Effect of host packed cell volume on the bloodmeal size of male tsetse flies, Glossina pallidipes

aematin contents of engorged, male tsetse flies, Glossina pallidipes Austen, were compared with the packed cell volumes of oxen on which they had fed. Haematin contents icnreased with packed cell volume up to packed cell volumes of approximately 30%. Haematin contents appeared to level off or decline with further increase in packed cell volume. These results support a model of blood-feeding in tsetse flies in which the rate of blood consumption decreases as packed cell volume increases, because of increase in blood viscosity, and tsetse are unable to compensate for the decrease in consumption rate by feeding for a longer time. After allowing for the effects of packed cell volume, bloodmeal sizes of tsetse increased with ox body temperature.     

Factors affecting the landing and feeding responses of the tsetse fly Glossina pallidipes to a stationary ox

In Zimbabwe, studies were made of the landing and feeding responses of Glossina pallidipes on an ox. Of the tsetse approaching an ox, ≈ 70% fed. Increasing densities of tsetse increased the grooming responses of the ox but had no significant effect on the percentage of tsetse that engorged. The landing site of tsetse on the ox varied with density, with ≈ 50% landing on the legs at low densities (< 20 flies per ox), compared to ≈ 80% at densities > 40 flies per ox. For male G. pallidipes , the mean bloodmeal size was 37 mg. The probability of feeding was negatively correlated with fat content, declining from 91% for flies with < 1 mg fat to < 50% for flies with > 4 mg fat. Bloodmeal size was also negatively correlated with fat content; the regression equation relating bloodmeal size and fat content indicated that the mean wet weight declined from 42 mg for flies with 1 mg of fat to 31 mg for flies with 5 mg of fat. For females, the probability of feeding was not significantly affected by age as determined by ovarian category but there was a paucity of young (ovarian category 0) flies attracted to the ox. Pregnancy status had no significant effect on the probability of feeding, but samples of flies attracted to the ox showed a relative dearth of females approaching larviposition and a preponderance just after.     

Nutritional levels of female tsetse Glossina pallidipes from artificial refuges

Female tsetse flies, Glossina pallidipes Austen, caught in artificial refuges were subjected to ovarian dissection and analysed for levels of fat, residual dry weight (RDW) and haematin. There were rather small proportions of flies in ovarian categories 0 and 1, in part due to large losses in the immature and teneral stages at the hottest time of year. The distribution of the female catch among pregnancy days was close to uniform. The wet and dry weights (WW and DW) and RDW of eggs, larvae and pupae increased by 0.821, 0.303 and 0.204 mg respectively, with each mm3 increase in volume. Water accounted for 71.7% of the fat-free WW, and fat for 32.7% of the DW. Between birth and ovulation, fat increased from 2 to 4 mg and RDW from 7 to 11 mg; thoracic RDW increased by 2.5mg and changed little thereafter. Fat levels increased 3.5mg by day 6 of pregnancy, but only 0.5 mg thereafter. Over the same periods RDW corrected to zero haematin (CRDW) increased by 1 and 8 mg respectively. Full-term fat and CRDW levels were 8.2 and 19.4 mg respectively. Cumulative haematin frequencies formed a smooth curve with a slope that increased continuously. The raw data were well fitted by a model where feeding rates increased exponentially and capture probability was independent of haematin content. The mean feeding interval was 60 h; feeding probabilities of >0.9/day were only found in flies that had failed to feed for>72 h. In early pregnancy, fat levels declined with haematin for flies that had fed>36 h previously; by days 5-7 fat levels were maintained at a constant high level for 60 h post-feeding. Fat-haematin graphs for female tsetse cannot be used to estimate rates of fat utilization. Traps sample tsetse with below-average fat and RDW in early and late pregnancy respectively. Refuge samples are less biased than those from traps; they give a better picture of the dynamics of pregnancy in normal flies and facilitate the explanation of existing anomalies.     

