- Email: [email protected]
Storage of nutriments by adult female Glossina morsitans and their transfer to the intra-uterine larva
J. Insect Physiol., 1976, vol. 22, pp. 1111 to 1115. Pergomon Press. Printed in Great Britain.
STORAGE OF NUTRIMENTS BY ADULT FEMALE GLOSSINA MORSITANS AND THEIR TRANSFER TO THE INTRA-UTERINE LARVA S. K. MOLOO Tsetse Research Laboratory, University of Bristol, School of Veterinary Science, Langford, Bristol BS18 7DU, England (Received 21 November 1975; revised 30 December
1975)
Abstract-Administration of U-W arginine, histidine, leucine, lysine, phenylalanine, threonine, tyrosine, or valine into the haemolymph of female Glossina morsirans on the first day of the pregnancy cycle was followed by radiometric analysis of the post-parturient larva. Radioactivity in the larva, expressed as a percentage of the administered activity, was low with histidine (0.3%) and arginine (2.3%) but higher with the other six amino acids (8.2% to 16.8%). 14C incorporation in the larval lipid was extremely low with arginine and histidine, but with the remaining six amino acids lipids showed the most 14C labelling. Radioactivity was detected in the larval amino acids corresponding to those injected into the female parents. Further radiometric study using labelled leucine showed that during the first 5 days of pregnancy surplus leucine was largely converted to lipids for larval growth. Thereafter, while the rate of leucine-derived i4C incorporation in the larval lipids declined rapidly that in the larval proteins increased. Implications are that female G. morsitans has a significant capacity to store nutriments derived from bloodmeals ingested during early pregnancy destined for larval development, and that normal growth of the intrauterine progeny is a function of optimum feeding throughout the pregnancy cycle.
INTRODUCTION
IN Glossina THEmode of reproduction is adenotrophic viviparity. At a maintenance temperature of 25”C, each pregnancy lasts for 9 to 10 days during which the female fly feeds at intervals upon vertebrate blood and the intra-uterine larva receives nutriments via the maternal uterine glands; the pregnancy cycle terminates with parturition of a fully-fed third instar larva (HOWMAN, 1954; DENLINGERand MA, 1974; LANGLEYand PIMLEY,1974). CMELIKet al. (1969) suggested that in pregnant female G. nwrsitans the nutritive secretion for larval development is elaborated from a single bloodmeal of 40mg and, although the supply of phenylalanine and tyrosine in this meal is inadequate to meet the demands for larval nutrition, other essential amino acids are supplied in quantities greatly exceeding the probable demand, the additional aromatics being derived from the earlier meals or from the symbionts. This suggestion infers that the female fly does not store essential amino acids, at least other than the aromatics, for larval development. LANGLEYand PIMLEY(1974, 1975) also suggested that this insect has little or no capacity to store nutriments for larval development, and that a large meal of about 60mg ingested on day 5 or 6 of the pregnancy period contributes most of the nutriments for larval growth. This hypothesis was based on indirect evidence and the possibility that the adult female fly has a good capability to store nutriments for larval development cannot be ruled out. In the
present study experimental methods were selected which provide direct evidence of the extent of nutrient storage by pregnant female G. morsitans, for the development of its intrauterine offspring. MATERIALS AND METHODS Flies G. morsitans were obtained from self-supporting goat-fed colony maintained in this laboratory (NASH et al., 1971). The experiments were undertaken with female flies in their second pregnancy cycle (MOLOO, 1976a). All experimental flies were fed on goats and kept at 25°C. Storage of amino acids and lipids
Female flies on the 1st day of pregnancy were individually injected through the thoracic cuticle with 2 ~1 of one of the labelled ammo acids (Radiochemical Centre, Amersham) shown in Table 1. Following larviposition 10 larvae were taken shortly after pupariation from each of the eight experimental groups, and were washed with 0.40/, lithium carbonate solution and then with several changes of distilled water to remove the adhering adult excreta. After drying the surface moisture, 5 puparia were individually solubilized in toto in 0.6 N NCS tissue solubilizer (Amersham/Se&e Corp.) at 50°C. The puparial shell did not solubilize completely in this solution. The samples were cooled to room temperature, brought to about
1111
1112
S. K. MOLOO
Table 1. Details of eight amino acids individually injected into female flies on the first day of second pregnancy cycle
U-14C amino acid Arginine Histidine Leucine Lysine HCl Phenylalanine Threonine Tyrosine Valine
Specific activity (mCi/m-mole) 318 324 311 318 411 232 522 265
Mol. wt at this specific activity 221 165 141 193 181 126.5 198 126
pH 7 with acetic acid, and total 14C-activity in each puparium and its contents were determined in 10ml of NE250 liquid scintillator (Nuclear Enterprises Ltd., Edinburgh) in a Nuclear Chicago counter (Model 6850). An internal standard was used to correct for any quenching, and all counts were corrected for background activity. This procedure of radioactivity measurement was adopted for all subsequent samples. The remaining five puparia from each group were chopped up in chloroform-methanol mixture (3:l) and lipids extracted for 48 hr. Radioactivity in the chloroform-soluble lipid fraction was determined as previously described (MOLOO, 1976a). After lipid extraction, the tissue debris from each sample was hydrolysed in 6N HCl in a sealed tube under nitrogen gas at 120°C for 24 hr. The hydrolysates were filtered (Whatman No. l), dried in uucuo, and each redissolved in 1 ml of 50% methanol. After passing through a membrane filter (Millipore, 0.45 pm), a 50 ~1 sample of each hydrolysate was taken for the determination of 14C-activity. Another 50 ~1 sample was subjected to two-dimensional paper chromatography, and 14C-activity in the amino acid that was originally injected into the corresponding pregnant female parents was determined in the manner previously described (Mom et al., 1974). Uptake of nutriments by in utero progeny Eight groups of female flies in different days of pregnancy, from 1 to 8, were individually injected with 2 pl of labelled leucine. Following parturition, 10 larvae were taken shortly after pupariation from each of the eight experimental groups, and five puparia were used to determine total 14C-activity in individual puparium and its contents while the remaining five were used to measure radioactivity in the lipid fraction and in the hydrolysate as described above. In another experiment, eight similar groups of seven female flies each were individually injected with 2 ~1 of labelled leucine. Twenty-four hr later, the uterine content of each fly was carefully removed, washed in several changes of physiological saline, and total 14C- labelling measured as described previously (Mo~oo, 1976a).
