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Temporal synthesis of cuticle proteins during larval development in Glossina morsitans
Comp. Biochem. Physiol. Vol. 105B,No. 2, pp. 309-316, 1993
0305-0491/93 $6.00+ 0.00 © 1993Pergamon Press Ltd
Printed in Great Britain
TEMPORAL SYNTHESIS OF CUTICLE PROTEINS D U R I N G LARVAL DEVELOPMENT IN G L O S S I N A M O R S I T A N S VINCENT O. OCHIENG,* ELLIE O. OSIR,~':~JAMESO. OCHANDA* and NORAH K. OLEMBO* *Department of Biochemistry, University of Nairobi, P.O. Box 30197, Nairobi, Kenya (Fax 803-360); and tThe International Centre of Insect Physiology and Ecology (ICIPE), P. O. Box 30772, Nairobi, Kenya (Received 21 October 1992; accepted 27 November 1992)
Abstract--l. Larval development in Glossina species occurs in utero with the mature third instar larva being deposited after a developmental period of 7 days. 2. In this study, the patterns of cuticular protein synthesis during larval development were analysed by two-dimensional gel electrophoresis. 3. From the results, four types of cuticle proteins were identified: those specific to larval, pupal and adult cuticles, and others common to all the stages. 4. Few cutieular proteins were synthesized between the first and second larval instars. By the third larval instar (two days before larviposition), a large number of proteins (Mr ~<30 kDa) were induced. These proteins persisted up to the brown pupal stage and showed a rapid decline thereafter. Most of the proteins with molecular weights Mr ~<30 kDa were undeteetable at apolysis (5 days after larviposition). 5. By day 15 of the pupal stage, the number of cuticle proteins was very small. The protein profile during the pupal stages remained relatively constant. This was probably due to the fact that the pupal cuticle does not provide any protection since it is itself enclosed at all times within the protective puparium.
INTRODUCTION The insect cuticle functions both as an exoskeleton and a barrier between the living tissue and the environment. Furthermore, the cuticle determines the shape and appearance of insects. Proteins are a major component of the cuticle constituting approximately 50% of the dry weight (Richards, 1978). The main factors that contribute to the mechanical properties of cuticle are organization of chitin microfibrils, length of chitin microfibrils, chitin-protein interactions, protein conformation and protein-protein interactions (Andersen, 1979). The properties of the various highly specialized cuticles are due mainly to protein-protein interactions, which can be influenced by subtle changes in the surrounding medium (Andersen, 1979). Proteins provide a great deal of diversity to the cuticle composition. It seems likely that they define much of the specialized nature of the cuticle both in different anatomical regions and at different metamorphic stages (Silvert, 1985; Willis, 1987). Following ecdysis, electrophoretic banding patterns from cuticular extracts change, both quantitatively and qualitatively, due to sclerotization and the synthesis and secretion of new proteins (Roberts and Willis, 1980a,b; Chihara et al., 1982) Proteins extracted from the cuticles of holometabolous insects at different developmental stages ~To whom correspondence should be addressed. Abbreviations--pI: isoelectric point; IEF: isoelectric focus-
ing; SDS: sodium dodecyl sulphate; PAGE: polyacrylamide gel electrophoresis.
usually differ, as determined electrophoretically or by amino acid composition analysis (Srivastava, 1970; Roberts and Willis, 1980a,b; Chihara et al., 1982; Sridhara, 1983). The tsetse fly and other viviparous insects have a unique mode of reproduction. The larva completes its entire development within the uterus of the female and is then larviposited as a mature third instar larva. Each pregnancy cycle, lasting from 9 to 10 days, culminates in the production of a single fully grown third instar larva which burrows into the soil and forms a puparium within a few hours of larviposition (Buxton, 1955). In this study cuticle proteins of different developmental stages of G. m. morsitans are examined by two-dimensional gel electrophoresis.
