VECTOR BIOLOGY (Entomology)

BIOLOGIE DU VECTEUR (Entomologie)

HYBRID STERILITY WITHIN GLOSSINA MORSITANS SUBSPECIES AND GLOSSINA SWYNNERTONI.

STERILITE HYBRIDE CHEZ LES SOUS-ESPECES DE GLOSSINA MORSITANS ET CHEZ GLOSSINA SWYNNERTONI

P. A. Olet¹ and A. S. Robinson²

¹Veterinary, Laboratories, P.O Kabete, Nairobi, Kenya

²FAO/IAEA Agriculture and Biotechnology Laboratory Agency’s Laboratories A-2444, Seibersdorf,

Austria.

Résumé

On a proposé la stérilité hybride et le satyrisme comme méthodes pour éliminer les populations de tsétsé. L’absence d’une barrière d’accouplement chez la sous-espèce de G. morsitans et l’espèce G. swynnertoni étroitement liées a fait d’elles des cibles pour une telle suppression. Dans la présente étude, des croisements réciproques de G. m. morsitans, G. m. centralis de la Tanzanie, G. m. centralis du Botswana et G. swynnertoni ont été réalisés. G. m. morsitans et G. m. centralis de la Tanzanie ont été testés pour leur compatibilité sexuelle. L’hybridation a réduit la fertilité des croisements d’au moins 50% et les croisements réciproques ont montré une hybridation asymétrique (HA). La plus efficace HA a été obtenue lorsque G. swynnertoni était croisée avec G. m. centralis du Botswana. Les mâles de G. swynnertoni provoquaient une sterilité partielle chez les femelles de G. m. centralis, tandis que le croisement réciproque était complètement stérile. Il n’y avait pas de HA dans le croisement entre G. m. morsitans et G. swynnertoni et entre les populations de G. m. centralis. Il y avait également une tendance des femelles hybrides à récouvrer leur fertilité avec le temps. G. m. morsitans et G. m. centralis étaient sexuellement compatibles avec une tendance aux femelles de G. m. morsitans de s’accoupler plus avec les mâles de G. m. centralis (satyrisme). Les femelles de G. m. centralis s’accouplaient au hasard avec les mâles de G. m. centralis et de G. m. morsitans. La femelle de G. m. morsitans s’accouplait de nouveau moins avec ses propres mâles lorsque le premier accouplement était fait avec des mâles de G. m. centralis. Le sperme utilisé par les femelles accouplées maintes fois était variable. En se basant sur ces données préliminaires, les populations de Glossina swynnertoni pourraient être éliminées par le lâcher des mâles de G. m. centralis du Botswana. En outre, les deux populations de G. m. centralis ne sont pas reproductivement isolées et les mâles de chacune des espèces pourront être utilisés pour la TIS. Même si les résultats montrent que les femelles deviennent réfractaires après accouplement avec les "satyrs", plusieurs analyses détaillées de l’ADN pour l’utilisation du sperme sont nécessaires si de multiples accouplements sont fréquents sur le terrain. L’analyse d’ADN Polymorphe à Amplification Aléatoire était utilisée pour évaluer la variabilité génétique de la population de G. swynnertoni en laboratoire et sur le terrain.