Injected amount, 2 pi/fly Radioactive concentration (&i/ml) 50
49 50 50 50 50 50 50
Radioactivity counts/min/2 ~1 1.75 1.75 2.14 1.75 1.90 1.80 1.90 2.05
x x x x x x x x
lo5 105 lo5 lo5 lo5 10’ lo5 lo5
Amount (pg) 0.07 0.05 0.05 0.06 0.04 0.05 0.04 0.05
Cumulative storage of nutriments
Four groups of pregnant female flies were individually injected with 2 ~1 of labelled leucine as follows. The first group received injections on day 1, the second on day 1 and 2, and the third and fourth groups respectively on the first 3 and 4 days of the pregnancy cycle. Following parturition, 10 larvae were taken shortly after pupariation from each group, and total 14C-activity in five puparia and that in the lipid fraction and in the hydrolysate in the remaining five puparia were determined as described above.
RESULTS Table 1 shows details of the U-14C amino acids used. Radioactivity expressed as counts/min/2 ~1 in each of the labelled materials is a mean of three measurements. The amount of each of the eight amino acids individually injected into females was extremely small in relation to the total amount of free amino acids present in the haemolymph (LANGLEY and PIMLEY, 1974), and hence the addition of such small amounts is unlikely to have affected the normal course of physiological events in the fly. Table 2 shows that radioactivity in the larval offspring (third instar) was relatively low after the labelled arginine and histidine injections of the female parents on the first day of pregnancy. Also, the recovery of total 14C-activity in the larva after histidine injection of the female parent was only 13% of that after the injection of arginine. In the case of the other amino acids, the recovery in the larval progeny was markedly higher and varied between 8.2 and 16.8% of the injected activity. Radioactivity was detected in both larval lipids and hydrolysate after parental injections of each of the 8 amino acids. Chromatographic and radiometric analyses of the hydrolysates revealed 14C labelling in larval amino acids corresponding to those injected into the female parents. Table 2 also shows that after the parental injection of the labelled arginine and histidine the proportions of radioactivity in the lipid fractions were extremely low, but in the case of the other six amino acids lipid fractions
Storage
of nutriments by adult female
Ghsim
1113
morsitans
Table 2. Total 14C-activity in the larval offspring and its distribution in the larval lipids and hydrolysate after injections of each of the eight U- ‘*C amino acids into female G. morsituns on the first day of the second pregnancy cycle “C-activity, counts/min/larva
U-14C amino acid
Third instar larva R + SE
Per cent of injected activity
Lipid fraction
Per cent of total recovery
Hydrolysate fraction
Per cent of total recovery
Arginine Histidine Leucine Lysine HCl Phenylalanine Threonine Tyrosine Valine
3946 k 368 482 f 23 35848 _+2521 25178 k 3298 17688 _+ 728 18518 + 2428 16960 + 994 16863 + 1138
2.3 0.3 16.8 14.4 9.3 10.3 8.9 8.2
144 85 25326 16060 9874 13859 10988 12590
6.2 19.6 88.6 87.0 79.7 84.0 86.2 85.6
2190 349 3270 2402 2517 2640 1760 2112
93.8 80.4 11.4 13.0 20.3 16.0 13.8 14.4
showed the most activity while 14C labelling in the hydrolysates were correspondingly low. Figure 1 shows that after the labelled leucine injections of the females on the first day of pregnancy, 14C-activity recovered in the larval progeny was about 14% of the injected activity. About the same level of activity was detected in the third instar larva after the parental injections on each of the following three days of pregnancy. Thereafter there was a marked increase in 14C-activity in the larval progeny
I
2
3
Age following first
4
5
larviposition,
6
7
6
days
Fig. 1. Radioactivity in the progeny, expressed as a percentage of the administered activity per fly, after haemocoelic injections of W4C leucine into G. morsitans on different days of their second pregnancy cycle. Vertical lines represent 95% confidence intervals for mean values. O--O, radioactivity in the intra-uterine progeny 24 hr after the labelled leucine injections of the female parents. M, radioactivity in the post-parturient third instar larva, and its distribution in the larval lipids (A-A) and hydrolysate (A-A) after such injections into similar females.
