Dynamics of the pregnancy cycle in the tsetse Glossina morsitans

J. Insect Phyriol., 1974, Vol. 20, pi. 1015 to 1026. Perganwn Press. printed in Greut Britain

DYNAMICS OF THE PREGNANCY CYCLE IN THE TSETSE GLOSSINA MORSITANS DAVID

L. DENLINGER

and WEI-CHUN

MA

International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya (Received 15 October 1973)

Abetract-A

normal pregnancy in tsetse involves the successful integration of larval development with maternal activity. At 25”C, ovulation in Glosska morst?mts occurs 1 br after the previous larviposition, the egg hatches on day 3-8 (l-57 mm length, O-09 mg dry wt.), ecdysis to second instar occurs on day 4.9 (2-3 mm, O-30 mg), the third instar cuticle is formed on day 6-8 (4.5 mm, 5-O mg), and parturition occurs on day 9-O (6.0 mm, 10-O mg). Melanization of the in utero third instar follows a regular sequence over a 2 day period. Parturition follows a circadian pattern with a peak 9 l-u after lights on (12 hr daily photophase). All instars receive nutriment from the female’s milk gland. During early pregnancy the rate of milk synthesis is greater than rate of uptake by the larva, thus causing expansion of the secretory reservoirs. After day 6, the volume of the secretory reservoirs decreases, but as is indicated by nuclear volume and larval growth the rate of synthesis remains high until day 8. Feeding activity of the adult female is maximal on day 1, levels off at 60 per cent up to day 6, and then declines sharply towards the end of pregnancy. Oircyte development proceeds in phase with larval development and thus minimizes a lag period between successive pregnancies. INTRODUCTION

THE EVOLUTIONof viviparity

in

the tsetse has resulted in a complex interaction between mother and progeny. Each pregnancy cycle (9 to 10 days) culminates in the production of a single fully grown third instar larva which burrows into the soil and forms a puparium within a few hours of larviposition (review by BUXTON,1955). The in utero development of the larva is made possible by the provision of nourishment from the milk gland, a modified female accessory gland. Similarly the mother’s ability to provide ‘milk is dependent upon her feeding activity. To maximize efficiency of the pregnancy cycle, larval development, ‘milk’ production, and adult feeding activity must be coordinated. Energy from the female is also channelled into development of the oiicyte which will become the larva in the following pregnancy cycle. Synchrony of oocyte and larval development improves the temporal efficiency of the system by minimixing the time between larviposition and ovulation of the next egg into the uterus. As a foundation for future work on abnormalities of tsetse reproduction, our investigation is designed to define the normal sequence of events in a pregnancy cycle of Glossina monitam morsitamWestw. 1015

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DAVID L. DENLINGERAND WBI-CHUNMA MATERIALS

AND

METHODS

Flies were obtained from the ICIPE laboratory colony, a colony that originated in 1968 from the Tsetse Research Laboratory at the University of Bristol and is supplemented fortnightly with pupae from Bristol. At emergence flies were placed in 18 x 8 x 4.5 cm cages made from PVC tubing covered with terylene netting. When in utero larvae developed externally visible black polypneustic lobes, the females were transferred to individual fly cages made from 4 x 6 cm polystyrene tubes covered at both ends with terylene netting (MEWS, 1970). Flies were placed on rabbit ears for 20 min each day (feeding time 10.00). With the exception of the feeding period, flies were kept in an environmental chamber (Percival Refrigeration Co.) at 25 f 0_5”C, ca. 75% r.h., and a daily 12 hr photophase (lights on at 8.00). Light intensity in the vicinity of the experimental animals was 100 lx. Due to the variability in time of first larviposition (MELLANBY, 1937), the experiments focused on the more regular second and third pregnancy cycles. Experimental flies were examined hourly for newly deposited larvae ; age of the female was based on the time since larviposition. Data from the second and third cycles are combined. Milk gland tissue examined histologically was fixed in Bouin’s fluid and after dehydration and clearance was embedded in Paraplast (melting point 56 to 57”C, Sherwood Medical Industries Inc.), sectioned at 7 q, and stained with either Ehrlich’s haemotoxylin-eosin or Mallory’s triple stain (modified after CARLETON, 1957). Surface areas were measured from camera lucida drawings with the aid of a planimeter. RESULTS

