies dissected). Our results show that reproductive disorders occur at any stage of the female pregnancy cycle. Amplifying these reproductive disorders using chemical compounds is proposed as a way of improving the efficacy of insecticide-impregnated targets (pour-on, traps and screens) used for tsetse control in rain forest areas.

In a previous study, comparison of the behaviour of teneral Glossina morsitans morsitans on waterbuck, Kobus defassa (a refractory host), and on two preferred hosts, buffalo, Syncerus caffer, and ox, Bos indicus, suggested the presence of allomones in the waterbuck odour. Examination of the volatile odours by coupled gas chromatography-electroantennographic detection showed that the antennal receptors of the flies detected constituents common to the three bovids (phenols and aldehydes), as well as a series of compounds specific to waterbuck, including C₈-C₁₃ methyl ketones, d-octalactone and phenols. In this study, behavioural responses of teneral G. m. morsitans to different blends of these compounds were evaluated in a choice wind tunnel. The flies’ responses to known or putative attractant blends (the latter comprising EAG-active constituents common to all three animals and those common to buffalo and ox, excluding the known tseste attractants, 4-methylphenol and 3-n-propylphenol), and to putative repellent (the blend of EAG-active compounds specific to the waterbuck volatiles), were different. A major difference related to their initial and final behaviours. When a choice of attractant blends (known or putative) and clean air was presented, flies initially responded by flying upwind toward the odour source, but later moved downwind and rested on either side of the tunnel, with some preference for the side with the odour treatments. However, when presented with a choice of waterbuck-specific blend (putative repellent) and clean air, the flies’ initial reaction appeared random; flies flew upwind on either side, but eventually settled down on the odourless side of the tunnel. Flies that flew up the odour plume showed an aversion behaviour to the blend. The results lend further support to previous indications for the existence of a tsetse repellent blend in waterbuck body odour and additional attractive constituents in buffalo and ox body odours.

In this news item, recent work on the tsetse symbiont Wigglesworthia glossinidia is described, and its possible relevance to the control of tsetse and the disease trypanosomiasis noted.

Fat body and hemocytes play a central role in cellular and humoral responses for systemic infections in invertebrates, similar to the mammalian liver and blood cells. Epithelial surfaces, in particular the midgut, participate in the initial local immune responses in order to aid in the generation of the terminal cytotoxic molecules that mediate non-self recognition. Here, we describe for the first time the immune responses of a cluster of cells at the foregut/midgut junction - known as proventriculus (cardia) in the medically and agriculturally important insect, tsetse fly (Glossina). We provide evidence for the transcriptional induction of the antimicrobial peptides attacin and defensin as well as for the reactive nitrogen intermediate (RNI) nitric oxide synthase (NOS) upon microbial challenge by either microinjection or feeding. The proventriculus from immune challenged flies also has higher NOS and nitric oxide (NO) activities as well as increased levels of the reactive oxygen intermediate, hydrogen peroxide (H₂O₂). In several vector pathogen systems, including tsetse flies and African trypanosomes, stimulation of systemic responses prior to pathogen acquisition has been shown to reduce disease transmission. Furthermore, the induction of systemic immune responses has been documented while pathogens are still differentiating within the midgut environment. While evidence for a close molecular communication between the local and systemic responses is accumulating, the molecular signals that mediate these interactions are at present unknown. Reactive intermediates such as NO or H₂O₂ may function as immunological signals for mediating the molecular communication between the different insect compartments. We discuss the putative role of the proventriculus in invertebrate immunity and specifically speculate on its significance for trypanosome transmission in tsetse.

Wigglesworthia glossinidia brevipalpis, the obligate bacterial endosymbiont of the tsetse fly Glossina brevipalpis, is characterized by extreme genome reduction and AT nucleotide composition bias. Here, multivariate statistical analyses are used to test the hypothesis that mutational bias and genetic drift shape synonymous codon usage and amino acid usage of Wigglesworthia. The results show that synonymous codon usage patterns vary little across the genome and do not distinguish genes of putative high and low expression levels, thus indicating a lack of translational selection. Extreme AT composition bias across the genome also drives relative amino acid usage, but predicted high-expression genes (ribosomal proteins and chaperonins) use GC-rich amino acids more frequently than do low-expression genes. The levels and configuration of amino acid differences between Wigglesworthia and Escherichia coli were compared to test the hypothesis that the relatively GC-rich amino acid profiles of high-expression genes reflect greater amino acid conservation at these loci. This hypothesis is supported by reduced levels of protein divergence at predicted high-expression Wigglesworthia genes and similar configurations of amino acid changes across expression categories. Combined, the results suggest that codon and amino acid usage in the Wigglesworthia genome reflect a strong AT mutational bias and elevated levels of genetic drift, consistent with expected effects of an endosymbiotic lifestyle and repeated population bottlenecks. However, these impacts of mutation and drift are apparently attenuated by selection on amino acid composition at high-expression genes.