reaching the maximum of about 30% of the administered activity into females on the seventh day of pregnancy. This was followed by only a slight decline. The 14C incorporation in the larval lipids was about the same following parental injections of the labelled leutine on each of the first 4 days of pregnancy. There was a detectable rise in such incorporation after parental injection on the fifth day of pregnancy followed by a rapid decline till the end of pregnancy. After the parental injections on the first day of pregnancy, the 14C labelling in the hydrolysate of the larval progeny was very low. Radioactivity in the hydrolysate increased very slowly with the parental injections on subsequent days until the fifth day of pregnancy. Thereafter, while 14C incorporation in the larval lipids declined rapidly that in the hydrolysate increased throughout the remainder of the pregnancy period. Figure 1 also shows 14C-activity in the intrauterine progeny 24 hr after the parental injections of labelled leucine on each of the first 8 days of pregnancy. During the first four days the egg in the uterus lacked radioactivity. Following eclosion, the first instar larva showed 14C labelling (day 4 to 5). During the next one to two days the uterus contained the second instar which showed markedly higher “C-activity compared with the previous instar. The most rapid uptake of leucine and its synthetic products occurred after ecdysis to the third instar which lasted for 2 to 3 days of late pregnancy. Table 3 shows that after labelled leucine injections of females on the first day of pregnancy, 14C-activity in the post-parturient third instar larva was about 14% of the injected activity. When such injections were repeated on the second day of pregnancy (Group 2), radioactivity in the larva increased by about 12.2% of the activity in the second injection. In the case of groups 3 and 4, the increase in 14C-activity over the previous counts in the larvae were respectively 23.2 and 29.0% of the last injected activity. In each group the 14C incorporation in the larval lipids was markedly higher than that in the hydrolysate. Also, there was a slight decre+e in the proportion of
S. K. MOLOO
1114
Table 3. Total “C-activity in the larval offspring and its distribution in the larval lipids and hydrolysate after varying the number of injections of U-‘4C leucine into female G. morsitnns during the first four days of the second pregnancy
cycle ‘V-activity,
Group
Days of pregnancy females injected at 2 Nfly
1 2 3 4
1 l-2 l-3 14
Third instar larva x + SE 30335 k 56421 k 106070 k 168121 k
2203 4168 5612 19191
counts/min/larva
Lipids
Per cent of total recovery
22878 36902 67047 110012
88.4 86.3 84.4 80.1
radioactivity in the lipid fraction with increasing number of labelled leucine injections from group 1 to 4, and a corresponding increase of that in the hydrolysates.
DISCUSSION The amino acids arginine, histidine, leucine, lysine, phenylalanine, threonine, and valine are essential in the diet of G. morsituns (Mo~oo et al., 1974) whereas
tyrosine is at least partially dispensable in that it can be synthesized from phenylalanine (Mom, 1975). Following parental injections with labelled leucine, lysine, phenylalanine, threonine, tyrosine, or valine on the first day of pregnancy, the proportion of radioactivity recovered from post-parturient larvae was higher in the lipid than in the hydrolysate fraction. Hence it is probable that these amino acids when transported to the haemolymph during digestion of bloodmeals taken during early pregnancy, in addition to supporting metabolism in the adult female, are converted to storage lipids and transferred as such, to the growing larva, during the second half of the pregnancy cycle (the first half of the pregnancy cycle is occupied by the developing embryo). It is thus apparent that lipids form the main food reserve of this insect (BURSELLet al., 1974). The present results also show a restricted lipid synthesis from arginine and histidine in G. nwrsitans. The storage of histidine for larval development was lower than that of arginine. This could partly explain the greater amount of histidine present in the adult excreta compared to that of arginine (BURSELL,1965; BAL~GUN, 1974), although there is also more histidine than arginine in the blood ingested (BURSELL,1965; Mom, 1976b). Administration of labelled leucine into maternal haemolymph on each of the first 8 days of pregnancy and, 24 hr later, measurement of radioactivity in the intra-uterine progeny revealed that during the first 4 days of pregnancy the uterus contains a cleidoic egg. After eclosion the first instar larva, which lasts for 1 day, feeds upon secretions from the maternal uterine glands, but the very low radioactivity in this
Hydrolysate 2992 5834 12408 27394
Per cent of total recovery 11.6 13.7 15.6 19.9
instar indicates its low demand for nutriment. The second instar, which lasts for nearly 2 days, has a much higher demand for nutriment but the most rapid transfer of nutriment occurs after the appearance of the third instar which grows for the last 2 to 3 days of pregnancy. When this pattern of nutrient uptake is compared with that of recorded radioactivity in the post-parturient third instar larva following administration of labelled leucine into females on different days of pregnancy, it becomes apparent that the female fly has a significant capacity to store nutriments derived from bloodmeals ingested during early pregnancy for larval development. About 14% of radioactivity injected into females during the first 4 days of pregnancy, is recoverable from the post-parturient larva. Injection on the 7th day of pregnancy results in recovery of 29.5%. The rate of nutrient uptake by the first instar is extremely low, and its recorded labelling 24 hr after maternal injection of radioactive leucine was only 0.7% of the administered activity. It is most likely that leucine and its synthetic products detected in the progeny after parental injection during early pregnancy were taken up by the intra-uterine larva largely after the appearance of the second instar early on the 6th day of pregnancy. Thus a significant proportion of the maternal nutriments derived from food ingested during the first 4 days of pregnancy probably serve to augment the nutrient store of the female parent for larval growth. Additional larval nutriments are derived from bloodmeals taken after the 4th day of pregnancy as indicated by the pattern of radioactivity in the post-parturient larva coincident with its progressive in utero growth. The present study shows that there was a cumulative increase in 14C-activity in the post-parturient larva with increasing number of labelled leucine injections of the female parents over the first 4 days of pregnancy. Again, i4C incorporation in the larval lipids was much higher than that in the respective hydrolysate. Since the nutrient transfer to the larva occurs in the main after the 5th day of pregnancy, the above cumulative pattern of radioactivity in the larva is probably a reflection of increasing storage
