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The nature of the establishment barrier for Trypanosoma brucei in the gut of Glossina pallidipes
364 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL ~EDICINE AND HYGIENE. Vol. 67. No. 3. 1973.
THE NATURE OF THE ESTABLISHMENT BARRIER FOR TRYPANOSOMA BRUCE/IN THE GUT OF GLOSSINA PALLIDIPES RUDOLF HARM_SEN*
Department of Zoology, University College Nairobi, Kenya Introduction The natural frequency of Trypanosoma brucei salivary gland infections in tsetse flies is extremely low. For instance, an extensive survey of 3 species of flies in Nigeria (LLoyD et al., 1924) carried out in different locations over several seasons, produced only 7 infected flies out of a total of over 16,000; this is an infection frequency of less than 0.04%. Such low frequencies are not the result of a rarity of infected vertebrates found in the feeding area of the fly, but seem to be caused by the failure of the trypanosomes in a blood meal to establish cyclical infections in the fly. The very low frequency found by LLOYDet al. (1924) may be partly the result of their sampling techniques (HARLEY, 1967), but even taking this into account it seems a valid generalization that natural infection rates are very low (HARLEY, 1966a; b). The frequency of flies to become cyclically infected after the taking of a blood meal from an infected vertebrate in the laboratory, under normal maintenance conditions, rarely exceeds 4% while much lower frequencies are reported: 0.25% (GORDON and MILLER,1961), 0.3-0.5% (TAYLOR,1932; BURTT, 1946; van H o o f et al., 1937). In the most general terms, one can say that the passage from vertebrate to fly is less of a hurdle to the trypanosome than the subsequent problems involved in survival in the fly. The questions to be asked are basically: what are the problems, and how does a small percentage of trypanosomes overcome them? An appreciable percentage of flies will show temporary or semi-permanent crop and/or gut infections at some stage after taking an infective blood meal, but never develop cyclical (i.e. salivary gland) infections (DUKE and MELLANBY,1936; DIPEOLUand ADAMS, 1971; HARLEY, 1971a). In these cases, obviously, the problem lies in some barrier to later stages of cyclical development. In many cases, however, the flies develop no lasting internal infections of any kind, and the problem seems to be part of the establishment and initial survival of the trypanosome in the fly's intestinal system. Such aspects as the species of vertebrate host, the strain of trypanosome, the number and morphological type of trypanosomes and the species and genetic constitution of the fly have been shown to have at times some, but usually only a minor *Present address: Biology Department, Queen's University, Kingston, Ontario, Canada. I wish to record my gratitude to the Director, Kenya Veterinary Research Laboratory, for supplying the G. pallidipes used in this work; to Dr. R. J. Onyango, Director East African Trypanosomiasis Research Organization for the strain of T. brucei used; to Dr. K. C. Willett, of W.H.O. for showing me his technique of removing the petritrophic membrane; to the Rockefeller Foundation for making a grant for research; to the Science Faculty, University College, Nairobi, through which this research was supported. In addition I must express my debt to my colleagues Prof. D. S. Kettle, Dr. M. J. Coe and Dr. T. R. Odhiambo of the Zoology Department with whom this work has been discussed.
RUDOLF HARMSEN
365
influence on the chance of establishing a cyclical infection in the fly. The only factor of major importance has been shown to be the age of the fly at the time of the infective feed. HARLEY(1971a) obtained infection rates as high as 24°/0 among male G. pallidipes that had taken their infective feed at an average age of 15 hours after emergence. The positive correlation between seasonal temperature and infection rate, first described by KllqGHORI%YORKEand LLOYD(1913) and corroborated by several other authors in more recent times (DUKE, 1933; BURTT, 1946), appears to have its foundation in the higher rate of fat-reserve consumption in the pupa reared at higher temperature (BURSELL,1960). This consumption of reserve food affects the fly by inducing it to feed earlier in adult life. Similarly, laboratory kept pupae, incubated at above average temperatures usually produce flies which will attain higher than average infection frequencies; again, the result of taking the first feed earlier in adult life. These aspects, therefore, are also brought back to the age of the fly at the time of the infective feed. This latter aspect is so important, that one can categorically state that except for extremely rare occasions only first feeds will ever result in salivary gland infections in tsetse flies (DUKE, 1935); furthermore, first feeds taken within 24 hours after emergence have a considerably higher chance of producing such infections in the fly than do later first feeds (WIJERS, 1958; HARLEY, 1971b). It appears, therefore, to be very much a matter of a barrier mechanism against trypanosome infections found in the mature adult tsetse flies, which is not fully developed until approximately 24 hours after emergence of the fly. Only flies which for some reason (usually depleted fat reserves as a result of above optimal pupal temperature) will feed before this barrier mechanism is installed will develop cyclical infections if infective trypanosomes were present in the meal. WIGGLESWORTH(1929) described the peritrophic membrane of the tsetse fly, and in so doing drew attention to this structure and its possible r61e in relation to the establishment and movements of trypanosomes in the fly gut. Some dipterous peritrophic membranes are well known to be completely impermeable to gut trypanosomatids (CHATTON, LINGERand L~GER, 1912). It became a point of regular recurrence in the literature to find speculations as to the actual movements of various species of trypanosomes from the endoperitrophic space to the ectoperitrophic space and vice versa (HoARE, 1931 ; TAYLOR, 1932; YORKE, MURGATROYDand HAWKING,1933; SOAP,E, 1935; LEWIS and LANGRIDGE, 1947; FAIRBAIRN,1958). LEWIS and LANGRIDGE(1947) suggested, and FAIRBAIRN(1958) confirmed that T. brucei actually penetrates the peritrophic membrane in the region of the proventriculus where possibly it is not fully polymerized and still semi-liquid. Despite considerable searching, however, Fairbairn failed to find evidence for an endo- to ecto-peritrophic penetration of the membrane at the time of establishment, only for an ecto- to endo-peritrophic penetration some days later. The establishment of ectoperitrophic infections remained seen as the result of "circumnavigation" of the membrane in the hind gut. Recently DIPEOLU and ADAM (1972) have reported a surprisingly high frequency of ecto-peritrophic infections shortly after feeding, in the posterior section of the midgut, suggesting a penetration or circumnavigation in this region. WILLETT(1966) has introduced the first working hypothesis which attempts to bring all the established facts together. In short, he suggests that in very young, immature flies the rate of growth of the membrane is such that endo-to ecto-peritrophic penetration takes place in the unusually long section of semi-liquid membrane. In mature flies this section is so much shorter, because of the slower growth of the membrane, that penetration becomes exceedingly rare. Although Willett's hypothesis is recognized as a major, constructive step towards understanding the nature of the barrier mechanism, it seems unlikely that such a minor