The response of tsetse flies to artificial baits in relation to age, nutritional and reproductive state

In various vegetation types in Zimbabwe, the catches of Glossina pallidipes Austen and G. morsitans morsitans Westw. (Diptera: Glossinidae) at a target baited with odour (acetone, 1-octen-3-ol and two phenols) were positively correlated with catches of the same species at an unbaited net. No correlation existed between target catches and hand net catches of tsetse flies sitting on the vegetation. G. pallidipes females caught at a target and at an unbaited net were older than those caught from vegetation. Of the female G. pallidipes caught at the target, 46% were in the first 3 days of pregnancy. Of those caught at the unbaited net, significantly fewer, 21%, were in this stage. G. pallidipes males caught from vegetation contained more fat (3.07±0.333 mg) than those caught at the unbaited net (2.06±0.339 mg) or at the target (2.19±0.218 mg). It is inferred that target catches consisted predominantly of tsetse which were already in flight when they sensed the stimuli from the target, and that target catches were biased towards female G. pallidipes in the first 3 days of pregnancy.     

The role of cattle as hosts of Glossina longipennisat Galana Ranch, south-eastern Kenya

Abstract. Glossina longipennis were recorded visiting and engorging on cattle in an enclosure and on a single ox in a crush using transparent electrocuting nets in an incomplete ring. Of the total flies caught, 3–6% of males and 5–6% of females in the total catches were engorged (a feeding success rate of up to 16.6% and 12.6%, respectively, depending on assumptions made about the proportion which had an opportunity to feed). Direct observation of tsetse from an observation pit showed 57% landing on the front legs, 13% on the hind legs, and 11 % on the belly of the host. The largest number of bloodmeals was taken from the front legs, although only 14% of landings there terminated in feeding; a higher proportion of the flies alighting on the hind legs and flank succeeded in feeding (28% and 21% respectively). Glossina longipennis were attracted to targets baited with ox odour from an underground pit in a dose-dependent manner. Odour of humans was much less attractive to G.longipennis than that of oxen (for equivalent biomass). Analysis of bloodmeal samples from tsetse caught in two sites on die ranch showed that G.longipennis preferentially feeds on suids, bovids and hippopotamus.     

Effect of host odours on trap catch composition of Glossina pallidipes in Kenya

The effect of odour attractants on the composition of samples of Glossina pallidipes Austen was investigated by comparing the age and nutritional status of flies caught in unbaited biconical traps with those caught in traps baited with cow urine and acetone. For both male and female flies, baited traps caught more flies with significantly higher fat content than did unbaited traps. Thus the samples from baited traps were more representative of the population as a whole: males showed a fuller range of the fat/haematin conditions known to occur in the field and proportionately more females were in later stages of the pregnancy cycle, than from unbaited traps.     

The use of discriminant analysis for the estimation of sampling biases for female tsetse

ABSTRACT.

• 1 In this paper we investigate whether the technique of discriminant analysis can be used to estimate sampling biases for female tsetse.
• 2 Discriminant analysis was first applied to laboratory samples of female tsetse, Glossina morshans morsitans Westwood, to test whether flies of known history could be assigned to the correct day of the pregnancy cycle on the basis of their fat, haematin and corrected residual dry weight.
• 3 Following the satisfactory results from the laboratory samples, the same technique was applied to field samples of G.m. centralis Machado captured by electric traps and hand nets in Zambia and of G.palpalis palpalis (Robineau-Desvoidy) captured in biconical traps at five sites in Ivory Coast. The results show that flies on day 1 of the pregnancy cycle were most likely to be caught, with a second peak of day-6 and day-7 flies, while very few day-8 or day-9 flies were caught.
• 4 These major peaks in fly trappability coincide with the known feeding habits of female tsetse, and indicate synchrony of feeding by many members of the population immediately after larviposition and again as the larva in utero moults from the second to third instar. G.palpalis is relatively more available at this later stage of its pregnancy cycle to the capture methods used than is G.morsitans. A third feed may be taken at a more variable point in the pregnancy cycle.
• 5 This method of estimating the sampling biases of female tsetse could allow an estimate of total population size, as long as the absolute sampling efficiency of flies on any one day of the pregnancy cycle could be established by, for example, mark-release-recapture experiments.

     

Physiological correlates of the availability of Glossina morsitans centralis Machado to different sampling methods

Abstract.