In utero development of progeny Females of varying ages after larviposition were dissected in CuZZiphora Ringer’s solution, the developmental stage was noted, and measurements of length and dry weight were recorded. Data in Fig. l(A) and l(B) show the normal sequence of development and the changes in progeny size and weight during a pregnancy cycle. Under our experimental conditions the mean f S.E. duration of a pregnancy cycle was 9.02 + 0.09 days (N = 175). Time of ovulation was determined from flies in which the time of larviposition ( + 2 min) was known. One hour after larviposition, 46.6 per cent of the females (N = 15) had ovulated; in two hours, 80-O per cent (N = 15) ; by 15 hr, ovulation had occurred in 92.3 per cent of the females (N = 26). The newly ovulated egg (l-57 f 0.01 mm length, N = 64) has a dry weight of O-086 f 0.001 mg (mean + S.E., N = 12). Eclosion of the first instar occurred in 50 per cent of the specimens (N = 14) by day 3.8. A first instar has not been observed before day 3.6. In histological sections milk has been observed in the gut of the first instar. Length of the larva increases to 2.3 mm at the time of ecdysis and dry weight increases three- to fourfold to O-30 mg. The first instar has a duration of 26 hr ; ecdysis to the second instar occurred in 50 per cent of the specimens (N = 12) on day 4.9.

THE PREGNANCY

s.ok_(Al

CYCLE IN GLOSSXNA MORSITANS

.

1017

Pupae

6.0 4.0 2.0

Time after larviposition, days

1. Dynamics of the pregnancy cycle in G. morsit~ns at 25 + O.S”C. (A) Time sequence of progeny development and length of individual larvae as a function of time. (B) Dry weight of progeny during pregnancy cycle. Each point represents a single individual. (C) Change in diameters of proximal and distal tubules of milk gland (mean and range, N = 20) and diameter of milk gland nucleus (mean ? SD., N = 20). (D) Percentage of adult females (N = 227) accepting a blood meal on different days of pregnancy cycle. (E) Growth of oiicyte during pregnancy cycle. Each point represents a single individual. FIG.

Over a 2 day period, day 4-9 to day 6.8, the second instar increases in length to 4.5 mm and dry weight increases to 5-O mg. Termination of the second instar stage is determined not by an ecdysis but by the formation of a third instar cuticle and the splitting of the second instar cuticle. Frequently the third instar remains partially ensheathed in the second instar cuticle until larviposition (BURSELL, 1955 ; BURSELL and JACKSON,1957) ; however, we confirm ROBERTS’(1972) observation that occasionally the exuvium of the second instar is also shed. Major

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DAVID L. DFJNLINGERAND WEI-CHUN MA

growth occurs during the third instar. Length increases 1.5 mm, width greatly increases, and dry weight doubles from 5.0 mg to 10.0 mg. Following parturition the length of the larva is reduced about 10 per cent as the larva contracts to form the puparium. Several useful landmarks can be used for assessing the relative age of in utero third instar larvae. Sclerotized mouth hooks, a feature unique to the third instar, are apparent from the onset of the stadium. Next, melanization of the polypneustic lobes begins as a faint grey granular spot at the point where the two lobes converge. The grey area becomes more dense and spreads over the entire lobes. Externally, black lobes are visible for at least 27 hr before larviposition; mean time is 34.7 hr (N = 43). While the proximal margin of the lobes is still white, the antennomaxillary processes turn black. Completion of melanization at the margin of the polypneustic lobes is followed by blackening of the anus. The last phase of the sequence involves a faint melanization of the entire body; a grey mottling begins anteriorly and progresses posteriorly. The colour changes of the in u&o third instar should be distinguished from the light brown to black colour change in the tanning and melanization of the third instar cuticle during pupariation. Deposition of the larva in a normal pregnancy is dependent not only upon the completion of developmental criteria, but also upon a component of circadian time. Experimental flies kept in a 12 hr daily photophase (lights on at 8.00) show a unimodal peak of larviposition in late afternoon (Fig. 2), 84.5 per cent of the larviposition (N = 317) occurred between 12.00 and 20.00; the acme of activity occurred between 16.00 and 17.00 and accounted for 18.3 per cent of the total larvipositions. Thus, if a larva is not developmentally prepared for larviposition until after the circadian time for larviposition in that day, the larva will be maintained in the uterus until the afternoon of the next day.