Storage of nutriments by adult female Glossiru~morsitans
of larval nutriments in the female fly throughout the first half of the pregnancy period. After the 5th day of pregnancy, while the rate of 14C incorporation in the larval lipids declined rapidly, that in the larval proteins increased concurrently, and this trend continued throughout the remainder of the pregnancy period. It would seem therefore that amino acids transported to the haemolymph during the most rapid larval growth are largely utilized for the synthesis of larval proteins. It is possible that the change from the synthesis of predominantly larval lipids from the bloodmeal amino acids during the first 5 days of pregnancy to that of larval proteins during the later period is also reflected in the composition of the uterine gland secretions. The cycle of milk gland activity described by DENLINGER and MA (1974) is of significance in this context. The secretion begins to accumulate in the reservoirs on the 3rd day of pregnancy, and such nutrient accumulation progressively increases reaching a maximum on the 6th day. It is possible that larval nutriments synthesized during the first 6 days are transported to these reservoirs, and since these nutriments comprise predominantly lipids the composition of secretion might be similar, that is more lipids than proteins. Thereafter the volume of the reservoirs decreases as the larva is nourished. During the last 3 days the rate of ‘milk’ discharge to the larva is greater than the rate of ‘milk’ synthesis, and since at this period of pregnancy there is a greater synthesis of proteins than lipids it is possible that the former nutriment predominates in the secretion. The present results can also be reconciled with the suggestion of LANGLEY and PIMLEY(1975) that composition of the secretion in terms of its gross nutrient content remains constant throughout the larval development. Accordingly, during early pregnancy the female fly undergoes a rapid lipogenic phase and lipids synthesized from bloodmeal-derived amino acids are stored. Later in pregnancy there is a rapid uptake of amino acids by uterine glands for de novo synthesis of larval proteins (TOBEand DAVEY.1974) and larval lipids taken by these glands during this period are partly derived from the storage lipids. Studies involving analyses of the uterine gland secretion at different times of pregnancy are required to ascertain which of the above alternative hypotheses is correct. CMELIK et al. (1969) and LANGLEYand PIMLEY (1974, 1975) have suggested that female G. nwrsitans has little or no storage capacity for larval nutriments and that nutritive secretion for larval development is elaborated mostly from a single bloodmeal ingested sometime in the middle of the pregnancy period. However, the present investigation has served to illustrate that this insect has a significant capacity to store
1115
nutriments derived from bloodmeals ingested during early pregnancy, and that normal growth of the intrauterine progeny is a function of optimum feeding by the female parent throughout the pregnancy cycle.
Acknowledgements-I thank Dr. A. M. JORDAN,Dr. P. A. LANGLEY,and Dr. J. D. GEE for helpful comments on the manuscript. The financial support of the Overseas Development Administration of the U.K. Foreign and Commonwealth Office is gratefully acknowledged.
REFERENCES BAL~GUNR. A. (1974) Studies on the amino acids of the tsetse fly, Glossina morsitans, maintained on in uirro and in uivo feeding systems. Camp. Biochem. Physiol. 49A, 215-222. BURSELLE. (1965) Nitrogenous waste products of the tsetse fly Glossina morsitans. J. Insect PhysioL 11, 993-1001. BURSELLE., BILLING K. C., HARGROVEJ. W.. MCCABE C. T., and SLACKE. (1974) Metabolism of the bloodmeal in tsetse fly. Acta trap. 31, 297-320. CMELIKS. H. W., BURSELL E., and SLACKE. (1969) Composition of the gut contents of third instar larvae (Glossina morsitans Westwood). Camp. Biochem. Physiol. 29, 447453.
DENLINGER D. L. and MA W. (1974) Dynamics of the pregnancy cycle in the tsetse Glossina morsitans. J. Insect Physiol. 20, 1015-1026.
HOFFMANR. (1954) Zur Fortpflanzungsbiologie und zur intra-uterinen Entwicklung von Glossina pulpalis. Acta trap. 11, l-57. LANGLEYP. L. and PIMLEYR. W. (1974) Utilization of LJ-14C amino acids and U-14C protein by adult GIossina morsitans during in utero development of larva. 3. Insect Physiol. 20, 2157-2170.
LANGLEYP. L. and PIMLEY R. W. (1975) Quantitative aspects of reproduction and larval nutrition in Glossina marsitans morsitans Westw. (Diptera, Glossinidae) fed in vitro. Bull. ent. Res. 65, 129-142.
MOLDY S. K. (1975) Amino acid synthesis in Glossina morsitans. Trans. R. Sot. trap. Med. Hyg. 69, 281. MOLXI S. K. (1976a) Nutrition of Glossina morsitans: metabolism of U-“‘C glucose during pregnancy J. Insect Physiol. 22, 195-200. MOLTO S. K. (1976b) Aspects of the nutrition of adult female Glossina morsitans during pregnancy. J. Insect Physiol. 22, 563-567.
Mom
S. K., LANGLEYP. L., and BALOGUNR. A. (1974) Amino acid synthesis from glucose-U-‘4C in Glossina morsitans. J. Insect Physiol. 20, 1807-1813.
NASH T.
A. M., JORDANA. M., and TREWERNM. A. (1971) Mass rearing of tsetse flies (Glossina spp.): recent advances. In Sterility Principle for Insect Control or Eradication, pp. 99-110. I.A.E.A. Vienna. TOBES. S. and DAVEYK. G. (1974) Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni. Tissue & Cell 6, 255-268.