• 1 The feeding cycle for male tsetse flies and the pregnancy cycle for females are used as frameworks for investigating the field behaviour of Glossina morsitans centralis Machado in Zambia, and hence the sampling biases of different capture devices.
• 2 Flies were collected in the field using hand nets and an electric back pack on foot patrols, and hand nets, hand-catching and an electric screen on landrover patrols. They were analysed for wing-fray, vein-length, chloroform-extractable fat and haematin. In addition, samples of laboratory-bred females killed daily during their second pregnancy cycle were analysed for the last two parameters.
• 3 The daily flying time of males is estimated from the fat—haematin curve to be about 32 min/day, which is identical to previous estimates for G. morsitans from Tanzania.
• 4 From the varying numbers and fat content of male flies caught by the different sampling methods during the course of the feeding cycle it is concluded that both haematin levels and fat reserves influence fly behaviour.
• 5 Comparison of the frequency distribution of wild-caught and laboratory-bred females along the corrected residual dry weight axis of the pregnancy cycle suggests that the results of combined electric-trap and hand-net sampling reflect the actual frequency distribution in the wild population at least for the first 7 days of the pregnancy cycle; but females in the last 2 days of their pregnancy cycle are apparently unavailable to sampling devices, perhaps because of their reduced flight activity.
• 6 The very variable percentage female catch (10.9–43.4%) returned by the different sampling methods is discussed in the light of the detailed analysis of the differential availability of male and female flies to each capture device.

     

Bloodmeal evacuation from the midgut of Glossina pallidipes in relation to tsetse foraging activity and trappabiIity

Abstract.

• 1 Foraging for bloodmeals is the most frequently recurrent and probably the most targetable of all activities that render tsetse vulnerable to interception with static trapping devices.
• 2 Surgical monitoring of the rnidgut every 24 h during three successive days of food deprivation, showed that a full bloodmeal, irrespective of its size or source, vacated the rnidgut of both sexes of Glossina pallidipes Austen in eight progressive stages.
• 3 Probing responsiveness in both sexes increased exponentially during the first four stages of their midgut evacuation, reaching a peak between stages 3 and 5. Thereafter it decreased, also exponentially.
• 4 Most G.pallidipes caught by NG2G traps baited with cow urine and acetone had midguts in the last three stages (6–8) of bloodmeal evacuation. The same was true of the majority of those that failed to feed on a calf shortly after entrapment.
• 5 The implications of the foregoing for tsetse foraging activity and trappability as well as for the potency of cattle urine and acetone as odour-bait for tsetse are discussed.

     

Post eclosion age predicts the prevalence of midgut trypanosome infections in Glossina

The teneral phenomenon, as observed in Glossina sp., refers to the increased susceptibility of the fly to trypanosome infection when the first bloodmeal taken is trypanosome-infected. In recent years, the term teneral has gradually become synonymous with unfed, and thus fails to consider the age of the newly emerged fly at the time the first bloodmeal is taken. Furthermore, conflicting evidence exists of the effect of the age of the teneral fly post eclosion when it is given the infected first bloodmeal in determining the infection prevalence. This study demonstrates that it is not the feeding history of the fly but rather the age (hours after eclosion of the fly from the puparium) of the fly when it takes the first (infective) bloodmeal that determines the level of fly susceptibility to trypanosome infection. We examine this phenomenon in male and female flies from two distinct tsetse clades (Glossina morsitans morsitans and Glossina palpalis palpalis) infected with two salivarian trypanosome species, Trypanosoma (Trypanozoon) brucei brucei and Trypanosoma (Nannomonas) congolense using Fisher's exact test to examine differences in infection rates. Teneral tsetse aged less than 24 hours post-eclosion (h.p.e.) are twice as susceptible to trypanosome infection as flies aged 48 h.p.e. This trend is conserved across sex, vector clade and parasite species. The life cycle stage of the parasite fed to the fly (mammalian versus insect form trypanosomes) does not alter this age-related bias in infection. Reducing the numbers of parasites fed to 48 h.p.e., but not to 24 h.p.e. flies, increases teneral refractoriness. The importance of this phenomenon in disease biology in the field as well as the necessity of employing flies of consistent age in laboratory-based infection studies is discussed.     