Summary

Hybrid sterility and satyrism have been proposed as methods to suppress tsetse populations. Lack of a mating barrier within the closely related G. morsitans subspecies and G. swynnertoni makes them targets for such suppression. In the present study, reciprocal crosses of G. m. morsitans, G. m. centralis from Tanzania, G. m. centralis from Botswana and G. swynnertoni were performed. G. m. morsitans and G. m. centralis Tanzania were tested for sexual compatibility. Hybridization reduced the fertility of the crosses by at least 50% and reciprocal crosses showed hybridization asymmetry (HA). The most significant HA was when G. swynnertoni were crossed with G. m.centralis from Botswana. G. swynnertoni males induced partial sterility in G. m. centralis females however the reciprocal cross was completely sterile. There was no HA in the cross between G. m. morsitans and G. swynnertoni and between the G. m. centralis populations. There was also a tendency for hybridized females to recover fertility with time. G. m. morsitans and G. m. centralis were sexually compatible with a tendency for G. m. morsitans females to mate more with G. m. centralis males (satyrism or negative assortative mating). Glossina m. centralis females mated randomly with both G. m. centralis and G. m. morsitans males. Female G. m. morsitans remated less with their own males when the first mating was with G. m centralis males. Sperm use by multiple mated females was variable. Based on these preliminary results, G. swynnertoni populations could be eliminated by the release of G. m. centralis males from Botswana. Furthermore, the two populations of G. m. centralis are not reproductively isolated and males of either species may be used for SIT. Though the results show that the females become refractory after mating with satyrs, a more detailed DNA analysis of sperm utilization is required if multiple mating is common in the field. Random Amplified Polymorphic DNA (RAPD) analysis was used to evaluate genetic variability in a laboratory and a field population of G. swynnertoni.

Background

Tsetse flies (Glossina) are well known vectors of African Trypanosomosis and their deadly effects on animals and humans are well documented. Hybridisation is an alternative genetic method of controlling tsetse flies and involves mating of closely related species. The hybridised females may become sterilised or produce male and female hybrids with various levels of sterility. In tsetse, there have been several extensive laboratory studies on this phenomenon stemming from the early observations made by Potts (1944) and Vanderplank (1944). Vanderplank (1947) in fact carried out the first field trial of genetic control for any pest insect. In tsetse, two strategies to use hybrid sterility can be considered: 1) the release of sterile hybrid males into a field population or 2) the release of males of one species into the field population of a second. The latter approach has been termed the use of “satyrs” (Ribeiro, 1988).

When considering using this phenomenon for control of an insect pest, several important questions have first to be answered.

· Is mating random between the different species in the field, i.e. are there pre-mating isolation barriers?

· What is the basis of the observed sterility, i.e. is it genetic, physiological or related to maternally transmitted factors?

· What is the physiological status of the “hybridised” female, i.e. will she tend to remate until she finds the “right” male?

· Will a multiply mated female use one type of sperm in preference to another?

In tsetse, important aspects need clarification before any field trial can be proposed. Major difficulties in answering the above questions relate to obtaining and colonising populations of tsetse and the uncertainty as to the genetic relationships of tsetse populations within a widely distributed species.

Objectives

1: Analyse hybridisation between G. swynnertoni and G. morsitans subspecies

In an attempt to validate the potential of hybrid sterility to control tsetse populations and cognisant of the fact that allopatry can cause sterility, G. m. centralis from Tanzania and Botswana were studied as different populations of the same species in regard to hybridisation with G. m. morsitans and G. swynnertoni.

2: Determine sperm use in multiple mated females and characterise genetic variability of G. swynnertoni populations.

Use of sperm in multiple mated females is an interesting biological phenomenon and if hybrid sterility is to be used to develop control methods for tsetse then it is important to assess how a female uses sperm from two taxa. The availability of easy to use molecular markers makes it possible to answer this question. Species-specific RAPD markers were identified for G. m. morsitans and G. m. centralis using laboratory colonies. For G. swynnertoni, RAPD markers were assayed in a long established laboratory colony and in a sample of flies trapped in the field. Selected markers were used to ascertain paternity in multiple mated females.

Protocol

All experimental flies were maintained under standard rearing conditions of 23-24°C and 65-80% RH. They were membrane fed on a diet of fresh frozen irradiated bovine blood. Females (2-4 days old) and males (7-14 days old) were mass mated in round cages (20cm diameter) when hybridising G. m. morsitans, G. m. centralis Tanzania, G. m. centralis Botswana and G. swynnertoni. Mating indices by Cayol et al., (1999) were used to assess sexual compatibility and competitiveness. Remating was done after 48h recovery. The females were observed up to 5^th ovulation cycle. Pupae produced were kept singly until emergence when they were frozen (-20°C) to await DNA analysis to determine paternity.