STORAGE OF NUTRIMENTS BY ADULT FEMALE GLOSSINA MORSITANS AND THEIR TRANSFER TO THE INTRA-UTERINE LARVA S. K. MOLOO Tsetse Research Laboratory, University of Bristol, School of Veterinary Science, Langford, Bristol BS18 7DU, England (Received 21 November 1975; revised 30 December
1975)
Abstract-Administration of U-W arginine, histidine, leucine, lysine, phenylalanine, threonine, tyrosine, or valine into the haemolymph of female Glossina morsirans on the first day of the pregnancy cycle was followed by radiometric analysis of the post-parturient larva. Radioactivity in the larva, expressed as a percentage of the administered activity, was low with histidine (0.3%) and arginine (2.3%) but higher with the other six amino acids (8.2% to 16.8%). 14C incorporation in the larval lipid was extremely low with arginine and histidine, but with the remaining six amino acids lipids showed the most 14C labelling. Radioactivity was detected in the larval amino acids corresponding to those injected into the female parents. Further radiometric study using labelled leucine showed that during the first 5 days of pregnancy surplus leucine was largely converted to lipids for larval growth. Thereafter, while the rate of leucine-derived i4C incorporation in the larval lipids declined rapidly that in the larval proteins increased. Implications are that female G. morsitans has a significant capacity to store nutriments derived from bloodmeals ingested during early pregnancy destined for larval development, and that normal growth of the intrauterine progeny is a function of optimum feeding throughout the pregnancy cycle.
INTRODUCTION
IN Glossina THEmode of reproduction is adenotrophic viviparity. At a maintenance temperature of 25”C, each pregnancy lasts for 9 to 10 days during which the female fly feeds at intervals upon vertebrate blood and the intra-uterine larva receives nutriments via the maternal uterine glands; the pregnancy cycle terminates with parturition of a fully-fed third instar larva (HOWMAN, 1954; DENLINGERand MA, 1974; LANGLEYand PIMLEY,1974). CMELIKet al. (1969) suggested that in pregnant female G. nwrsitans the nutritive secretion for larval development is elaborated from a single bloodmeal of 40mg and, although the supply of phenylalanine and tyrosine in this meal is inadequate to meet the demands for larval nutrition, other essential amino acids are supplied in quantities greatly exceeding the probable demand, the additional aromatics being derived from the earlier meals or from the symbionts. This suggestion infers that the female fly does not store essential amino acids, at least other than the aromatics, for larval development. LANGLEYand PIMLEY(1974, 1975) also suggested that this insect has little or no capacity to store nutriments for larval development, and that a large meal of about 60mg ingested on day 5 or 6 of the pregnancy period contributes most of the nutriments for larval growth. This hypothesis was based on indirect evidence and the possibility that the adult female fly has a good capability to store nutriments for larval development cannot be ruled out. In the
present study experimental methods were selected which provide direct evidence of the extent of nutrient storage by pregnant female G. morsitans, for the development of its intrauterine offspring. MATERIALS AND METHODS Flies G. morsitans were obtained from self-supporting goat-fed colony maintained in this laboratory (NASH et al., 1971). The experiments were undertaken with female flies in their second pregnancy cycle (MOLOO, 1976a). All experimental flies were fed on goats and kept at 25°C. Storage of amino acids and lipids
Female flies on the 1st day of pregnancy were individually injected through the thoracic cuticle with 2 ~1 of one of the labelled ammo acids (Radiochemical Centre, Amersham) shown in Table 1. Following larviposition 10 larvae were taken shortly after pupariation from each of the eight experimental groups, and were washed with 0.40/, lithium carbonate solution and then with several changes of distilled water to remove the adhering adult excreta. After drying the surface moisture, 5 puparia were individually solubilized in toto in 0.6 N NCS tissue solubilizer (Amersham/Se&e Corp.) at 50°C. The puparial shell did not solubilize completely in this solution. The samples were cooled to room temperature, brought to about
1111
1112
S. K. MOLOO
Table 1. Details of eight amino acids individually injected into female flies on the first day of second pregnancy cycle
U-14C amino acid Arginine Histidine Leucine Lysine HCl Phenylalanine Threonine Tyrosine Valine
Specific activity (mCi/m-mole) 318 324 311 318 411 232 522 265
Mol. wt at this specific activity 221 165 141 193 181 126.5 198 126
pH 7 with acetic acid, and total 14C-activity in each puparium and its contents were determined in 10ml of NE250 liquid scintillator (Nuclear Enterprises Ltd., Edinburgh) in a Nuclear Chicago counter (Model 6850). An internal standard was used to correct for any quenching, and all counts were corrected for background activity. This procedure of radioactivity measurement was adopted for all subsequent samples. The remaining five puparia from each group were chopped up in chloroform-methanol mixture (3:l) and lipids extracted for 48 hr. Radioactivity in the chloroform-soluble lipid fraction was determined as previously described (MOLOO, 1976a). After lipid extraction, the tissue debris from each sample was hydrolysed in 6N HCl in a sealed tube under nitrogen gas at 120°C for 24 hr. The hydrolysates were filtered (Whatman No. l), dried in uucuo, and each redissolved in 1 ml of 50% methanol. After passing through a membrane filter (Millipore, 0.45 pm), a 50 ~1 sample of each hydrolysate was taken for the determination of 14C-activity. Another 50 ~1 sample was subjected to two-dimensional paper chromatography, and 14C-activity in the amino acid that was originally injected into the corresponding pregnant female parents was determined in the manner previously described (Mom et al., 1974). Uptake of nutriments by in utero progeny Eight groups of female flies in different days of pregnancy, from 1 to 8, were individually injected with 2 pl of labelled leucine. Following parturition, 10 larvae were taken shortly after pupariation from each of the eight experimental groups, and five puparia were used to determine total 14C-activity in individual puparium and its contents while the remaining five were used to measure radioactivity in the lipid fraction and in the hydrolysate as described above. In another experiment, eight similar groups of seven female flies each were individually injected with 2 ~1 of labelled leucine. Twenty-four hr later, the uterine content of each fly was carefully removed, washed in several changes of physiological saline, and total 14C- labelling measured as described previously (Mo~oo, 1976a).