A model for blood meal digestion and fat metabolism in male tsetse flies (Glossinidae)

Abstract. Fat and haematin levels of mature male Glossina morsitans morsitans Westwood were estimated at different times after feeding at temperatures between 15 and 30°C. Flies were kept (largely inactive) in 7.5 × 2.5 cm tubes, or in actograph cages, where flight activity increased with time after feeding. Haematin excretion was modelled as a series of three first order reactions, all with the same rate parameter. The model accounted for > 98% of the variance in mean haematin in each of seven experiments; the rate parameter increased linearly with temperature and activity level. A similar approach was adopted for modelling fat metabolism. The rate coefficients of lipogenesis increased with temperature, and that for lipolysis with temperature, activity level and their interaction. All experiments were analysed simultaneously to provide equations predicting haematin or fat levels for all times, for active or inactive flies, and for temperatures between 15 and 30°C. Haematin exhibited large variations between individuals, but for active flies the expected haematin content at a given time varied little between flies kept at 25 and at 30°C. In inactive flies kept at 25°C, lipogenesis peaked at ≈ 24 h and lipolysis at ≈ 48 h. For active flies the times were 12 and 24 h, respectively; both rates were about twice as high as in inactive flies. Active flies produced (up to 1 mg) more fat out of a given size of blood meal than inactive flies. Curves of fat content against logarithm of haematin content differed little with temperature, and can therefore be useful for comparative studies of field populations of tsetse.     

Suppressive action of Samorin on the cyclical development of pathogenic trypanosomes in Glossina morsitans centralis

Male Glossina sexually sterilized by gamma-irradiation are as efficient vectors of trypanosomiasis as fertile males. An attempt was made, using isometamidium chloride (Samorin), to interfere with the cyclical development of trypanosomes in sterile males, destined for use in the sterile insect release (SIR) method of tsetse eradication. The infection rate with mature Trypanosoma congolense Broden was effectively reduced in sterile male Glossina morsitans centralis Machado, when the flies were fed on an infected goat 2 days after they were fed as tenerals on an in vitro bloodmeal containing 8 micrograms Samorin/ml blood. The infection rates with mature T.vivax Ziemann and T.brucei brucei Plimmer & Bradford were completely suppressed at this drug dose. Whensterile teneral males were fed on a bloodmeal containing 12 micrograms/ml Samorin and given the infected bloodmeal 10 days later, infections by mature T.vivax, T.congolense and T.b.brucei were completely suppressed. Hence in the management of a tsetse eradication programme utilizing the SIR method, it is recommended that the sterile teneral male tsetse should, prior to release, be given a bloodmeal containing Samorin at 12-15 micrograms/ml blood. This will effectively suppress future disease transmission.     

Age‐specific changes in sperm levels among female tsetse (Glossina spp.) with a model for the time course of insemination

The age, insemination and ovulation status of tsetse flies Glossina pallidipes Austen (n = 154369) and Glossina morsitans morsitans Westwood (n = 19659), captured over 11 years in Zimbabwe, are assessed by ovarian dissection. Instantaneous rates of insemination increase exponentially with age in both species; 90% insemination levels are reached after 5 days post‐emergence in G. m. morsitans and 7 days in G. pallidipes, varying little with season. More than 95% of both species have ovulated by the age of 8 days and 99% by 12 days. Older flies that have not ovulated are > 100‐fold more likely to be caught in October and November than in other months. A 500‐fold decrease in trap catches did not result in any detectible decrease in the probability of females being inseminated. The proportion of partially filled spermathecae rises for approximately 6 days then declines, consistent with some flies having mated more than once. For flies caught on electric nets, with wings undamaged during capture, wing‐fray data are used to extend ovarian age estimates up to 11 ovulations. Among these flies, the volume of sperm in the spermathecae declines little in flies that have ovulated up to seven times; thereafter, it declines by approximately 1% per ovulation. The time course of insemination and the mating frequency of females are important considerations in modelling tsetse fly populations, as well as for the dynamics of interventions involving the release of genetically‐modified insects, which should not be seriously compromised by the limited levels of polyandry currently observed.     

Effects of two blood-feeding regimes on mortality and female reproduction in a laboratory colony of stable flies,Stomoxys calcitrans

Abstract. Stable flies (Stomoxys calcitrans L.) deprived of a bloodmeal until 3 days post-emergence had higher mortality rates than control flies fed from the day of emergence. Fat bodies of deprived females required one more bloodmeal to reach maximum size, and maximum size was smaller, than fat bodies of control females. Ovarian development did not commence prior to feeding in deprived flies, and proceeded more slowly thereafter, resulting in a one bloodmeal delay in egg maturation in deprived flies. Deprived females produced fewer (54.7, SD 2.8) eggs than controls (75.9, SD 3.7) and eggs from deprived females were smaller (mean length 684.0 μrn) than control females' eggs (mean length 1165.7 μm).     