Time

of day,

hr

FIG. 2. Circadian pattern of larviposition recorded from females of G. morsitans reared in individual containers at 25 k O-ST and under a daily 12 hr photophase (lights on at 8.00).

FIG. 3. Daily variation in the milk gland during a pregnancy cycle of G. morsitans. Cross-sections through distal tubules on day 1 (a), day 2 (b), day 3 (c), day 4 (d, e), day 5 (f, g), day 6 (h), day 7 (i, j), day 8 (k), and day 9 or day 0 (1). Scale 20 pm.

THE PREGNANCY

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Growth of the progeny during the pregnancy cycle is exponential. Demands upon the female for nutrition begin at eclosion of the first instar on day 4 and reach a maximum late in day 6. During the 6 days of larval development enough nutriment is channelled through the female’s milk gland to produce a larva of 29.92 f 0.51 mg wet wt. (mean + S.E., N = 53). The cycle thus culminates in the production of a fully grown larva that has within it the nutritional potential for development of an adult fly. Cycle of milk gland activity Milk gland activity was quantified from histological preparations by measuring tubule and nucleus diameters as indices of glandular activity. The cell number in the gland tubules remains constant throughout the pregnancy cycle as does the volume of the milk gland lumen. Volumetric changes in tubule diameter are due to variation in the quantity of ‘milk’ stored in the secretory reservoir (MA and DENLINGER,1973) of the secretory cells. Earlier reports (HOFFMAN,1954) suggested that changes in the volume of the milk gland were due to changes in lumen diameter and alternating cycles of breakdown and proliferation of tubule cells. The distal tubules which are characterized by a narrow lumen and a lack of spines on an almost invisible cuticular intima are at a minimum external diamater of 30 to 35 F on the day of larviposition (Fig. 1C). Tubule diameter changes little within the next 3 days. On day 0 to 1 of the pregnancy cycle the cells of the milk gland are still in the act of regression from the previous pregnancy. The gland cytoplasm is vacuolated and basally involuted around the nucleus (Fig. 3). These features start to disappear on day 2. Although the secretory reservoirs are still small on day 3 a size increase is apparent, thus indicating that secretion is beginning to accumulate. At eclosion of the first instar (day 4), the reservoirs are round in shape and the diameter of the tubules has increased to 50 pm. A maximum diameter of 90 to 100 cun is reached on day 6 when the larva is a second instar, and subsequently the milk gland volume decreases rapidly towards the end of the pregnancy cycle. Masses of small round lipid vacuoles (MA et al., 1973) appear in the cytoplasm about 1 day before larviposition. At larviposition the gland has a typically involuted appearance. The proximal tubules which are characterized by having a wider lumen and a thick spinose intima show a similar pattern of volumetric change although the scale is a magnitude of 10 to 20 p greater. The nuclei of the gland celIs also undergo volumetric changes during a pregnancy cycle (Fig. 1C). Diameter of the nucleus doubles from a minimum in the early phase of pregnancy to the maximum size seen in days 5 to 8. It is interesting that the nuclear size does not decrease until after day 8. The secretory reservoirs have already undergone considerable shrinkage by this time. This suggests that during days 6 to 8 the gland is still actively secreting but during this period the feeding rate of the larva exceeds the rate of ‘milk’ production. Evidence for a continued high rate of ‘milk’ synthesis is provided by the continued weight increase of the progeny during this period.