DNA extraction and RAPD amplification

The DNA from hybrids and parents was extracted using a modified method of Bender et al., (1983). PCR based RAPD reactions were carried out following the methods developed by Welsh and McClelland (1990) and Williams et al., (1990).

Results

Crosses

Males and females of the different taxa readily mated with each other except those involving G. swynnertoni males confined with either G. m. centralis (T) females or G. m. morsitans females All the crosses and the reciprocal crosses were able to inseminate. One of the reciprocal crosses always had a higher fertility hybridisation asymmetry (HA) cross was fully sterile. The populations of G. m. centralis from Tanzania and Botswana were sexually compatible. There was apparently no sex ratio distortion, as F₁ ratios for the crosses did not differ from the Mendelian ratio of 1:1 at 5 % level of significance. Fertility in most cases increased with the increasing age of the females. Fertility was delayed more when hybridisation was with G. m. morsitans males. However the females appeared to overcome the incompatibility and fertility was recovered at the 3^rd and 4^th ovulation cycles.

G. m. morsitans and G. m. centralis (T) males were equally competitive when no choice was offered. G. m. morsitans females preferred G. m. centralis (T) males when there was choice (satyrism) but remated less with G. m. morsitans males when the first mating was with G. m. centralis (T) males. There was no difference in the tendency of G. m. centralis (T) females to remate, regardless of the male mating sequence.

Primer selection

Oligonucleotide 10-mer primer set #100/1 from University of British Columbia (UBC) were used in a RAPD based PCR to identify molecular markers specific for G. m. morsitans, G. m. centralis and G. swynnertoni. Eighty primers were tested on male DNA but only 28 were selected for further analysis. Only diagnostic bands that were intense and highly consistent were used for the paternity analysis in the offspring of multiple mated females. Primers # 46, 53 and 66 produced polymorphic bands specific for G. m. morsitans, G. m. centralis and G. swynnertoni. Primers # 46 and 66 produced intense profiles that could be used to differentiate G. m. morsitans from G. m. centralis.

Characterisation of offspring from multiple mating

The table summarises the data of the paternity analysis. Glossina m. morsitans females use sperm from their own males following multiple mating with G. swynnertoni and G. m. morsitans males, independent of the order of mating. However, when G. m. morsitans females are multiply mated with G. m. morsitans males and G. m. centralis(T) males, sperm use depends on the order of mating. If the first male is G. m. morsitans then all progeny are sired by this male and there is no sperm displacement but if the first male is G. m. centralis(T) then mixed progeny are produced following some sperm displacement. This latter pattern is also found with G. m. centralis(T) females multiply mated with G. m. morsitans and G. m. centralis(T) males.

Five out of 10 primers gave polymorphic bands that showed Interpopulation variation between field collected and laboratory adapted G. swynnertoni. The frequency of some of the bands appeared to be predominant in one of the populations, an indication of unique traits that may be specific for that population. A thorough analysis is still required for major genetic differences.

Discussion

The use of the SIT as a component for tsetse intervention programmes is gaining increasing support within the tsetse community and eradication campaigns for large areas of Africa is being planned. The results reported here make a contribution towards improving the efficiency of the SIT and highlight some natural sterility mechanisms that could complement the implementation of conventional SIT technology. The feasibility of releasing G. m. centralis Botswana males into G. swynnertoni habitat has been strongly suggested. G. m. morsitans males can also be used to eradicate G. m. centralis Botswana and G. swynnertoni. The results have suggested that observation of females should extend beyond 3^rd ovulation cycles before being declared sterile. The two G. m. centralis populations are not reproductively isolated and males of either population could be used to eliminate the other. The preference of G. m. morsitans females for G. m. centralis males and the reluctance to mate a second time with their own males seem to support the theory of satyrism (Ribeiro 1988) and may suggest further evolutionary dynamics for quality males within G. morsitans complex. The use of RAPD polymorphism has been demonstrated to identify species and subspecies and also determine paternity. These easy to use molecular markers singly or in combination with other techniques are valuable tools to assess genetic compatibility between laboratory and field flies and may be manipulated to produce high quality males before SIT release programme.