Factors Affecting the Propensity of Tsetse Flies to Enter Houses and Attack Humans Inside: Increased Risk of Sleeping Sickness in Warmer Climates

Background

Sleeping sickness, or human African trypanosomiasis, is caused by two species of Trypanosoma brucei that are transmitted to humans by tsetse flies (Glossina spp.) when these insects take a bloodmeal. It is commonly assumed that humans must enter the normal woodland habitat of the flies to become infected, but recent studies found that tsetse frequently attack humans inside buildings. Factors affecting human/tsetse contact in buildings need identification.

Methodology/Principal Findings

In Zimbabwe, tsetse were allowed access to a house via an open door. Those in the house at sunset, and those alighting on humans in the house during the day, were caught using hand-nets. Total catches were unaffected by: (i) the presence of humans in the house and at the door, (ii) wood smoke from a fire inside the house or just outside, (iii) open windows, and (iv) chemicals simulating the odor of cattle or of humans. Catches increased about 10-fold with rising ambient temperatures, and during the hottest months the proportion of the total catch that was taken from the humans increased from 5% to 13%. Of the tsetse caught from humans, 62% consisted of female G. morsitans morstans and both sexes of G. pallidipes, i.e., the group of tsetse that normally alight little on humans. Some of the tsetse caught were old enough to be effective vectors.

Conclusion/Significance

Present results confirm previous suggestions that buildings provide a distinctive and important venue for transmission of sleeping sickness, especially since the normal repellence of humans and smoke seems poorly effective in such places. The importance of the venue would be increased in warmer climates.     

Feeding frequency in relation to reproduction in Glossina morsitans morsitans and G.pallidipes

Abstract Measurements of residual haematin in males of Glossina morsitans morsitans Westwood reared in the laboratory at 25^oC suggest that blood meal digestion is completed in 4 or 5 days after feeding. However, a high proportion of haematin is present as faecal matter 2 days after feeding and it is concluded that digestion is completed sooner than indicated by the regression of logio haematin on time. Therefore, low levels of residual haematin in field-caught tsetse provide no indication of the frequency with which they feed. For this reason the effects of feeding frequency upon various reproductive parameters in the laboratory have been examined. It is concluded that the best performance is achieved by G.m. morsitans females which ingest four blood meals per inter-larval period and that for a similar performance in G.pallidipes five blood meals are required. The extent to which such feeding frequencies are a reflection of feeding activity in the field are discussed in terms of the biochemical requirements to maintain a reproductive adult female tsetse in positive energy balance.     

A Neglected Aspect of the Epidemiology of Sleeping Sickness: The Propensity of the Tsetse Fly Vector to Enter Houses

Background

When taking a bloodmeal from humans, tsetse flies can transmit the trypanosomes responsible for sleeping sickness, or human African trypanosomiasis. While it is commonly assumed that humans must enter the normal woodland habitat of the tsetse in order to have much chance of contacting the flies, recent studies suggested that important contact can occur due to tsetse entering buildings. Hence, we need to know more about tsetse in buildings, and to understand why, when and how they enter such places.

Methodology/Principal Findings

Buildings studied were single storied and comprised a large house with a thatched roof and smaller houses with roofs of metal or asbestos. Each building was unoccupied except for the few minutes of its inspection every two hours, so focusing on the responses of tsetse to the house itself, rather than to humans inside. The composition, and physiological condition of catches of tsetse flies, Glossina morsitans morsitans and G. pallidipes, in the houses and the diurnal and seasonal pattern of catches, were intermediate between these aspects of the catches from artificial refuges and a host-like trap. Several times more tsetse were caught in the large house, as against the smaller structures. Doors and windows seemed about equally effective as entry points. Many of the tsetse in houses were old enough to be potential vectors of sleeping sickness, and some of the flies alighted on the humans that inspected the houses.

Conclusion/Significance

Houses are attractive in themselves. Some of the tsetse attracted seem to be in a host-seeking phase of behavior and others appear to be looking for shelter from high temperatures outside. The risk of contracting sleeping sickness in houses varies according to house design.     