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DAVID L. DJSNLINGER AND WEI-CHUN MA

Adult feeding activity The cycles of larval development and cyclic activity in the milk gland are integrated with a cycle of feeding activity in the adult female. The percentage of flies (N = 227) that took a blood meal during a daily 20-min exposure to rabbit ears is recorded throughout the pregnancy cycle in Fig. l(D). In our experiments, no feeding was recorded on the day of larviposition. However, with our procedure the blood meal was offered in the morning and the peak of larviposition occurred in late afternoon: thus our data for day 0 represents females still carrying fully grown larvae that are ready to be deposited. Feeding activity was maximal on day 1 and then fell to a fairly constant rate of around 60 per cent on day 2 up to day 6. As the larva becomes larger during the late second and third instars feeding activity decreases sharply. The finite space available within the abdomen of the female prohibits engorgement during the later phases of pregnancy. The low feeding activity in late pregnancy and the complete absence of feeding on the day of larviposition contribute to the major peak of feeding on day 1; the feeding on day 1 presumably makes a major contribution to the energy demands of the adult female. An appreciable amount of energy is not channelled into ‘milk’ synthesis until day 4. Since the major growth of the larva occurs after a decline in the recruitment of exogenous energy, it is apparent that storage of ‘milk and ‘milk precursors is an essential component contributing to the feasibility of tsetseviviparity. Oiicyte a%vebpent During a pregnancy cycle energy is also demanded for development of the oiicyte that will be ovulated into the uterus in the following pregnancy cycle. As is seen in Fig. l(E), at larviposition the length of the oiicyte is 0.15 mm. The point of inflection on the exponential growth curve occurs around day 5. The mean maximum size of 1.57 mm (see above section on in utero development) was obtained occasionally by day 6, and by an increasingly greater number of individuals during the following 3 days. Chorionation was observed in the first individuals on day 7. Synchronization of oocyte development with in utero development of the larva maximizes uterine occupancy and hence reproductive potential by eliminating a time lag between parturition and ovulation. The rate of oijcyte development is accelerated if the in utero progeny is not viable. For example, one fly which was dissected at age 4.8 days should have contained a first instar larva and an oocyte of about 0.90 mm. Instead the uterus contained a partially deflated, non-viable egg and the oocyte was 140 mm, a size not normally attained until day 7. Several similar examples have been observed, thus indicating the existence of a feedback mechanism that governs the rate of oijcyte development as a function of the success of the pregnancy. The potential of the entire 9 day pregnancy cycle will thus not be wasted on an unsuccessful pregnancy. DISCUSSION

Although literature on the adult tsetse is voluminous, information on earlier developmental stages is minimal (review by BUXTON, 1955). The inaccessibility