Acknowledgement

This work was supported by a Fellowship from International Atomic Energy Agency

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SOME OBSERVATION ON ECOLOGY OF GLOSSINA FUSCIPES FUSCIPES IN BHAR EL JEBAL STATE, SUDAN

QUELQUES REMARQUES SUR L’ECOLOGIE DE GLOSSINA FUSCIPES FUSCIPES DANS L’ETAT DE BHAR EL JEBAL, AU SOUDAN

Mohammed¹, Y.O.; Mohammed-Ahmed², M.M.; El-Rayah³, I.E.; Suad³. M.S.; Malik, K.H.⁴

¹ Central Veterinary Research Laboratories (CVRL), Ministry of Science and Technology.

² Faculty of Veterinary Science, Sudan University for Science and Technology.

³ Tropical Medicine Research Institute, Ministry of Science and Technology.

⁴ Faculty of Veterinary Science, University of Khartoum.

Résumé

Une enquête préliminaire a été menée dans l’Etat de Bhar El-Jebal pour évaluer l’incidence, la répartition et la diversité des espèces de tsétsé, en utilisant des pièges Epsilon et biconiques non-appâtés. Les pièges ont été posés le long de divers types de végétation existante. Les résultats obtenus ont confirmé la présence de Glossina fuscipes fuscipes dans la région. L’on a découvert que cette espèce s’est répandue au-delà de la limite nord que l’on a connue auparavant.

Des données précises sur le type de végétation, la densité apparente et le taux de mouches ténérales de G. f. fuscipes ont été évaluées en utilisant des pièges Epsilon et biconiques non-appâtés pour l’échantillonnage des glossines. On a également évalué l’effet de l’emplacement des pièges par rapport au type de végétation sur la capture de mouches et l’efficacité des pièges. Les sites de contact mouche/homme ont aussi été bien identifiés.

L’écologie de la région a fait l’objet d’une description relative au climat, au type de végétation, au système de drainage et aux animaux sauvages et domestiques.

On a proposé des méthodes de lutte susceptibles d’être essayées dans la région, y compris la chimio-stérilisation du réservoir-homme par des techniques de détection et de traitement, et par la réduction des risques de contact mouche/homme en posant des pièges imprégnés de deltaméthrine le long des cours d’eau et de la végétation riveraine, à raison de 5 pièges par kilomètre linéaire.

Summary

A preliminary survey of tsetse flies (Diptera: Glossinidae) and Human African Trypanosomosis (HAT) was conducted in active foci of sleeping sickness in Bhar El-Jebal State, Southern Sudan. The survey was carried out during the rainy and dry seasons of the years 2000 and 2001, respectively. Tsetse flies were caught in biconical traps and Epsilon traps. Trypanosomosis was detected serologically in human volunteers of all age groups using Card Agglutination Test for Trypanosomosis (CATT) and Card Indirect Antigen Test for Trypanosomosis (CIATT) simultaneously. Only Glossina fuscipes fuscipes was caught, mainly in biconical traps. Catch size was up to 6.4/trap/day in riverine gallery forest, riverine thickets and hedges in farmland bordering watercourses. The prevalence of HAT in the area ranged between 18.5-80% and 29.7-100% with CATT and CIATT, respectively. These results are discussed in relation to human/tsetse contact and control of sleeping sickness in the area.