366
ESTABLISHMENT BARRIER ]FOR T. B R U C E I IN GUT OF G. P A L L I D I P E S
mechanical hurdle by itself could not be compensated for by the trypanosome. Whatever the barrier mechanism is, it exerts a major mortality pressure on the trypanosome population at a time of considerable selective importance. It is, therefore, suggested that the barrier mechanism involves a much more basic adaptation of the trypanosome to life inside the tsetse alimentary system than a mere ability to penetrate a certain section of the fly's peritrophic membrane. On the other hand, the relationship between infection rate and membrane growth is most likely more than coincidental. The purpose of the present study is firstly to investigate the growth, structure and position of the peritrophic membrane of the tsetse fly during the first 48 hours of adult life, particularly in relation to feeding activity. The intention of this part of the work is to relate the localization of the trypanosomes to their survival. The distribution a n d movement of the blood meal in the alimentary system of the fly and its relation to the growth and size of the peritrophic membrane was therefore given special consideration. Secondly, any establishment barrier must act upon the trypanosome, and it was, therefore, considered essential to study some aspects of the trypanosome during the initial period inside the tsetse gut. With these questions in mind, do the trypanosomes react to their new environment, and is this reaction related to their localization in the invertebrate host? It was decided to study certain properties of a representative enzyme system as an indicator of the trypanosomes reactivity. Materials and methods
All work was done with Glossina pallidipes Aust. Most specimens were field collected by the staff of the Kenya Department of Veterinary Services in the Kiboko region. Some were obtained from the East African Trypanosomiasis Research Organization in Tororo, Uganda. All tsetses arrived into our laboratory as puparia of unknown age, and were incubated on arrival in darkness at 25°C. or 30°C. and 60% r.h. Adults were kept in individual 1 × 3 inch tubes in the same incubator. Flies were fed on healthy or infected hooded laboratory rats as required. The trypanosomes all originated from a single isolate of Trypanosoma brucei Plimmer and Bradford taken from a cow in Eastern Uganda; it is not known whether the specimens are of brucei brucei or of brucei rhodesiense. A single passage through a rat produced a sufficiently high parasitaemia for the preparation of over 100 samples of one stabilate which were stored in deep frozen state (CmqNINGHAM,LUMSDE~ and WEBBER,1963; LUMSDENand HARDY, 1965). The distribution of the blood meal within the alimentary system of the tsetse fly was studied on thick (100 ~t) serial sections of the entire fly. Flies, at various intervals after complete feeds were instantly frozen in a dry-ice alcohol mixture, and subsequently fixed in ice-cold alcoholic Bouln's. This fixation technique prevented any unnatural movement of blood at the time of death or shortly after. Specimens were embedded in high melting point paraffin wax and sectioned at 100 ~z. The sections were stuck in sequence directly on to transparent cellotape just before cutting. Unstained sections of this kind display the layout of the alimentary system and the distribution of the bloodmeal when viewed under the microscope at relatively low power. This technique was not adequate for a study of the peritrophic membrane. A dissecting technique of living or freshly killed flies has been developed by WILLVTTin 1965, (pers. commun.; 1966) which makes it possible to remove the entire, undamaged membrane from the gut of the fly. It was possible to extricate membranes containing entire blood meals after introducing only minor modifications into Willett's technique. The cytochemical analysis of general and specific phosphatases of trypanosomes at different stages has been described in a separate communication centered on the development of the relevant techniques (H~a~MSEN, 1973a). Peritrophic membrane and distribution of bloodmeal
Experimental Batches of puparia of G. pallidipes were kept in the laboratory and checked at 15 minute intervals throughout the day for newly emerged flies. Individual flies were killed and the
RUDOLF HARMSEN
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peritrophic membrane dissected out of the gut at periods of growth varying from 0-80 hours. The length of the membrane was measured in each case and recorded as a function of the growth period after emergence. The resultant growth curve (Fig. 1) shows a linear relationship for the first 30 hours of approximately 1 mm./hour c0rtoborating WILLETT'S (1966) results, but this stage is followed by a gradually declining rate'until the membrane reaches a maximum length of 5 0 ~ 0 mm. at 80 hours.
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T h e growth rate is slightly higher at 25°C. than at 30°C. This probably indicates that 30°C. is somewhat above the physiologically optimal temperature for G. pallidipes. If during the period between 30-80 hours the fly is fed, the original I mm./hour growth rate of the peritrophic membrane is resumed. There is no indication of a sudden "spirmaretlike" rapid growth during and immediately after feeding as suggested by WIGGLESWORTH (1929), nor is there any indication that the growth rate shortly after feeding is higher in young than in older flies~ as suggested by WILLETT (1966). Instantaneous freezing of flies interrupted during the feeding period has shown that an initial small amount of blood goes into the dorsal straight part of the midgut (Fig. 2) where it is trapped inside the peritrophic membrane. The remainder of the feed is deposited in the crop which becomes greatly distended. T h e amount of the initial blood volume which enters the midgut depends on the length of the peritrophic membrane; the blood never penetrates beyond the "tied-off" end of the membrane (Fig. 2). Examination of thick serial sections has shown that the rate of transfer of blood from the crop to the midgut during the post-feeding period also depends on the age of the fly; i.e. length of the peritrophic membrane (Fig. 3). I n fully mature (over 30 hours in age) flies the peritrophic membrane is long enough to extend into the 2nd midgut coil (Fig. 2) and is capable of containing nearly the entire blood meal. I n these flies the blood would be completely transferred from the crop to the midgut within 20 minutes. I n younger flies the membrane only extends into the straight dorsal part or into the first coil of the midgut (Fig. 2) and can only accommodate a smaller part of the blood meal. In these flies, only after the blood in the midgut becomes dehydrated can more be transferred from the crop. Also the further growth of the peritrophic membrane will allow a further transfer of blood. The crop in very young flies, may take 3-6 hours to empty.