Trap efficiency for Glossina pallidipes (Diptera: Glossinidae) at Nguruman, south-west Kenya

An incomplete ring of electric nets was evaluated as a means of estimating trap efficiency for Glossina spp. This methodology assumes flies approach the trap directly, and then enter, or leave directly in random directions. These results showed that the ratio of the number of flies intercepted on the outside of the electric nets to the number on the inside was lower than predicted by this single-approach behavioural model. Moreover, an incomplete ring of nets around a trap reduced trap catch more than the model predicted. These inconsistencies were greater early in the day, and greater for females than for males. It is suggested that flies may make several approaches to a trap before entering or departing. This mixes arriving and departing flies on each side of the electric nets. Use of a complete ring of nets around a trap to estimate trap efficiency entails fewer behavioural assumptions. Catches at a complete ring around a trap were compared to catches in a trap without nets, replicated in a cross-over design. The efficiency of an odour baited NG2G trap was estimated to be 58% for males and 37% for females. Biconical traps were much less efficient. Both trap types were less efficient in the early morning, suggesting entry response is temperature dependent. The NG2G trap was more efficient for non-teneral nulliparous females than for other ages. For both trap types there was little difference between mean fat content of approaching and trapped males, but the mean fat content of trapped females was lower than that of approaching females.     

Tracking the feeding patterns of tsetse flies (Glossina genus) by analysis of bloodmeals using mitochondrial cytochromes genes

Tsetse flies are notoriously difficult to observe in nature, particularly when populations densities are low. It is therefore difficult to observe them on their hosts in nature; hence their vertebrate species can very often only be determined indirectly by analysis of their gut contents. This knowledge is a critical component of the information on which control tactics can be developed. The objective of this study was to determine the sources of tsetse bloodmeals, hence investigate their feeding preferences. We used mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) gene sequences for identification of tsetse fly blood meals, in order to provide a foundation for rational decisions to guide control of trypanosomiasis, and their vectors. Glossina swynnertoni were sampled from Serengeti (Tanzania) and G. pallidipes from Kenya (Nguruman and Busia), and Uganda. Sequences were used to query public databases, and the percentage identities obtained used to identify hosts. An initial assay showed that the feeds were from single sources. Hosts identified from blood fed flies collected in Serengeti ecosystem, included buffaloes (25/40), giraffes (8/40), warthogs (3/40), elephants (3/40) and one spotted hyena. In Nguruman, where G. pallidipes flies were analyzed, the feeds were from elephants (6/13) and warthogs (5/13), while buffaloes and baboons accounted for one bloodmeal each. Only cattle blood was detected in flies caught in Busia and Uganda. Out of four flies tested in Mbita Point, Suba District in western Kenya, one had fed on cattle, the other three on the Nile monitor lizard. These results demonstrate that cattle will form an integral part of a control strategy for trypanosomiasis in Busia and Uganda, while different approaches are required for Serengeti and Nguruman ecosystems, where wildlife abound and are the major component of the tsetse fly food source.     

Olfactory and behavioural responses of tsetse flies, <Emphasis Type="Italic">Glossina</Emphasis> spp., to rumen metabolites

Herbivores provide tsetse flies with a blood meal, and both wild and domesticated ruminants dominate as hosts. As volatile metabolites from the rumen are regularly eructed with rumen gas, these products could serve tsetse flies during host searching. To test this, we first established that the odour of rumen fluid is attractive to hungry Glossina pallidipes in a wind tunnel. We then made antennogram recordings from three tsetse species (G. pallidipes morsitans group, G. fuscipes palpalis group and G. brevipalpis fusca group) coupled to gas chromatographic analysis of rumen fluid odour and of its acidic, mildly acidic and neutral fractions. This shows tsetse flies can detect terpenes, ketones, carboxylic acids, aliphatic aldehydes, sulphides, phenols and indoles from this biological substrate. A mixture of carboxylic acids at a ratio similar to that present in rumen fluid induced behavioural responses from G. pallidipes in the wind tunnel that were moderately better than the solvent control. The similarities in the sensory responses of the tsetse fly species to metabolites from ruminants demonstrated in this study testify to a contribution of habitat exploitation by these vertebrates in the Africa-wide distribution of tsetse.