THE PREGNANCY CYCIB

IN GLOTSZNA MORSZTANS

1023

of the tsetse larva has apparently retarded our knowledge of very basic aspects of its biology; yet a better understanding of the larval stage and the demands made by it upon the systems of the adult female could lead to innovative practical consequences. Some of our findings on in utero development in Gloss&a conflict with earlier reports, but environmental and species different may account for some variability in the timing of developmental events. In G. pulpalis, MELLANBY(1937) reports that the egg descends into the oviduct in 15 to 18 hr after the previous larviposition. By contrast, HOFFMANN(1954) reports that ovulation in G. pulpalis occurs about 2 hr after larviposition. Our results with G. molsitatLFat 25 + 0.5”C demonstrate that ovulation occurs 1 to 2 hr after larviposition. Earlier reports suggest that the first instar lasts only a few hours (ROUBAUD,1909) and that this instar is incapable of feeding due to mouth blockage by the oral egg tooth (BURTT and JACKSON, 1950). ROBERTS(1971) shows that blockage of the mouth by the egg tooth does not occur. We find that the first instar lasts about 26 hr and during this time the larva actively feeds. Milk can be observed in the gut and the dry weight gain of 0.21 mg during this time represents a three- to fourfold increase in weight by end of the first instar. The third instar has also been described as a non-feeding stage (BURSELLand JACKSON,1957), but we find that the third instar represents the stage of greatest weight increase, as is true in other cyclorrhaphous Diptera. The time of termination of the pregnancy cycle is dependent upon a circadian component. In our experiments, using a daily 12 hr photophase, the peak of h&position activity occurred in late afternoon (9 hr after lights on). Using G. morsituns that had emerged from wild puparia, PHELPS and JACKSON(1971) also found the peak of larviposition under a daily 12 hr photophase to occur 9 hr after lights on. By contrast, NASH and TREWERN(1972) find a morning peak of larviposition (5 hr after lights on) in G. morsitans. The differences between our results and the results of Nash and Trewern are particularly intriguing since our colony originated and is fortnightly supplemented with flies from their colony at Bristol. Thus a genetic difference is not a likely explanation of the observed disparity. Our experimental conditions are similar to the conditions of Phelps and Jackson in that flies were maintained singly and light intensity was about 100 lx. The experiments of Nash and Trewem were based on groups of flies kept in larger cages, and light intensity was low (10-15 lx). BURTT (1952) demonstrated a difference in the larviposition pattern that depended upon the characteristics of the fly containers: flies kept singly in 3 x 1 in. tubes larviposited later than flies kept together in larger cages. It is interesting to note that larvae which are deposited early in the day have a longer free-living larval stage before puparium formation than larvae which are deposited late in the day (FINLAYSON,1967) ; thus the time duration of the last few hours before formation of the puparium is constant whether the time is spent in utero or as a free-living larva. It is difficult to determine if either larviposition rhythm represents the natural situation for G. morsitans. In the field (CARPENTER,1911) k&position in G. pa@& was observed most frequently during the hotter hours of the day and it may be

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DAZEDL. DENLINGER AND WEI-CHUNMA

adaptive for larviposition to occur during this time. Pupae are sensitive to high temperature and may sometimes be killed by temperatures found in breeding sites (PHELPS and BURROWS,1969). Likewise, a site of high atmospheric humidity is preferred for pupariation (FINLAYSON,1967) and appears to be advantageous for pupal survival (DEAzEvEDo and PINHAO, 1969). Thus if larviposition were to occur in the afternoon when soil temperature is highest and humidity is lowest larvae would be more likely to select a suitable pupariation site that would not be exposed to lethal temperatures and unfavourable humidity. Although the timing of the natural circadian peak of larviposition remains unresolved, it is apparent that a rhythm of parturition does exist. This implies that the act of parturition is dependent upon more than the mere attainment of a critical size by the larva. There is an ability to hold the larva for several hours if necessary. Whether the circadian trigger for parturition comes from the female or the larva is unknown. The timing of parturition is extremely important for the reproductive success of the species. Premature larviposition can result in the abortion of a larva that cannot survive (MELLANBY,1937; SAuNnERs, 1972) whereas if a larva is maintained in the uterus beyond a critical time, pupariation can occur in &YO (ROUBAUD, 1909; SIMPSON,1918; ROBINSON,1964; NASH et al., 1967; BOYLE, 1971). Information on the status of the in utero progeny must be relayed to the adult female. If death occurs in an embryo or larva the female must be capable of emptying the uterus. The sense of timing is also extremely important in the provision of ‘milk’ for the larva. ‘Milk’ must be available when the larva hatches and must increase in quantity as the larva grows. Although earlier reports suggest that ‘milk’ secretion is restricted to the early phase of pregnancy (BURSELLand JACKSON,1957 ; HOFFMANN,1954) our results imply that secretion continues until nearly the end of the pregnancy cycle. ‘Milk’ begins to accumulate in the secretory reservoirs (MA and DENLINGER,1973) shortly before the time of eclosion, and up to day 6 the rate of ‘milk’ synthesis exceeds the rate of secretion. After day 6 the volume of the secretory reservoirs (and thus the milk gland tubules) decreases; however, during this phase the larva undergoes its major weight increase. Thus ‘milk’ is still being released but the rate of ‘milk’ discharge is now greater than the rate of ‘milk’ synthesis, and hence the tubules are no longer as large. The large nuclear volume of the secretory cells in late pregnancy may be correlated with the continued demand for a high rate of ‘milk’ synthesis. Ultrastructural observations support the concept of a continued high rate of milk synthesis for 2 days after the peak in secretory reservoir volume has been reached (MA et al., 1973). Control of the milk gland poses intriguing questions which are at the very crux of the tsetse mother-larva relationship. The accessory glands of some other female insects are known to be under regulation by the corpora allata (DE WILDE, 1964). Synchronization of the pregnancy cycle by the tsetse’s endocrine system seems likely. Development of the o&yte which will become the progeny in the next pregnancy proceeds in synchrony with the cycle of milk synthesis and larval development. This is suggestive of a common endocrine mechanism regulating the two