368
ESTABLISHMENT BARRIER FOR T B R U C E I I N GUT OF G. P . 4 L L I D I P E S
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FIG. 2. Distribution of bloodmeal in the gut of Glossina pallidipes. A. Immediately after feeding in a 24 hour old fly. B. After completion of crop-emptying in a 40 hour old fly (see Fig. 3). C. Pedtrophic membrane of 32 hour old, unfed fly. D. Pedtrophic membrane of 52 hour old fly, 30 minutes after feeding, pv--proventriculus, cd--crop duct, tmgm thoracic midgut, amgmabdominal midgut, ds---dorsal straight part of amg, fc--first coil of amg, sc--second coil of amg. Discussion At this stage of the assessment of the situation, 2 possible methods of establishment of extra-peritrophic infections of rrypanosomes in the tsetse fly can bc excluded: firstly, in young flies the membrane does not grow faster during the period of crop-gut blood transfer, thus WILLETT'Stheory (1966) of easier penetration seems unlikely; secondly, the blood never penetrates beyond the peritrophic membrane during or shortly after feeding; it is always completely retained within the membrane. Only in flies of over 60 hours old
RUDOLF
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does the bloodmeal and its trypanosome population penetrate to the distal end of the midgut, but even then, it remains within the peritrophic membrane.
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FIG. 3. Crop emptying rate for Glossina pallidipes, related to age of the fly at time of feeding. We have, however, a situation which shows considerable differences between young flies and older flies. The blood meal (including any possible trypanosomes) is, partly at least, retained in the crop for a much longer period in young flies, and its movement from crop to gut is much slower. The crop emptying rate as plotted against age in Fig. 3 shows that the critical 24 hour age (with a relatively high infection chance under 24 hours and a considerably lower infection chance in flies over 24 hours) corresponds with a 45-75 minute period of blood retention in the crop. 2 possible mechanisms could link the crop emptying rate with a chance of infectivity. The first one, more or less an extension of Willett's theory, would assume an increased chance of penetration of the semi-liquid proximal part of the peritrophic membrane by trypanosomes under conditions of slow flow. A rate of flow which would empty the crop in less than 45 minutes would be too fast to allow trypanosomes a chance of adhesion and penetration. Slower rates would increase this chance markedly. This theory cannot be rejected at the moment, but both its simplicity and supporting evidence for the second hypothesis render it unlikely. The second hypothesis is based on the actual time spent by the trypanosome inside the crop rather than the midgut of the tsetse fly. The crop internal environment is very different from the midgut. There is probably no dehydration, no digestive enzyme activity and no clotting or coagulation of the blood proteins. It appeared a distinct possibility that trypanosomes need a certain period of time in an environment other than the vertebrate blood stream and yet less "hostile" than the tsetse midgut in order to adapt physiologically tolife in the fly. The fly crop is just such an environment, and in young flies the period spent by the trypanosomes in the crop is much longer than in older flies. Since infection rates go down rapidly in flies of over 24 hours of age, and crop emptying at that age takes 45-75 minutes, this hypothetical
370
ESTABLISHMENT BARRIER FOR 2". B R U C E I I N GUT OF 12. P A L L I D I P E S
adjustment period of the trypanosome 'is postulated to take somewhere around 60 minutes. Trypanosoma brucei occurs in 2 distinct shapes in the bloodstream of mammals: the growth (slender) form and the transmission (stumpy) form. Only the latter is capable of survival in the fly or in culture media (REICHENOW,1921 ; WIJERSand WILLETT,1960). The transformation from growth to transmission forms is caused by an unknown density dependent factor (HARMSEN,in preparation, b) and involves a major change in enzyme inventory (VICKERMAN,1965). It is, therefore, to be considered a pre-adaptation to life in the fly. The stumpy form as found in the vertebrate host, however, is not identical to the fly-gut form, neither morphologically nor biochemicaliy (FLYNN and BOWMAN, 1970). ! If the adjustment period hypothesis is correct it must be postulated that a second physiological (and probably enzymological) transformation takes place in those stumpy trypanosomes which are inside the crop of the fly; that this transformation takes 45-60 minutes to complete, and that this transformation is necessary before the trypanosome can survive in the midgut of the fly, and there develop into the typical fly gut-form.
Temperature sensitivity of trypanosome metabolic enzymes Experimental If the trypanosomes pass through a physiological transformation during the first hour after entry into the crop of the fly, it should be possible to note consistent differences in activity level, location and/or chemical species of a number of arbitrarily chosen enzyme types. A number of cytochemical techniques for general and specific phosphatases (PEARSE, 1961) have been adapted to produce consistent and distinct results with Trypanosoma brucei(HARMSEN,in preparation, a). It was found that both acid and alkaline phosphatase activity levels and localization showed minor changes on host-transfer. These enzyme systems are, in most organisms, summations of a fairly large number of eazym~es and consequently not very suitable for the study of minor differences. The 2 specific phosphatases studied: glucose-6-phosphatase and 5-nucleotidase are much more promising in this respect, especially glucose-6-phosphatase, since it showed a clear increase in intensity and a shift in localization within 1 hour of host transfer (HARMSEI%in preparation, a) and appeared to be a suitable indicator enzyme of a possible metabolic transformation. It appeared desirable to show not only a quantitative change in enzyme activity level and a change in localization, but also a switch in isozyme species. The trypanosome inside the vertebrate is exposed to a fairly constant 37°C. temperature. In the tsetse fly on the other hand a 20-25°C. temperature prevails. For any poikilothermal organism, the total enzyme complex has to be able to withstand changes in the temperature. Parasitic organisms which are transferred from mammals to invertebrates are exposed to sudden, drastic changes in temperature at the time of host transfer. One could predict that any arbitrarily chosen enzyme will show either a wide range of near optimal temperatures or a definite number of alternative isozyme species, each with its own, more narrowly defined optimal temperatures. The above situation has been investigated for glucose-6-phosphatase. 6 categories of trypanosomes were compared: those directly from the rat's peripheral circulatory system, and 5 groups taken from the fly crop, 10 minutes, 30 minutes, 60 minutes,. 2 hours and 3 hours after feeding of the fly. Smears prepared from these trypanosomecontaining blood samples were exposed after formalin fixation to a temperature of 40°C.