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. 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. 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.

EP and GPEET procyclin, the major surface glycoproteins of procyclic forms of Trypanosoma brucei, are truncated by proteases in the midgut of the tsetse fly Glossina morsitans morsitans. We show that soluble extracts from the midguts of teneral flies contain trypsin-like enzymes that cleave the N-terminal domains from living culture-derived parasites. The same extract shows little activity against a variant surface glycoprotein on living bloodstream form T. brucei (MITat 1.2) and none against glutamic acid/alanine-rich protein, a major surface glycoprotein of Trypanosoma congolense insect forms although both these proteins contain potential trypsin cleavage sites. Gel filtration of tsetse midgut extract revealed three peaks of tryptic activity against procyclins. Trypsin alone would be sufficient to account for the cleavage of GPEET at a single arginine residue in the fly. In contrast, the processing of EP at multiple sites would require additional enzymes that might only be induced or activated during feeding or infection. Unexpectedly, the pH optima for both the procyclin cleavage reaction and digestion of the trypsin-specific synthetic substrate Chromozym-TRY were extremely alkaline (pH 10). Direct measurements were made of the pH within different compartments of the tsetse digestive tract. We conclude that the gut pH of teneral flies, from the proventriculus to the hindgut, is alkaline, in contradiction to previous measurements indicating that it was mildly acidic. When tsetse flies were analysed 48 h after their first bloodmeal, a pH gradient from the proventriculus (pH 10.6 ± 0.6) to the posterior midgut (pH 7.9 ± 0.4) was observed.

An ultrastructural study of the heart of the tsetse fly Glossina morsitans and of several other species of cyclorraphan flies revealed that the ventral region of the heart of adult flies is supported by a muscular septum not present in the larval stage. The pericardial septum of the adult heart is composed laterally of alary muscles and a central longitudinal muscle that extends the length of the abdominal aorta, whereas the larval heart is supported ventrally only by alary muscles and strands of connective tissue. Thus, unlike the larval stage, and the heart of other insects, the pericardial septum of adult cyclorraphan flies contains a central band of longitudinal muscle that, along with the alary muscle, forms a large pericardial sinus lying between the septum and the heart. Neurosecretory nerves arising from the lateral nerves of the thoracico-abdominal ganglion extend dorsad to the pericardial septum, where they form neuromuscular junctions on the muscle fibres of the pericardial septum or traverse the septum terminating in the pericardial sinus, thereby creating one of the largest neurohaemal organs in these flies. In the tsetse fly, some of the neurosecretory fibres also extend between the muscle fibres of the myocardium, and release their material into the lumen of the heart.

Glossina pallidipes is a vector of African trypanosomiasis. Here we characterize eight new polymorphic microsatellite loci in 288 G. pallidipes sampled from 12 Kenya populations. The number of alleles per locus ranged from four to 36 with a mean of 20.5 ± 10.1. Expected single locus heterozygosities varied from 0.044 to 0.819. Heterozygosity averaged 0.616 ± 0.246. No linkage disequilibrium was found. We also report results in eight other tsetse species estimated by using the primers developed in G. pallidipes. The primers worked best in G. swynnertoni and G. austeni and worst in G. m. morsitans and G. m. submorsitans.

Teneral Glossina palpalis gambiensis (Diptera: Glossinidae) were infected with a culture of procyclic forms of Trypanosoma brucei gambiense using a single-bloodmeal membrane feeding technique. The infection was monitored by analysing the saliva (mature infection) and anal drop (midgut infection) of each fly at different post-infection times both by microscopic observation and polymerase chain reaction (PCR). Amplification revealed many more positive anal drops than microscopy. The monitoring showed that the installation of T. b. gambiense in Glossina took place at least 11 days after the infection and that maturation occurred after 29 days. It also reflected precisely the parasitic status of each tsetse fly as determined by the dissection, microscopic examination and PCR amplification of the midguts and salivary glands 47 days post-infection. Twice as many tsetse flies with mature salivary glands infection were revealed by PCR than by microscopic examination, but the two techniques gave exactly the same results regarding the proportion of flies with midgut infection. This study also demonstrated the ability of natural non-infective procyclic forms of T. b. gambiense to colonise the midgut and subsequently establish in the salivary glands of G. p. gambiensis.