THEPREGNANCYCYCLEINGLQSSINA

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systems

concurrently. By contrast, some viviparous cockroaches (EN-, 1970) and the fleshfIy Su~c@hugu u~gyrostomu (Possmms et d., 1967) display a restraint on egg maturation during the gestation period. Egg maturation and ‘milk’ production are in turn dependent upon the nutrients made available from the blood meal of the female. pupal weight can be directly correlated with the total amount of blood imbibed during a pregnancy cycle (BOYLE, 1971), but the meals are not evenly spaced throughout the cycle. As pregnancy advances increasingly less space is available for gut expansion; feeding activity declines sharply after day 6. Engorgement late in the pregnancy can cause parturition difficulties that may lead to in utero pupariation (NASHet aZ., 1967). The marked growth of the larva during the late phase of pregnancy is thus not a direct result of continued blood feeding by the female, but is dependent upon the female’s ability to store the products of digestion. Depletion of unreplenished energy stores in late pregnancy contributes to the major peak of feeding activity immediately following parturition. The proper timing and integration of numerous other events contribute to the maintenance of a normal pregnancy. Fertilization of the egg can proceed only after the opening of the spermathecal valve (ROBERTS,1973). ROBERTS(1972) has also reported sequential changes in the shape of the uterine wall during the course of pregnancy; however, doubt has been cast (TOBE and DA~EY, 1971; ROBERTS, 1972) on the earlier idea that the choriothete undergoes cycles of activity correlated with eclosion and larval ecdysis (HOFF-, 1954; BUR~ELLand JACKSON,1957). Discovery of new dynamic aspects of the pregnancy cycle will continue to reveal the precise integration of the events that have evolved in the unique mother-larva relationship in GZostinu. Interference with one of the interdependent links could have obvious consequences for the reproductive success of the female. AcknowZe&ments-We thankProfessor JANDE WILDE,Professor THONU~R. ODHLWBO, and Dr. JULIAN SHEPHBRDfor their critical comments on the manuscript. The research was supported in part by a National Institutes of Health Fellowship to D. L. D. from the Institute of Allergy and Infectious Diseases.

REFERENCES Azsvmo J. F. DE and PINIUO R. DA C. (1964) The maintenance of a laboratory colony of Glostina morsitanssince 1959. Bull. Wbi Hlth Org. 31,835-841. BOYLE J. A. (1971) Effect of blood intake of GZosJiM Austin Newst. on pupal weights in successive reproductive cycles. Bull. mt. Res. 61, l-5. BUR~BLLE. (1955) The polypneustic lobes of the tsetse larva (Glossina, Diptera). PYOC. R. Sot. (B) 144,275-286. BUR~BLL E. and JACKSONC. H. N. (1957) Notes on the choriothete and mik gland of Gloss&a and Hippobosca(Diptera). Proc. R. ent. Sot. Lord (A) 32, 30-34. BURRT E. (1952) The occurrence in nature of tsetse pupae (Gloska mymertoni Austen). Acta trop. 9, 304-344. BURTT E. and JACKKIN C. H. N. (1950) Illustrations of tsetse larvae. Bull. ent. Res. 41, 523-527.