Introduction

Human African Trypanosomosis (HAT) transmitted by tsetse flies causes considerable human suffering, varying degree of mortality and serious loss of efficiency wherever it occurs and may result in migration and depopulation with consequent disruption of all human-activities. Southern Sudan has suffered a series of successive HAT epidemics since the last century (Bloss, 1960; Morris, 1961; Hutchinson, 1975; Snow, 1983). Considerable efforts and resources were mobilized to keep the disease under-control. Thus the disease had been nearly eradicated as a result of a sophisticated systemic diagnosis and treatment efforts undertaken over five years (Snow, 1999). However, there has been a massive resurgence due to population displacement and collapse of health system caused by the latest outbreak of the civil war, which had started in 1983. The current epidemic of HAT has affected people in all three States of the Equatorial Region (Snow, 1999), with prevalence ranging from 19.5-30.3% (El-Rayah, et al., 1999). It is accepted that these figures are actually underestimates due to difficulties in accession, surveillance and diagnosis. It must also be accepted that the problems presented by prevalence of tsetse flies are not those of diseases alone, but also the fundamental negative effects on natural resources and investment. In tropical Africa, land is a basic resource of a country although its fixed productivity can usually be improved by integrated settled system of farming, an ideal that is impossible to achieve in presence of tsetse flies.

In the Sudan, the land which is deprived of agricultural activities and improvement of farming system as a result of tsetse infestation is about 300,000 km²; most of this land lies in Southern Sudan South of latitude 10^o30N4 (Lewis, 1949; Buxton, 1955; Ford, 1963; Razig and Yagi, 1972; Ford and Katondo, 1977). Although Katondo (1984) had suggested the retreat of Glossina fuscipes fuscipes about 154 km to the south, no data or confirmation were presented. It is highly probable that the latter author might have suspected that the fly belt had contracted as a consequence of drought and subsequent environmental changes. To date no systemic survey to define the distribution of tsetse flies in Southern Sudan has ever been done since Lewis (1949). Here we propose to fill this gap in our knowledge by conducting extensive surveys of tsetse in the Southern region, starting with Bhar El-Jebal State. The latter State has recently suffered serious epidemics of HAT in various locations around Juba, its capital city.

Materials and methods

Study area (Map-1)

The study area lies around Juba town (30^o20-31^o45E and 4^o40-5^o00N) about 1200 km south of Khartoum. The most dominant feature of the area is River Bhar El-Jebal, which links the White Nile through Uganda with lake Victoria in East Africa. The soil is of the loamy and loamy-clay types transected by a vast number of streams or rivers. These watercourses generally originate from the Nile/ Congo water shed.

The climate is hot throughout the year with the rains falling between March and November. The annual rainfall ranges between 600 - 800mm.

Two main types of vegetation predominate: (1) open savanna woodland (Wilson, et al., 1963) comprised of the scrubs Acacia nilotica, A. sielerana, A. seyal and A. mellifera and the deciduous trees of Anogeissus leiocarpus and Combretum glutinosum. Around several watercourses evergreen trees of Ficus religiose, Borassus aethiopum, Ziziphus spina-christi, Tamarindus indica and Khaya senegalensis flourish. The main grasses are of the Imperata, Panicum and Andropogon species : (2) riverine gallery forest and riverine thickets comprised mainly of Ficus religiose, Tamarindus indica, Azadirachta indica, Anogeissus leiocarpus altogether with Combretum spp and various climbers and grasses. This riverine vegetation is however interrupted here and there by small numerous plots for subsistence farming and several villages. In most cases these farms contain hedges which were deemed suitable for tsetse flies. The wild animals as detected by dung, spoor and sightings were comprised of Hippopotamus (Hippopotamus amphibious), Monitor Lizard (Varanus niloticus niloticus), Nile Crocodile (Crocodylus niloticus), Warthog (Phacochoerus aethiopicus), Tsessebe (Damaliscus lanatus), Bushbuck (Tragelaphus scriptus), Waterbuck (Kobus defassa), Vervet Monkey (Cercopithecus aethiops), Baboon (Papio ursinus), Wildcat (Felis lybica), Cheetah (Acinomyx jubatus) and Porcupine (Hystrix africae-australis).