Injected amount, 2 pi/fly Radioactive concentration (&i/ml) 50
49 50 50 50 50 50 50
Radioactivity counts/min/2 ~1 1.75 1.75 2.14 1.75 1.90 1.80 1.90 2.05
x x x x x x x x
lo5 105 lo5 lo5 lo5 10’ lo5 lo5
Amount (pg) 0.07 0.05 0.05 0.06 0.04 0.05 0.04 0.05
Cumulative storage of nutriments
Four groups of pregnant female flies were individually injected with 2 ~1 of labelled leucine as follows. The first group received injections on day 1, the second on day 1 and 2, and the third and fourth groups respectively on the first 3 and 4 days of the pregnancy cycle. Following parturition, 10 larvae were taken shortly after pupariation from each group, and total 14C-activity in five puparia and that in the lipid fraction and in the hydrolysate in the remaining five puparia were determined as described above.
RESULTS Table 1 shows details of the U-14C amino acids used. Radioactivity expressed as counts/min/2 ~1 in each of the labelled materials is a mean of three measurements. The amount of each of the eight amino acids individually injected into females was extremely small in relation to the total amount of free amino acids present in the haemolymph (LANGLEY and PIMLEY, 1974), and hence the addition of such small amounts is unlikely to have affected the normal course of physiological events in the fly. Table 2 shows that radioactivity in the larval offspring (third instar) was relatively low after the labelled arginine and histidine injections of the female parents on the first day of pregnancy. Also, the recovery of total 14C-activity in the larva after histidine injection of the female parent was only 13% of that after the injection of arginine. In the case of the other amino acids, the recovery in the larval progeny was markedly higher and varied between 8.2 and 16.8% of the injected activity. Radioactivity was detected in both larval lipids and hydrolysate after parental injections of each of the 8 amino acids. Chromatographic and radiometric analyses of the hydrolysates revealed 14C labelling in larval amino acids corresponding to those injected into the female parents. Table 2 also shows that after the parental injection of the labelled arginine and histidine the proportions of radioactivity in the lipid fractions were extremely low, but in the case of the other six amino acids lipid fractions
Storage
of nutriments by adult female
Ghsim
1113
morsitans
Table 2. Total 14C-activity in the larval offspring and its distribution in the larval lipids and hydrolysate after injections of each of the eight U- ‘*C amino acids into female G. morsituns on the first day of the second pregnancy cycle “C-activity, counts/min/larva
U-14C amino acid
Third instar larva R + SE
Per cent of injected activity
Lipid fraction
Per cent of total recovery
Hydrolysate fraction
Per cent of total recovery
Arginine Histidine Leucine Lysine HCl Phenylalanine Threonine Tyrosine Valine
3946 k 368 482 f 23 35848 _+2521 25178 k 3298 17688 _+ 728 18518 + 2428 16960 + 994 16863 + 1138
2.3 0.3 16.8 14.4 9.3 10.3 8.9 8.2
144 85 25326 16060 9874 13859 10988 12590
6.2 19.6 88.6 87.0 79.7 84.0 86.2 85.6
2190 349 3270 2402 2517 2640 1760 2112
93.8 80.4 11.4 13.0 20.3 16.0 13.8 14.4
showed the most activity while 14C labelling in the hydrolysates were correspondingly low. Figure 1 shows that after the labelled leucine injections of the females on the first day of pregnancy, 14C-activity recovered in the larval progeny was about 14% of the injected activity. About the same level of activity was detected in the third instar larva after the parental injections on each of the following three days of pregnancy. Thereafter there was a marked increase in 14C-activity in the larval progeny
I
2
3
Age following first
4
5
larviposition,
6
7
6
days
Fig. 1. Radioactivity in the progeny, expressed as a percentage of the administered activity per fly, after haemocoelic injections of W4C leucine into G. morsitans on different days of their second pregnancy cycle. Vertical lines represent 95% confidence intervals for mean values. O--O, radioactivity in the intra-uterine progeny 24 hr after the labelled leucine injections of the female parents. M, radioactivity in the post-parturient third instar larva, and its distribution in the larval lipids (A-A) and hydrolysate (A-A) after such injections into similar females.