(c) Distribution, ecology, behaviour, population studies

[See also 27: no. 12669.]

A density-dependent model is used to describe the dynamics of an open population of tsetse flies. Immigration (or emigration) takes place when the total population is below (or above) a biologically determined threshold value. The population is also subjected to birth and death rates, as well as to the risk of being trapped (continuously or intermittently). During trapping the population decreases toward a ‘low’ equilibrium population and when trapping ceases the population starts recovering and increases toward a ‘high’ equilibrium. The model is fitted using data collected on trapped flies in four experiments. The first one was conducted with ‘intermittent trapping’ (i.e. several trapping-recovery cycles) on Glossina fuscipes fuscipes in the Central African Republic (Bangui area). In the other experiments, trapping data on Glossina palpalis palpalis was collected in ‘aggregate’ form over several days at a time. Two of these were in Congo-Brazzaville (Bouenza area) and one in the Ivory Coast (Vavoua focus). Estimates are derived for the low and high equilibrium values as well as the trapping rate. The estimated effect of sustained trapping is to reduce the population to low equilibrium values that are 85-87 percent lower than the levels without trapping. The effects of the natural intrinsic growth and of the migration flows cannot be estimated separately because in the model they are mathematically indistinguishable.

Observations on the parasitism of Glossina palpalis palpalis by Hexamermis glossinae were carried out over a period of one year by catching flies at Abengourou, Aboisso and Daloa (forested area of Côte d’Ivoire). No parasite was observed in 2 168 Glossina palpalis palpalis caught at Abengourou and in 9 732 caught at Aboisso. At Daloa, dissections of 7 341 Glossina revealed 1.75 percent parasitised flies. All the worms were located in the abdominal cavity, loosely intertwined with the internal organs. Males had a higher infestation rate than females (2.68 percent as against 1.26 percent, respectively). The parasites were more abundant among the nulliparous flies (2.30 percent) than among the young parous flies (1.19 percent) and the old parous flies (0.52 percent). The majority of infected flies were caught at the beginning of the rainy season (5.17 percent) and few were caught in the dry season (0.23 percent). The low parasitic infection rate observed here indicates a minimal effect on the population dynamics of the vector of sleeping sickness in Côte d’Ivoire.

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.

In tsetse both sexes feed exclusively on the blood of vertebrates for a few minutes every 2-3 days. Tsetse flies seek cover from high temperatures to conserve energy and plants provide shelter for tsetse in all the biotopes they occupy. Recently, tsetse have taken cover in plantations and under the invasive bush Lantana camara that has invaded large areas of the tsetse fly belt of Africa. Flies from such refugia are implicated in sleeping sickness epidemics. In a wind tunnel we show that both foliage and an extract of volatiles from foliage of L. camara attract three tsetse spp. from different habitats: Glossina fuscipes fuscipes (riverine), G. brevipalpis (sylvatic) and G. pallidipes (savannah). Gas chromatography analysis of volatiles extracted from leaves and flowers of L. camara coupled to electroantennograme recordings show that 1-octen-3-ol and b-caryophyllene are the major chemostimuli for the antennal receptor cells of the three tsetse spp. studied. A binary mixture of these products attracted these flies in the wind tunnel. The gas chromatography linked electroantennograme analysis of the L. camara extracts also show that the antennal receptor cells of the three tsetse spp. respond similarly to groups of volatiles derived from the major biosynthetic and catabolic pathways of plants, i.e. to mono- and sesquiterpenes, to lipoxidation products and to aromatics. Mixtures of these plant volatiles also attracted tsetse in the wind tunnel. These findings show that tsetse flies have conserved a strong sensitivity to volatile secondary products of plants, underlining the fundamental role of vegetation in tsetse survival.

Studies were made of infection rates of trypanosomes in the tsetse fly Glossina morsitans morsitans when maintained in vivo (rabbits) or in vitro on high quality, gamma-irradiated, sterile defibrinated bovine blood, obtained from the Entomology Unit of the IAEA. For both Trypanosoma congolense and T. b. brucei, in vitro maintenance significantly reduced the proportion of flies that developed mature metacyclic trypanosome infections.