RUDOLF HARMSEN
371
for a variety of times, up to 90 minutes. After this exposure, the slides were all incubated in the normal way for glucose-6-phosphatase (HaaMSEN, in preparation, a) and the intensity of enzyme activity was noted. The results as tabulated in Table I, show that the glucose-6-phosphatase of the trypanosomes appears to become more temperature labile after about 1 hour in the fly crop. TABLE I. The effect of exposure to 40°C. for varying periods of time on trypanosome glucose-6-phosphatase activity in trypanosomes during various stages of adjustment to the invertebrate host. Origin of trypanosome fly crop 10 minutes
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90 ÷ ÷ ÷ strong activity ÷ ÷ weak but definite activity ÷ weak activity, uncertain - no activity It can be concluded that not only does the total level of activity and the intracellular localization change shortly after host transfer, but also the properties of the enzyme change. Whether this involves the synthesis of an alternative isozyme species or not cannot be stated on the basis of present data. Conclusions
When a population of T. brucei reaches a certain density (different for different strains) the first transformation occurs. The rapidly reproducing, slender trypanosomes metamorphose into non-reproducing stumpy, transmission forms. The major enzymic change which takes place during this transformation is well documented and fully appreciated (VICKERMalq, 1965). It is suggested here, that this transformation produces a trypanosome capable of survival in the fly crop, but not in the fly gut. The second transformation takes place immediately after the host transfer and may well be triggered by a simple environmental stimulus such as cooling. It is apparent that the entire transformation from the growth form to the fly-gut form can take place neither in the vertebrate blood stream nor in the insect. The so-called stumpy form, must therefore be considered as a semiTdormant intermediate which can survive in the vertebrate at 37°C. and in the fly crop at room temperature, but cannot reproduce in either environment.
372
ESTABLISHMENT BARRIERFOR 7". B R U C E I IN GUT OF G. P A L L I D I P E S
The second transformation (in the crop) takes approximately 45-60 minutes. It is, therefore, essential that trypanosomes stay in the crop for at least that time, if they are subsequently to survive in the gut. Thus, only in flies, with an incompletely formed peritrophic membrane (which results in a reduced crop to gut blood-transfer rate) can one expect any significant survival of trypanosome infections. Flies with incompletely formed peritrophic membranes are immature flies. The often observed 24 hour threshold can now be explained as follows. Flies younger than 24 hours have a peritrophic membrane of less than 24 mm. in length; such a membrane can only contain an entire blood meal after a period of growth and dehydration of the initial input which lasts for at least 60 minutes, the time required for completion of the second transformation. There is no direct evidence that the chance of complete cyclical survival in the fly is enhanced by a 60 minute or longer period in the fly crop; the closest to direct evidence can be found in the reported increase in cyclical infection rate for flies fed on trypanosome culture forms (GORDON and MILLER, 1961). There are several weaknesses in this argument. Firstly, the evidence supports the theory, but does not exclude other possible explanations. There is still a possibility of a direct mechanical effect, and possibly other, more complex factors may contribute. Secondly, a number of questions cannot be answered as yet. Foremost among these is the question of how an ectoperitrophic infection is established. Willett's theory included an answer to this; the present theory does not. Outward penetration of the proximal portion of the membrane remains a possibility but no evidence exists. Circumnavigation of the distal end of the membrane in the hindgut is not a very plausible concept as the hindgut environment appears to be highly toxic to brucei trypanosomes (WILLErT, pers. commun., 1965), and the bloodmeal has never been observed to extend beyond the distal end o f the peritrophic membrane. Penetration of the peritrophic membrane in the distal end of the midgut seems a possibility in the light of recent observations by DIPEOLU and ADAM (1972) but again, no direct evidence is available. One more problem must be mentioned in some detail. In a sizeable number of flies which have been fed on an infective vertebrate but which have not developed cyclical salivary gland infections, temporary or chronic crop and/or gut infections have been reported (DuI~ and M~I~LAN~Y, 1936). These infections indicate that a secondary barrier, one that operates after successful establishment, but before metamorphosis to metacyclic forms also exists.
Summary The low survival rate of Trypanosoma brucei in tsetse flies is interpreted as in part at least the result of an establishment barrier. This barrier appears to be less active in young flies than in older flies. The growth rate of the peritrophic membrane is 1 mm./hour during the first 30 hours after emergence, and also after feeding. The distribution of the blood meal in relation to the growth of the peritrophic membrane makes it appear unlikely that the membrane itself is the barrier mechanism. A postulated adjustment period for trypanosomes in the crop of young flies (with an incomplete peritrophic membrane) is supported by cytochemical evidence concerning an enzymic transformation within 1 hour of feeding. This phenomenon implies a double transformation for successful host transfer: one (well established) in the vertebrate host sometime before transfer, a second (new) in the fly, immediately after transfer. The destruction of non-transformed trypanosomes in the mid-gut (after leaving the crop) of mature tsetse flies is considered to be the main establishment barrier.