reaching the maximum of about 30% of the administered activity into females on the seventh day of pregnancy. This was followed by only a slight decline. The 14C incorporation in the larval lipids was about the same following parental injections of the labelled leutine on each of the first 4 days of pregnancy. There was a detectable rise in such incorporation after parental injection on the fifth day of pregnancy followed by a rapid decline till the end of pregnancy. After the parental injections on the first day of pregnancy, the 14C labelling in the hydrolysate of the larval progeny was very low. Radioactivity in the hydrolysate increased very slowly with the parental injections on subsequent days until the fifth day of pregnancy. Thereafter, while 14C incorporation in the larval lipids declined rapidly that in the hydrolysate increased throughout the remainder of the pregnancy period. Figure 1 also shows 14C-activity in the intrauterine progeny 24 hr after the parental injections of labelled leucine on each of the first 8 days of pregnancy. During the first four days the egg in the uterus lacked radioactivity. Following eclosion, the first instar larva showed 14C labelling (day 4 to 5). During the next one to two days the uterus contained the second instar which showed markedly higher “C-activity compared with the previous instar. The most rapid uptake of leucine and its synthetic products occurred after ecdysis to the third instar which lasted for 2 to 3 days of late pregnancy. Table 3 shows that after labelled leucine injections of females on the first day of pregnancy, 14C-activity in the post-parturient third instar larva was about 14% of the injected activity. When such injections were repeated on the second day of pregnancy (Group 2), radioactivity in the larva increased by about 12.2% of the activity in the second injection. In the case of groups 3 and 4, the increase in 14C-activity over the previous counts in the larvae were respectively 23.2 and 29.0% of the last injected activity. In each group the 14C incorporation in the larval lipids was markedly higher than that in the hydrolysate. Also, there was a slight decre+e in the proportion of
S. K. MOLOO
1114
Table 3. Total “C-activity in the larval offspring and its distribution in the larval lipids and hydrolysate after varying the number of injections of U-‘4C leucine into female G. morsitnns during the first four days of the second pregnancy
cycle ‘V-activity,
Group
Days of pregnancy females injected at 2 Nfly
1 2 3 4
1 l-2 l-3 14
Third instar larva x + SE 30335 k 56421 k 106070 k 168121 k
2203 4168 5612 19191
counts/min/larva
Lipids
Per cent of total recovery
22878 36902 67047 110012
88.4 86.3 84.4 80.1
radioactivity in the lipid fraction with increasing number of labelled leucine injections from group 1 to 4, and a corresponding increase of that in the hydrolysates.
DISCUSSION The amino acids arginine, histidine, leucine, lysine, phenylalanine, threonine, and valine are essential in the diet of G. morsituns (Mo~oo et al., 1974) whereas
tyrosine is at least partially dispensable in that it can be synthesized from phenylalanine (Mom, 1975). Following parental injections with labelled leucine, lysine, phenylalanine, threonine, tyrosine, or valine on the first day of pregnancy, the proportion of radioactivity recovered from post-parturient larvae was higher in the lipid than in the hydrolysate fraction. Hence it is probable that these amino acids when transported to the haemolymph during digestion of bloodmeals taken during early pregnancy, in addition to supporting metabolism in the adult female, are converted to storage lipids and transferred as such, to the growing larva, during the second half of the pregnancy cycle (the first half of the pregnancy cycle is occupied by the developing embryo). It is thus apparent that lipids form the main food reserve of this insect (BURSELLet al., 1974). The present results also show a restricted lipid synthesis from arginine and histidine in G. nwrsitans. The storage of histidine for larval development was lower than that of arginine. This could partly explain the greater amount of histidine present in the adult excreta compared to that of arginine (BURSELL,1965; BAL~GUN, 1974), although there is also more histidine than arginine in the blood ingested (BURSELL,1965; Mom, 1976b). Administration of labelled leucine into maternal haemolymph on each of the first 8 days of pregnancy and, 24 hr later, measurement of radioactivity in the intra-uterine progeny revealed that during the first 4 days of pregnancy the uterus contains a cleidoic egg. After eclosion the first instar larva, which lasts for 1 day, feeds upon secretions from the maternal uterine glands, but the very low radioactivity in this
Hydrolysate 2992 5834 12408 27394
Per cent of total recovery 11.6 13.7 15.6 19.9
instar indicates its low demand for nutriment. The second instar, which lasts for nearly 2 days, has a much higher demand for nutriment but the most rapid transfer of nutriment occurs after the appearance of the third instar which grows for the last 2 to 3 days of pregnancy. When this pattern of nutrient uptake is compared with that of recorded radioactivity in the post-parturient third instar larva following administration of labelled leucine into females on different days of pregnancy, it becomes apparent that the female fly has a significant capacity to store nutriments derived from bloodmeals ingested during early pregnancy for larval development. About 14% of radioactivity injected into females during the first 4 days of pregnancy, is recoverable from the post-parturient larva. Injection on the 7th day of pregnancy results in recovery of 29.5%. The rate of nutrient uptake by the first instar is extremely low, and its recorded labelling 24 hr after maternal injection of radioactive leucine was only 0.7% of the administered activity. It is most likely that leucine and its synthetic products detected in the progeny after parental injection during early pregnancy were taken up by the intra-uterine larva largely after the appearance of the second instar early on the 6th day of pregnancy. Thus a significant proportion of the maternal nutriments derived from food ingested during the first 4 days of pregnancy probably serve to augment the nutrient store of the female parent for larval growth. Additional larval nutriments are derived from bloodmeals taken after the 4th day of pregnancy as indicated by the pattern of radioactivity in the post-parturient larva coincident with its progressive in utero growth. The present study shows that there was a cumulative increase in 14C-activity in the post-parturient larva with increasing number of labelled leucine injections of the female parents over the first 4 days of pregnancy. Again, i4C incorporation in the larval lipids was much higher than that in the respective hydrolysate. Since the nutrient transfer to the larva occurs in the main after the 5th day of pregnancy, the above cumulative pattern of radioactivity in the larva is probably a reflection of increasing storage