Methods

Tsetse surveys

Tsetse flies were caught in unbaited biconical traps (Challier, et al., 1977) and Epsilon traps (FAO, 1992). In each vegetation type (that is riverine gallery forest, riverine thicket, open savanna woodland, farmland and villages) traps were placed 200m apart for three-five days. Catches were collected every 24 hours. Captured flies were counted, sexed and examined to separate tenerals from non-tenerals. The mean catch per trap per day in the collection periods represented the apparent density of flies in the area.

HAT surveillance

A preliminary surveillance for Human African Trypanosomosis (HAT) was carried out to cover the area surveyed for tsetse infestation. At each village visited, 5-37 people, according to the population density, were randomly sampled. Blood samples were collected from basilic vein. Serum was separated 24 hours after blood collection. Sero-diagnostic techniques, Card Agglutination Test for Trypanosomosis (CATT) and Card Indirect Antigen Test for Trypanosomosis (CIATT), were used to assess the infection rate by detecting circulating trypanosomal internal antibodies and antigens (Mangnus, et al., 1978; Nantulya, 1997).

Results

Tsetse flies

Only Glossina f. fuscipes was caught mainly in biconical traps. The latter trap caught up to 6 times more flies than the Epsilon trap which was alternated with it for two days in each location during both the dry and rainy seasons. However, more G. f. fuscipes was caught during the rainy than in the dry season (Table-1). The results obtained indicated a further north distribution of the fly from its previously known limit of a distance of 18 miles along the River Bhar El-Jebal and 30 miles along Luri Stream (Map-2).

Trap catches show that G. f. fuscipes was strictly restricted to riverine vegetation including riverine gallery forest, riverine thicket and riverine farmland (Table-2). The fly was, however, apparently absent from inside the villages and the open savanna woodland.

Human African Trypanosomosis (HAT)

Table (3) shows the prevalence rate (43.9% and 48.1% for CATT and CIATT, respectively) of HAT in the inhabitants of the various villages visited, together with the apparent density of G. f. fuscipes in the nearest watering site to each village. The prevalence rate ranged between 18.5-80% using CATT and 29.7-100% using CIATT. Clearly there was no relationship between the density of tsetse flies and the presence of antibodies/antigens against HAT in the local people. Nor was there any significant discrepancy in the prevalence rate of the disease between the various age groups of the people so far examined (Table-4).

Discussion

Since the area under tsetse infestation has more than one Glossina species, mainly G. f. fuscipes and G. morsitans submorsitans (Wilson, et al., 1963), two different trap designs, biconical and Epsilon, were used. Biconical trap is the one most frequently used for sampling tsetse flies of Palpalis group while Epsilon trap is the one recommended for survey and monitoring of Morsitans group (Challier, et al., 1977; FAO, 1992). The biconical trap was found to be 6 times more effective than Epsilon (P< 0.05) for trapping G. f. fuscipes. The result obtained confirmed the presence of G. f. fuscipes in the study area (Ford and Katondo, 1977). However, the fly was caught further north of its previously known limit (Wilson, et al., 1963); a fact that is in contrast with Katondo's (1984) suggestion. The northern spread of the fly recorded here might be a result of availability of the preferred host, Monitor Lizard (Mohamed-Ahmed and Mihok, 1999) and forest extension resulting from the latest civil war that forced people to leave the farmland and agricultural activities. Odulaja and Mohamed Ahmed found that G. f. fuscipes in Kenya fed almost exclusively on Monitor Lizard. On the other hand this same tsetse species (Glossina. f. fuscipes) was reported to be able to adjust to changing habitats in Uganda (Okoth, 1982). The area that is recently occupied by the fly is potentially productive and this creates negative pressure for land utilization. Moreover, due to the current war, people are now settled in hamlets along the infested watercourses where bush clearance is not practiced for security reasons. Such a circumstance has led to the creation of suitable habitats for tsetse flies to survive and HAT transmission to thrive.