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REFERENCES BtmS~LL, E. (1960). Bull. ent. Res., 51, 583. B~TT, E. (1946). Ann. trop. Med. Parasit., 40, 18. CHATTON, 1~.., L~GER, A. & LINGER,M. (1912). C. r. Soc. Biol., 72, 453. CLr~mNGbU~W,M. P., LUMSDEN, W. H. R. & WFBBF~, W. A. F. (1963). Exp. Parasit.j 14, 280. DIPEOLU, O. O. & ADAM, K. M. G. (1972). Trans. R. Soc. trop. Med. Hyg., 6 6 , 337. DUKE, H. L. (1933). Ann. trop. Med. Parasit., 27, 437. - (1935). Ibid., 29, 131. & M~LL~'BY, K. (1936). Ibid., 30, 29. FAmB~m% H. (1958). Ibid., 52, 18. F L ~ , I. W. & BOWMAN, I. B. R. (1970). Trans. R. Soc. trop. Med. Hyg., 64, 175. GORDON, R. M. & MILLER, J. K. (1961). Nature, Lond., 191, 1317. HARLEY, J. M. B. (1966a). Bull. ent. Res., 56, 595. - (1966b). Ibid., 57, 23. - (1967). Entomologia exp. appl., 10, 240. (1971a). Ann. trop. Med. Parasit., 65, 185. (1971b). Ibid., 65, 191. H~mMSEN,R. Cytochemical localization of general and specific phosphatases in Trypanosoma brucd. In preparation. (a) •. Evolutionary aspects of respiratory and antigenic variation in parasitic trypanosomes. In preparation. (b) Ho~mE, C. A. (1931). Trans. R. Soc. trop. Med. Hyg., 2,5, 57. - (1935). Red. Tray. 25 Anniv. sci. Pavlovsky, 1909-34, Moscow, 367-371. HOOF, M. J. J. L. VAN,H ~ g A ~ , C. & PEEL, E. (1937). Ann. Soc. bdge M3d. trop., 17, 249. KINGHORN,A., YoRr~, W. & LLOYD~L. (1913). Ann. trop. Med. Parasit., 7, 183. LEWIS, E. A. & L~GRIDGE, W. P. (1947). Ibid., 41, 6. LLOYD, L., JOHNSON,W. B., YOmqG, W. A. & MORRISON, H. (1924). Bull. ent. Res., 15, 1. LUMSDEN, W. H. R. & H~a~y, G. J. C. (1965). Nature, Lond., 205, 1032. PEPmSE,A. G. E. (1961). Histochemistry Theoretical and Applied. London: J. and A. ChurchiU. REICa-mNOW,E. (1921). Z. Hyg. InfektKrankh., 94, 266. TAYLOR, A. W. (1932). Parasitology, 24, 401. V I C e - - N , K. (1965). Nature, Lond., 208, 762. WIC~LESWORTH,V. B. (1929). Parasitology, 21, 288. WIJERS, D. J. B. (1958). Ann. trop. Med. Parasit., 52, 385. & WIT LETT, K. C. (1960). Ibid., 54, 341. WILLETT, K. C. (1966). Exp. Paradt., 18, 290. YORKE, W., MURGATRO~D, F. & HAWr~N~, F. (1933). Ann. trop. Med. Parasit., 27, 347.
THE NATURE OF THE ESTABLISHMENT BARRIER FOR TRYPANOSOMA BRUCE/IN THE GUT OF GLOSSINA PALLIDIPES RUDOLF HARM_SEN*
Department of Zoology, University College Nairobi, Kenya Introduction The natural frequency of Trypanosoma brucei salivary gland infections in tsetse flies is extremely low. For instance, an extensive survey of 3 species of flies in Nigeria (LLoyD et al., 1924) carried out in different locations over several seasons, produced only 7 infected flies out of a total of over 16,000; this is an infection frequency of less than 0.04%. Such low frequencies are not the result of a rarity of infected vertebrates found in the feeding area of the fly, but seem to be caused by the failure of the trypanosomes in a blood meal to establish cyclical infections in the fly. The very low frequency found by LLOYDet al. (1924) may be partly the result of their sampling techniques (HARLEY, 1967), but even taking this into account it seems a valid generalization that natural infection rates are very low (HARLEY, 1966a; b). The frequency of flies to become cyclically infected after the taking of a blood meal from an infected vertebrate in the laboratory, under normal maintenance conditions, rarely exceeds 4% while much lower frequencies are reported: 0.25% (GORDON and MILLER,1961), 0.3-0.5% (TAYLOR,1932; BURTT, 1946; van H o o f et al., 1937). In the most general terms, one can say that the passage from vertebrate to fly is less of a hurdle to the trypanosome than the subsequent problems involved in survival in the fly. The questions to be asked are basically: what are the problems, and how does a small percentage of trypanosomes overcome them? An appreciable percentage of flies will show temporary or semi-permanent crop and/or gut infections at some stage after taking an infective blood meal, but never develop cyclical (i.e. salivary gland) infections (DUKE and MELLANBY,1936; DIPEOLUand ADAMS, 1971; HARLEY, 1971a). In these cases, obviously, the problem lies in some barrier to later stages of cyclical development. In many cases, however, the flies develop no lasting internal infections of any kind, and the problem seems to be part of the establishment and initial survival of the trypanosome in the fly's intestinal system. Such aspects as the species of vertebrate host, the strain of trypanosome, the number and morphological type of trypanosomes and the species and genetic constitution of the fly have been shown to have at times some, but usually only a minor *Present address: Biology Department, Queen's University, Kingston, Ontario, Canada. I wish to record my gratitude to the Director, Kenya Veterinary Research Laboratory, for supplying the G. pallidipes used in this work; to Dr. R. J. Onyango, Director East African Trypanosomiasis Research Organization for the strain of T. brucei used; to Dr. K. C. Willett, of W.H.O. for showing me his technique of removing the petritrophic membrane; to the Rockefeller Foundation for making a grant for research; to the Science Faculty, University College, Nairobi, through which this research was supported. In addition I must express my debt to my colleagues Prof. D. S. Kettle, Dr. M. J. Coe and Dr. T. R. Odhiambo of the Zoology Department with whom this work has been discussed.