Storage of nutriments by adult female Glossiru~morsitans
of larval nutriments in the female fly throughout the first half of the pregnancy period. After the 5th day of pregnancy, while the rate of 14C incorporation in the larval lipids declined rapidly, that in the larval proteins increased concurrently, and this trend continued throughout the remainder of the pregnancy period. It would seem therefore that amino acids transported to the haemolymph during the most rapid larval growth are largely utilized for the synthesis of larval proteins. It is possible that the change from the synthesis of predominantly larval lipids from the bloodmeal amino acids during the first 5 days of pregnancy to that of larval proteins during the later period is also reflected in the composition of the uterine gland secretions. The cycle of milk gland activity described by DENLINGER and MA (1974) is of significance in this context. The secretion begins to accumulate in the reservoirs on the 3rd day of pregnancy, and such nutrient accumulation progressively increases reaching a maximum on the 6th day. It is possible that larval nutriments synthesized during the first 6 days are transported to these reservoirs, and since these nutriments comprise predominantly lipids the composition of secretion might be similar, that is more lipids than proteins. Thereafter the volume of the reservoirs decreases as the larva is nourished. During the last 3 days the rate of ‘milk’ discharge to the larva is greater than the rate of ‘milk’ synthesis, and since at this period of pregnancy there is a greater synthesis of proteins than lipids it is possible that the former nutriment predominates in the secretion. The present results can also be reconciled with the suggestion of LANGLEY and PIMLEY(1975) that composition of the secretion in terms of its gross nutrient content remains constant throughout the larval development. Accordingly, during early pregnancy the female fly undergoes a rapid lipogenic phase and lipids synthesized from bloodmeal-derived amino acids are stored. Later in pregnancy there is a rapid uptake of amino acids by uterine glands for de novo synthesis of larval proteins (TOBEand DAVEY.1974) and larval lipids taken by these glands during this period are partly derived from the storage lipids. Studies involving analyses of the uterine gland secretion at different times of pregnancy are required to ascertain which of the above alternative hypotheses is correct. CMELIK et al. (1969) and LANGLEYand PIMLEY (1974, 1975) have suggested that female G. nwrsitans has little or no storage capacity for larval nutriments and that nutritive secretion for larval development is elaborated mostly from a single bloodmeal ingested sometime in the middle of the pregnancy period. However, the present investigation has served to illustrate that this insect has a significant capacity to store
1115
nutriments derived from bloodmeals ingested during early pregnancy, and that normal growth of the intrauterine progeny is a function of optimum feeding by the female parent throughout the pregnancy cycle.
Acknowledgements-I thank Dr. A. M. JORDAN,Dr. P. A. LANGLEY,and Dr. J. D. GEE for helpful comments on the manuscript. The financial support of the Overseas Development Administration of the U.K. Foreign and Commonwealth Office is gratefully acknowledged.
REFERENCES BAL~GUNR. A. (1974) Studies on the amino acids of the tsetse fly, Glossina morsitans, maintained on in uirro and in uivo feeding systems. Camp. Biochem. Physiol. 49A, 215-222. BURSELLE. (1965) Nitrogenous waste products of the tsetse fly Glossina morsitans. J. Insect PhysioL 11, 993-1001. BURSELLE., BILLING K. C., HARGROVEJ. W.. MCCABE C. T., and SLACKE. (1974) Metabolism of the bloodmeal in tsetse fly. Acta trap. 31, 297-320. CMELIKS. H. W., BURSELL E., and SLACKE. (1969) Composition of the gut contents of third instar larvae (Glossina morsitans Westwood). Camp. Biochem. Physiol. 29, 447453.
DENLINGER D. L. and MA W. (1974) Dynamics of the pregnancy cycle in the tsetse Glossina morsitans. J. Insect Physiol. 20, 1015-1026.
HOFFMANR. (1954) Zur Fortpflanzungsbiologie und zur intra-uterinen Entwicklung von Glossina pulpalis. Acta trap. 11, l-57. LANGLEYP. L. and PIMLEYR. W. (1974) Utilization of LJ-14C amino acids and U-14C protein by adult GIossina morsitans during in utero development of larva. 3. Insect Physiol. 20, 2157-2170.
LANGLEYP. L. and PIMLEY R. W. (1975) Quantitative aspects of reproduction and larval nutrition in Glossina marsitans morsitans Westw. (Diptera, Glossinidae) fed in vitro. Bull. ent. Res. 65, 129-142.
MOLDY S. K. (1975) Amino acid synthesis in Glossina morsitans. Trans. R. Sot. trap. Med. Hyg. 69, 281. MOLXI S. K. (1976a) Nutrition of Glossina morsitans: metabolism of U-“‘C glucose during pregnancy J. Insect Physiol. 22, 195-200. MOLTO S. K. (1976b) Aspects of the nutrition of adult female Glossina morsitans during pregnancy. J. Insect Physiol. 22, 563-567.
Mom
S. K., LANGLEYP. L., and BALOGUNR. A. (1974) Amino acid synthesis from glucose-U-‘4C in Glossina morsitans. J. Insect Physiol. 20, 1807-1813.
NASH T.
A. M., JORDANA. M., and TREWERNM. A. (1971) Mass rearing of tsetse flies (Glossina spp.): recent advances. In Sterility Principle for Insect Control or Eradication, pp. 99-110. I.A.E.A. Vienna. TOBES. S. and DAVEYK. G. (1974) Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni. Tissue & Cell 6, 255-268.