In the present study G. f. fuscipes was trapped only in the riverine vegetation which includes, riverine gallery forest, riverine thickets and riverine farmlands. This finding agrees with those reported by Mohamed-Ahmed and Mynholds (1997). Trap performance increased when they were placed at less than 5m from the edges of riverine vegetation. This observation confirmed those reported by Hargrove and Vale (1980), Dransfield, et al., (1982) and Mohamed-Ahmed and Mynholds (1997). This information is useful for effective placement of traps during control of G. f. fuscipes. The fact that the fly is living within a narrow margin of its threshold of existence makes it amenable to control and eradication by the use of selective bush clearance and trapping techniques. Places where people collect water, do their fishing, washing, farming or foot paths are the main sites of human-fly-contact and HAT transmission. Thus control activities should be focused on water sources.

The sensitivity and efficiency of the serological tests, CATT and CIATT, had been assessed and recommended for detecting infection with Trypanosoma brucei gambiense and for active surveillance (Enyoru, et al., 1997; Akol, et al., 1997; WHO, 1998). The results obtained show high prevalence of infection with HAT in the study area although the apparent density of the tsetse flies is low. However, sleeping sickness epidemics were reported to occur even at very low density of the fly vector (Jordan, 1974; Lancien, et al., 1990). These results confirmed serologically the presence of the disease in the study area. The prevalence decreased tremendously with the corresponding decrease of the chances of fly-man-contact. Data analysis showed that there was no significant (P> 0.05) relationship between exposure to disease and the density of tsetse flies, nor with age of the so far examined people. This finding agrees with that reported by Snow (1999).

Control approaches suggested for implementation in the area, to keep the disease under-control include: chemo-sterilisation of host-reservoir by a combination of case-detection, use of both passive surveillance and active screening; and treatment of infected individuals with a suitable drug; reduction of fly population density. Use of traps impregnated with Deltamethrin in the vegetation habitats infested with G. f. fuscipes in densities of 5 trap/linear kilometer, was reported to be effective (Odulaja and Mohamed-Ahmed, 1997) and should be tried here. Bush clearance is recommended to make the habitat unsuitable for tsetse to survive.

Recommendations

1-In order to focus effort on communities with the highest prevalence of HAT and define the extent of the problem, active surveillance of areas closely associated with tsetse in previous surveys should be carried out.

2-Glossina f. fuscipes is distributed further north of its previously known zone. It occupies more potentially productive land than previously believed; and it is associated with and encountered only along riverine woodland. These are the places where people do their washing. Footpaths that pass along the dense riverine and water course vegetation are other points of contact as are farms situated at river-banks. These are the main sites where man - tsetse contact and transmission of HAT occur.

3-Case detection and treatment, and vector control using Biconical traps impregnated with insecticide sited at G. f. fuscipes habitats, and selective clearing technique should be implemented simultaneously to control the disease.

4-The spreading of awareness of HAT and the role tsetse plays in its transmission should be undertaken in all the communities living in infested areas so that they may be fully involved in the implementation of the control strategies.

Table 1: The locations where Glossina fuscipes fuscipes were encountered and its apparent density +SD.

Location	Season	Apparent density
Luri	Wet-hot	-
	Dry-hot	0.33+0.33/trap/day
Khor Lado	Wet-hot	4.33+0.88^ns/trap/day
	Dry-hot	3.00+0.58/trap/day
Rejaf West	Wet-hot	1.33+0.88^ns/trap/day
	Dry-hot	0.33+0.33/trap/day
Logo West	Wet-hot	6.5+1.76^s/trap/day
	Dry-hot	1.00+0.41/trap/day
Nyori	Wet-hot	5.00+1.15^s/trap/day
	Dry-hot	2.00+0.58/trap/day
Logo East	Wet-hot	-
	Dry-hot	4.00+1.15/trap/day

ns: not significant

s: significant (P<0.05)

Table 2: The mean catch of Glossina fuscipes fuscipes + SD in various vegetation types in the study area.

Vegetation type

Apparent density