RUDOLF HARMSEN
365
influence on the chance of establishing a cyclical infection in the fly. The only factor of major importance has been shown to be the age of the fly at the time of the infective feed. HARLEY(1971a) obtained infection rates as high as 24°/0 among male G. pallidipes that had taken their infective feed at an average age of 15 hours after emergence. The positive correlation between seasonal temperature and infection rate, first described by KllqGHORI%YORKEand LLOYD(1913) and corroborated by several other authors in more recent times (DUKE, 1933; BURTT, 1946), appears to have its foundation in the higher rate of fat-reserve consumption in the pupa reared at higher temperature (BURSELL,1960). This consumption of reserve food affects the fly by inducing it to feed earlier in adult life. Similarly, laboratory kept pupae, incubated at above average temperatures usually produce flies which will attain higher than average infection frequencies; again, the result of taking the first feed earlier in adult life. These aspects, therefore, are also brought back to the age of the fly at the time of the infective feed. This latter aspect is so important, that one can categorically state that except for extremely rare occasions only first feeds will ever result in salivary gland infections in tsetse flies (DUKE, 1935); furthermore, first feeds taken within 24 hours after emergence have a considerably higher chance of producing such infections in the fly than do later first feeds (WIJERS, 1958; HARLEY, 1971b). It appears, therefore, to be very much a matter of a barrier mechanism against trypanosome infections found in the mature adult tsetse flies, which is not fully developed until approximately 24 hours after emergence of the fly. Only flies which for some reason (usually depleted fat reserves as a result of above optimal pupal temperature) will feed before this barrier mechanism is installed will develop cyclical infections if infective trypanosomes were present in the meal. WIGGLESWORTH(1929) described the peritrophic membrane of the tsetse fly, and in so doing drew attention to this structure and its possible r61e in relation to the establishment and movements of trypanosomes in the fly gut. Some dipterous peritrophic membranes are well known to be completely impermeable to gut trypanosomatids (CHATTON, LINGERand L~GER, 1912). It became a point of regular recurrence in the literature to find speculations as to the actual movements of various species of trypanosomes from the endoperitrophic space to the ectoperitrophic space and vice versa (HoARE, 1931 ; TAYLOR, 1932; YORKE, MURGATROYDand HAWKING,1933; SOAP,E, 1935; LEWIS and LANGRIDGE, 1947; FAIRBAIRN,1958). LEWIS and LANGRIDGE(1947) suggested, and FAIRBAIRN(1958) confirmed that T. brucei actually penetrates the peritrophic membrane in the region of the proventriculus where possibly it is not fully polymerized and still semi-liquid. Despite considerable searching, however, Fairbairn failed to find evidence for an endo- to ecto-peritrophic penetration of the membrane at the time of establishment, only for an ecto- to endo-peritrophic penetration some days later. The establishment of ectoperitrophic infections remained seen as the result of "circumnavigation" of the membrane in the hind gut. Recently DIPEOLU and ADAM (1972) have reported a surprisingly high frequency of ecto-peritrophic infections shortly after feeding, in the posterior section of the midgut, suggesting a penetration or circumnavigation in this region. WILLETT(1966) has introduced the first working hypothesis which attempts to bring all the established facts together. In short, he suggests that in very young, immature flies the rate of growth of the membrane is such that endo-to ecto-peritrophic penetration takes place in the unusually long section of semi-liquid membrane. In mature flies this section is so much shorter, because of the slower growth of the membrane, that penetration becomes exceedingly rare. Although Willett's hypothesis is recognized as a major, constructive step towards understanding the nature of the barrier mechanism, it seems unlikely that such a minor
366
ESTABLISHMENT BARRIER ]FOR T. B R U C E I IN GUT OF G. P A L L I D I P E S
mechanical hurdle by itself could not be compensated for by the trypanosome. Whatever the barrier mechanism is, it exerts a major mortality pressure on the trypanosome population at a time of considerable selective importance. It is, therefore, suggested that the barrier mechanism involves a much more basic adaptation of the trypanosome to life inside the tsetse alimentary system than a mere ability to penetrate a certain section of the fly's peritrophic membrane. On the other hand, the relationship between infection rate and membrane growth is most likely more than coincidental. The purpose of the present study is firstly to investigate the growth, structure and position of the peritrophic membrane of the tsetse fly during the first 48 hours of adult life, particularly in relation to feeding activity. The intention of this part of the work is to relate the localization of the trypanosomes to their survival. The distribution a n d movement of the blood meal in the alimentary system of the fly and its relation to the growth and size of the peritrophic membrane was therefore given special consideration. Secondly, any establishment barrier must act upon the trypanosome, and it was, therefore, considered essential to study some aspects of the trypanosome during the initial period inside the tsetse gut. With these questions in mind, do the trypanosomes react to their new environment, and is this reaction related to their localization in the invertebrate host? It was decided to study certain properties of a representative enzyme system as an indicator of the trypanosomes reactivity. Materials and methods
All work was done with Glossina pallidipes Aust. Most specimens were field collected by the staff of the Kenya Department of Veterinary Services in the Kiboko region. Some were obtained from the East African Trypanosomiasis Research Organization in Tororo, Uganda. All tsetses arrived into our laboratory as puparia of unknown age, and were incubated on arrival in darkness at 25°C. or 30°C. and 60% r.h. Adults were kept in individual 1 × 3 inch tubes in the same incubator. Flies were fed on healthy or infected hooded laboratory rats as required. The trypanosomes all originated from a single isolate of Trypanosoma brucei Plimmer and Bradford taken from a cow in Eastern Uganda; it is not known whether the specimens are of brucei brucei or of brucei rhodesiense. A single passage through a rat produced a sufficiently high parasitaemia for the preparation of over 100 samples of one stabilate which were stored in deep frozen state (CmqNINGHAM,LUMSDE~ and WEBBER,1963; LUMSDENand HARDY, 1965). The distribution of the blood meal within the alimentary system of the tsetse fly was studied on thick (100 ~t) serial sections of the entire fly. Flies, at various intervals after complete feeds were instantly frozen in a dry-ice alcohol mixture, and subsequently fixed in ice-cold alcoholic Bouln's. This fixation technique prevented any unnatural movement of blood at the time of death or shortly after. Specimens were embedded in high melting point paraffin wax and sectioned at 100 ~z. The sections were stuck in sequence directly on to transparent cellotape just before cutting. Unstained sections of this kind display the layout of the alimentary system and the distribution of the bloodmeal when viewed under the microscope at relatively low power. This technique was not adequate for a study of the peritrophic membrane. A dissecting technique of living or freshly killed flies has been developed by WILLVTTin 1965, (pers. commun.; 1966) which makes it possible to remove the entire, undamaged membrane from the gut of the fly. It was possible to extricate membranes containing entire blood meals after introducing only minor modifications into Willett's technique. The cytochemical analysis of general and specific phosphatases of trypanosomes at different stages has been described in a separate communication centered on the development of the relevant techniques (H~a~MSEN, 1973a). Peritrophic membrane and distribution of bloodmeal
Experimental Batches of puparia of G. pallidipes were kept in the laboratory and checked at 15 minute intervals throughout the day for newly emerged flies. Individual flies were killed and the
RUDOLF HARMSEN
367
peritrophic membrane dissected out of the gut at periods of growth varying from 0-80 hours. The length of the membrane was measured in each case and recorded as a function of the growth period after emergence. The resultant growth curve (Fig. 1) shows a linear relationship for the first 30 hours of approximately 1 mm./hour c0rtoborating WILLETT'S (1966) results, but this stage is followed by a gradually declining rate'until the membrane reaches a maximum length of 5 0 ~ 0 mm. at 80 hours.