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BUXTONP. A. (1955) The Natural History of Tsetse Flies. London School of Hygiene and Tropical Medicine, Memoir No. 10. H. K. Lewis, London. CARLETONH. M. (1957) HistologicuZ Technique. Oxford University Press. CARPENTERG. D. H. (1911) Progress report of investigations into the bionomics of GZoss+za palpalis. Rep. sleep. Sickn. Commn. R. Sot. 12, 79-l 11. ENGELMANNF. (1970) The PhysioZogy of Insect Reproduction. Pergamon Press, Oxford. FINLAYSONL. N. (1967) Behaviour and regulation of puparium formation in the larva of the tsetse fly Glossina nwrsitans orientalis Vanderplank in relation to humidity, light and mechanical stimuli. Bull. ent. Res. 57, 301-313. HOFFMANNR. (1954) Zur FortpfIanzungsbiologie und zur intrauterinen Entwicklung von Glossina paipalis. Acta trap. 11, l-57. MA W. C. and DENLINGERD. L. (1974) Secretory discharge and microflora of the milk gland in tsetse flies. Nature, Lond. 247, 301-303. MA W. C., DENLINGER D. L., SMITH D. S. and J#RLFoRs, U. (1974) Cytological modulations of the milk gland during a pregnancy cycle in G. morsitans. In press. MELLANBY H. (1937) Experimental work on reproduction in the tsetse fly, G. pulpalis. Parasitology 29, 131-141. MEWS A. R. (1970) Some new techniques for the maintenance of Gloss&a in the laboratory. In Tsetse Fly Breeding under Laboratory Conditions and its Practical Application (Ed. by DE AZEVBDO J. F.), pp. 145-148. Junta de Investiga@es do Ultramar, Lisbon. NASH T. A. M., JORDANA. M., and BOYLE J. A. (1967) A method of maintaining Glossinu austeni Newst. singly, and a study of the feeding habits of the female in relation to larviposition and pupal weight. Bull. ent. Res. 57, 327-336. NASH T. A. M. and m M. A. (1972) Hourly distribution of larviposition by Gloss&a austeni Newst. and G. morsitafis mwitans Westw. (Dipt., Glossinidae). Bull. ent. Res. 61, 673-700. PHELPS R. J. and BURROWSP. M. (1969) Lethal temperatures for puparia of Glossinn morsituns orientalis. Entomologia exp. appl. 12, 23-32. PHELPS R. J. and JACKSONP. J. (1971) Factors influencing the moment of larviposition and eclosion in Glostima morsitans orientalis Vanderplank (Diptera: Muscidae). J. ent. Sot. sth Afr. 24, 145-157. Posso~p~s B., CHARBONNIERE J., and RALIOSAB. 0. (1967) fivolution des cellules neuros&r&rices de la pars intercerebralis, croissance des ovocytes et ovovivipariid chez Sarcophaga argyrostoma (Dipt. Cyclorrhaphe). Ann. Sot. ent. Fr. 3, 359-599. ROBERTSM. J. (1971) The functional anatomy of the head in the larvae of the tsetse fly, Gloss&a austeni Newstead (Diptera, Glossinidae). Entomologist 104, 190-203. ROBERTSM. J. (1972) The role of the choriothete in tsetse flies. Parasitology 64, 23-36. ROBERTSM. J. (1973) The control of fertilization in tsetse flies. Ann. trap. Med. Purasit. 67, 117-123. ROBINSONG. G. (1964) Abnormality in tsetse fly. Trans. R. Sot. trop. Med. Hyg. 58, 579. ROUBAUDE. (1909) La Gloss&a palpalis R. Desv.: sa biologie, son rble dans l’&idogie des trypanosomiases. Thesis No. 1344. University of Paris. SAUNDERS D. S. (1972) The effects of starvation on the length of the interlarval period in the tsetse fly Glostina morsitans orientalis Vanderplank. J. Ent. (A) 46,197-202. SIMPSONJ. J. (1918) Bionomics of tsetse and other parasitological notes in the Gold Coast. Bull. ent. Res. 8, 193-214. TOBB S. S. and DA~EY K. G. (1971) The choriothete of GIossina austeni Newst. Bull. ent. Res. 61, 363-368. DE WILDE J. (1964) Reproduction-Endocrine control. In The PhysioZogy of Insecta (Ed. by ROCK~TEINM.) 1, 59-90. Academic Press, New York.