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T h e growth rate is slightly higher at 25°C. than at 30°C. This probably indicates that 30°C. is somewhat above the physiologically optimal temperature for G. pallidipes. If during the period between 30-80 hours the fly is fed, the original I mm./hour growth rate of the peritrophic membrane is resumed. There is no indication of a sudden "spirmaretlike" rapid growth during and immediately after feeding as suggested by WIGGLESWORTH (1929), nor is there any indication that the growth rate shortly after feeding is higher in young than in older flies~ as suggested by WILLETT (1966). Instantaneous freezing of flies interrupted during the feeding period has shown that an initial small amount of blood goes into the dorsal straight part of the midgut (Fig. 2) where it is trapped inside the peritrophic membrane. The remainder of the feed is deposited in the crop which becomes greatly distended. T h e amount of the initial blood volume which enters the midgut depends on the length of the peritrophic membrane; the blood never penetrates beyond the "tied-off" end of the membrane (Fig. 2). Examination of thick serial sections has shown that the rate of transfer of blood from the crop to the midgut during the post-feeding period also depends on the age of the fly; i.e. length of the peritrophic membrane (Fig. 3). I n fully mature (over 30 hours in age) flies the peritrophic membrane is long enough to extend into the 2nd midgut coil (Fig. 2) and is capable of containing nearly the entire blood meal. I n these flies the blood would be completely transferred from the crop to the midgut within 20 minutes. I n younger flies the membrane only extends into the straight dorsal part or into the first coil of the midgut (Fig. 2) and can only accommodate a smaller part of the blood meal. In these flies, only after the blood in the midgut becomes dehydrated can more be transferred from the crop. Also the further growth of the peritrophic membrane will allow a further transfer of blood. The crop in very young flies, may take 3-6 hours to empty.
368
ESTABLISHMENT BARRIER FOR T B R U C E I I N GUT OF G. P . 4 L L I D I P E S
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FIG. 2. Distribution of bloodmeal in the gut of Glossina pallidipes. A. Immediately after feeding in a 24 hour old fly. B. After completion of crop-emptying in a 40 hour old fly (see Fig. 3). C. Pedtrophic membrane of 32 hour old, unfed fly. D. Pedtrophic membrane of 52 hour old fly, 30 minutes after feeding, pv--proventriculus, cd--crop duct, tmgm thoracic midgut, amgmabdominal midgut, ds---dorsal straight part of amg, fc--first coil of amg, sc--second coil of amg. Discussion At this stage of the assessment of the situation, 2 possible methods of establishment of extra-peritrophic infections of rrypanosomes in the tsetse fly can bc excluded: firstly, in young flies the membrane does not grow faster during the period of crop-gut blood transfer, thus WILLETT'Stheory (1966) of easier penetration seems unlikely; secondly, the blood never penetrates beyond the peritrophic membrane during or shortly after feeding; it is always completely retained within the membrane. Only in flies of over 60 hours old
RUDOLF
HARMSEN
369
does the bloodmeal and its trypanosome population penetrate to the distal end of the midgut, but even then, it remains within the peritrophic membrane.
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FIG. 3. Crop emptying rate for Glossina pallidipes, related to age of the fly at time of feeding. We have, however, a situation which shows considerable differences between young flies and older flies. The blood meal (including any possible trypanosomes) is, partly at least, retained in the crop for a much longer period in young flies, and its movement from crop to gut is much slower. The crop emptying rate as plotted against age in Fig. 3 shows that the critical 24 hour age (with a relatively high infection chance under 24 hours and a considerably lower infection chance in flies over 24 hours) corresponds with a 45-75 minute period of blood retention in the crop. 2 possible mechanisms could link the crop emptying rate with a chance of infectivity. The first one, more or less an extension of Willett's theory, would assume an increased chance of penetration of the semi-liquid proximal part of the peritrophic membrane by trypanosomes under conditions of slow flow. A rate of flow which would empty the crop in less than 45 minutes would be too fast to allow trypanosomes a chance of adhesion and penetration. Slower rates would increase this chance markedly. This theory cannot be rejected at the moment, but both its simplicity and supporting evidence for the second hypothesis render it unlikely. The second hypothesis is based on the actual time spent by the trypanosome inside the crop rather than the midgut of the tsetse fly. The crop internal environment is very different from the midgut. There is probably no dehydration, no digestive enzyme activity and no clotting or coagulation of the blood proteins. It appeared a distinct possibility that trypanosomes need a certain period of time in an environment other than the vertebrate blood stream and yet less "hostile" than the tsetse midgut in order to adapt physiologically tolife in the fly. The fly crop is just such an environment, and in young flies the period spent by the trypanosomes in the crop is much longer than in older flies. Since infection rates go down rapidly in flies of over 24 hours of age, and crop emptying at that age takes 45-75 minutes, this hypothetical
370
ESTABLISHMENT BARRIER FOR 2". B R U C E I I N GUT OF 12. P A L L I D I P E S
adjustment period of the trypanosome 'is postulated to take somewhere around 60 minutes. Trypanosoma brucei occurs in 2 distinct shapes in the bloodstream of mammals: the growth (slender) form and the transmission (stumpy) form. Only the latter is capable of survival in the fly or in culture media (REICHENOW,1921 ; WIJERSand WILLETT,1960). The transformation from growth to transmission forms is caused by an unknown density dependent factor (HARMSEN,in preparation, b) and involves a major change in enzyme inventory (VICKERMAN,1965). It is, therefore, to be considered a pre-adaptation to life in the fly. The stumpy form as found in the vertebrate host, however, is not identical to the fly-gut form, neither morphologically nor biochemicaliy (FLYNN and BOWMAN, 1970). ! If the adjustment period hypothesis is correct it must be postulated that a second physiological (and probably enzymological) transformation takes place in those stumpy trypanosomes which are inside the crop of the fly; that this transformation takes 45-60 minutes to complete, and that this transformation is necessary before the trypanosome can survive in the midgut of the fly, and there develop into the typical fly gut-form.
Temperature sensitivity of trypanosome metabolic enzymes Experimental If the trypanosomes pass through a physiological transformation during the first hour after entry into the crop of the fly, it should be possible to note consistent differences in activity level, location and/or chemical species of a number of arbitrarily chosen enzyme types. A number of cytochemical techniques for general and specific phosphatases (PEARSE, 1961) have been adapted to produce consistent and distinct results with Trypanosoma brucei(HARMSEN,in preparation, a). It was found that both acid and alkaline phosphatase activity levels and localization showed minor changes on host-transfer. These enzyme systems are, in most organisms, summations of a fairly large number of eazym~es and consequently not very suitable for the study of minor differences. The 2 specific phosphatases studied: glucose-6-phosphatase and 5-nucleotidase are much more promising in this respect, especially glucose-6-phosphatase, since it showed a clear increase in intensity and a shift in localization within 1 hour of host transfer (HARMSEI%in preparation, a) and appeared to be a suitable indicator enzyme of a possible metabolic transformation. It appeared desirable to show not only a quantitative change in enzyme activity level and a change in localization, but also a switch in isozyme species. The trypanosome inside the vertebrate is exposed to a fairly constant 37°C. temperature. In the tsetse fly on the other hand a 20-25°C. temperature prevails. For any poikilothermal organism, the total enzyme complex has to be able to withstand changes in the temperature. Parasitic organisms which are transferred from mammals to invertebrates are exposed to sudden, drastic changes in temperature at the time of host transfer. One could predict that any arbitrarily chosen enzyme will show either a wide range of near optimal temperatures or a definite number of alternative isozyme species, each with its own, more narrowly defined optimal temperatures. The above situation has been investigated for glucose-6-phosphatase. 6 categories of trypanosomes were compared: those directly from the rat's peripheral circulatory system, and 5 groups taken from the fly crop, 10 minutes, 30 minutes, 60 minutes,. 2 hours and 3 hours after feeding of the fly. Smears prepared from these trypanosomecontaining blood samples were exposed after formalin fixation to a temperature of 40°C.
RUDOLF HARMSEN
371
for a variety of times, up to 90 minutes. After this exposure, the slides were all incubated in the normal way for glucose-6-phosphatase (HaaMSEN, in preparation, a) and the intensity of enzyme activity was noted. The results as tabulated in Table I, show that the glucose-6-phosphatase of the trypanosomes appears to become more temperature labile after about 1 hour in the fly crop. TABLE I. The effect of exposure to 40°C. for varying periods of time on trypanosome glucose-6-phosphatase activity in trypanosomes during various stages of adjustment to the invertebrate host. Origin of trypanosome fly crop 10 minutes
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90 ÷ ÷ ÷ strong activity ÷ ÷ weak but definite activity ÷ weak activity, uncertain - no activity It can be concluded that not only does the total level of activity and the intracellular localization change shortly after host transfer, but also the properties of the enzyme change. Whether this involves the synthesis of an alternative isozyme species or not cannot be stated on the basis of present data. Conclusions
When a population of T. brucei reaches a certain density (different for different strains) the first transformation occurs. The rapidly reproducing, slender trypanosomes metamorphose into non-reproducing stumpy, transmission forms. The major enzymic change which takes place during this transformation is well documented and fully appreciated (VICKERMalq, 1965). It is suggested here, that this transformation produces a trypanosome capable of survival in the fly crop, but not in the fly gut. The second transformation takes place immediately after the host transfer and may well be triggered by a simple environmental stimulus such as cooling. It is apparent that the entire transformation from the growth form to the fly-gut form can take place neither in the vertebrate blood stream nor in the insect. The so-called stumpy form, must therefore be considered as a semiTdormant intermediate which can survive in the vertebrate at 37°C. and in the fly crop at room temperature, but cannot reproduce in either environment.
372
ESTABLISHMENT BARRIERFOR 7". B R U C E I IN GUT OF G. P A L L I D I P E S
The second transformation (in the crop) takes approximately 45-60 minutes. It is, therefore, essential that trypanosomes stay in the crop for at least that time, if they are subsequently to survive in the gut. Thus, only in flies, with an incompletely formed peritrophic membrane (which results in a reduced crop to gut blood-transfer rate) can one expect any significant survival of trypanosome infections. Flies with incompletely formed peritrophic membranes are immature flies. The often observed 24 hour threshold can now be explained as follows. Flies younger than 24 hours have a peritrophic membrane of less than 24 mm. in length; such a membrane can only contain an entire blood meal after a period of growth and dehydration of the initial input which lasts for at least 60 minutes, the time required for completion of the second transformation. There is no direct evidence that the chance of complete cyclical survival in the fly is enhanced by a 60 minute or longer period in the fly crop; the closest to direct evidence can be found in the reported increase in cyclical infection rate for flies fed on trypanosome culture forms (GORDON and MILLER, 1961). There are several weaknesses in this argument. Firstly, the evidence supports the theory, but does not exclude other possible explanations. There is still a possibility of a direct mechanical effect, and possibly other, more complex factors may contribute. Secondly, a number of questions cannot be answered as yet. Foremost among these is the question of how an ectoperitrophic infection is established. Willett's theory included an answer to this; the present theory does not. Outward penetration of the proximal portion of the membrane remains a possibility but no evidence exists. Circumnavigation of the distal end of the membrane in the hindgut is not a very plausible concept as the hindgut environment appears to be highly toxic to brucei trypanosomes (WILLErT, pers. commun., 1965), and the bloodmeal has never been observed to extend beyond the distal end o f the peritrophic membrane. Penetration of the peritrophic membrane in the distal end of the midgut seems a possibility in the light of recent observations by DIPEOLU and ADAM (1972) but again, no direct evidence is available. One more problem must be mentioned in some detail. In a sizeable number of flies which have been fed on an infective vertebrate but which have not developed cyclical salivary gland infections, temporary or chronic crop and/or gut infections have been reported (DuI~ and M~I~LAN~Y, 1936). These infections indicate that a secondary barrier, one that operates after successful establishment, but before metamorphosis to metacyclic forms also exists.
Summary The low survival rate of Trypanosoma brucei in tsetse flies is interpreted as in part at least the result of an establishment barrier. This barrier appears to be less active in young flies than in older flies. The growth rate of the peritrophic membrane is 1 mm./hour during the first 30 hours after emergence, and also after feeding. The distribution of the blood meal in relation to the growth of the peritrophic membrane makes it appear unlikely that the membrane itself is the barrier mechanism. A postulated adjustment period for trypanosomes in the crop of young flies (with an incomplete peritrophic membrane) is supported by cytochemical evidence concerning an enzymic transformation within 1 hour of feeding. This phenomenon implies a double transformation for successful host transfer: one (well established) in the vertebrate host sometime before transfer, a second (new) in the fly, immediately after transfer. The destruction of non-transformed trypanosomes in the mid-gut (after leaving the crop) of mature tsetse flies is considered to be the main establishment barrier.
RUDOLF HARMSEN
373
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