MATERIALSAND METHODS Experimental insects
Tsetse flies, Glossina morsitans morsitans Westwood, were supplied by the Insect and Animal Breeding Unit of the International Centre of Insect Physiology and Ecology. They were reared on a 12 light: 12 dark photoperiod at 70-80% relative humidity and 25°C. The insects were fed daily on rabbit ears. Pregnant flies were dissected to obtain first and second instar larvae. Third instar larvae (2 days before larviposition) were forcibly expelled from the uterus by applying slight pressure on the female abdomen. Some flies were left to larviposit normally
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and the pupae selected at different stages. The rest of the pupae were kept until adult emergence. Preparation o f cuticles
Whole cuticles were dissected from larval, pupal and adult stages in tsetse Ringer solution (Kiely and Riddiford, 1985a,b). Larvae were anaesthetized in water and placed on a microscope slide. After removing the polyneustic lobes from the anterior region using a scalpel, the larvae were opened and the viscera removed. The integument was then scraped in tsetse Ringer solution from the body wall musculature. The pupal cuticle separates from puparium 5 days after white puparium formation. For adults, only the abdominal cuticles were used. In this case, cuticles were freed of contaminating tissue under a dissecting microscope using a previously described procedure (Hackman and Goldberg, 1971). The dissected cuticles were rinsed three times in tsetse Ringer solution, blotted dry on a filter paper and stored at - 7 0 ° C until analysis. Extraction o f cuticle proteins
The extraction of cuticle proteins was carried out as previously described (Kiely and Riddiford, 1985 a,b). Briefly, samples were homogenized at 4°C using 100 pl/insect of 6.25 mM Tris-HC1, pH 6.8, 1% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co., St Louis, MO). The protein content of extracts was determined using the bicinchoninic acid assay (BCA; Pierce, Rockford, IL). Bovine serum albumin (Fraction V; Calbiochem, San Diego, CA) was used as the protein standard. Gel electrophoresis
Two-dimensional gel electrophoresis was carried out according to O'Farrell et al. (1977) as modified by Jones (1980). Protein samples were separated by isoelectric focusing (IEF) gels in the first dimension using a 4:1 ratio of pH 5-7 and pH 3-10 ampholines (LKB, Pharmacia, Bromma, Sweden). The gels (11 cm) were cast in Pyrex tubes (140 x 30 mm, i.d.). The samples were prepared in IEF sample buffer (9.5 M urea, 2% (w/v) NP-40, 2% ampholines composed of 1.6%, pH range 5-7, and 0.4%, pH range 3-10, and 5% fl-mercaptoethanol). The cathode and anode solutions were 10mM NaOH and 20mM H3PO4, respectively. The gels were prefocused according to the following schedule for each gel: (a) 20 V for 25 min; (b) 30 V for 30 min; (c) 40 V for 30 min. The extruded gels were placed in 1 ml of equilibrium soaking solution (O'Farrell, 1977). After shaking gently for 30 min at room temperature, the gels were kept at - 70°C until used. For the second dimension, the SDS-polyacrylamide gel electrophoresis (SDSPAGE) was used (Laemmli, 1970). The running gel consisted of 8.5% polyacrylamide overlaid with 3% stacking gel. Individual proteins were resolved as discrete spots that were visualized after silver staining (O'Farrell et al., 1977).
RESULTS In order to ensure that the cuticles used in this study were free of contaminating tissue and haemolymph, the cuticles were thoroughly cleaned and then rinsed several times. Examination under a dissecting microscope was carried out to ascertain that the cuticles were clean. Furthermore, the haemolymph protein bands were different from those of the cuticle proteins. Initial attempts to separate the cuticular proteins were carried out by the nonequilibrium pH gradient gel (NEPHGE) system which involved the use of pH 3.5-10 ampholines in the first dimension. This system gave a poor separation since approximately two-thirds of the protein spots could not be seen. However, the use of isoelectric focusing, instead of NEPHGE in the first dimension, gave a good resolution of the proteins. Using this system, very striking changes were revealed in the patterns of cuticle proteins during larval development. The most interesting of these was the disappearance of some polypeptides as well as the appearance of others as the larva developed into an adult. From these results, four types of cuticle proteins were identified, namely, those specific to larval, pupal and adult cuticles, and others common to the stages. The first and second instar larvae exhibited the same protein profiles except for proteins 4, 15 and 16, which appeared by day 1 (second instar) and disappeared by day 5 (pupal stage) (Figs 1 and 2). By the third larval instar stage (2 days before larviposition), a large number of proteins appeared (Fig. 3). For example, a group of acidic proteins (16-20 and 26) with molecular weights of less than Mr ~ 30 kDa appeared in large amounts, persisted up to the brown pupal stage and thereafter showed a rapid decline, becoming undetectable at apolysis (5 days after larviposition) (Fig. 6). Protein 18, present in early third instar larvae, was not visible by the late third instar. Similarly, protein 19 which, in early third instar, was present in large amounts, was found only in trace amounts in late third instar, and became undetectable by the brown pupal stage. Proteins 5-6 and 9 appeared by early third instar larvae and persisted up to the adult stage. A number of proteins (20-30 and 42) with molecular weights of M r ~ 4 5 - 6 6 k D a , appeared by early third instar. These proteins increased in amounts by late third instar larvae (larviposition) and attained the highest amounts by the brown pupal stage. The proteins then decreased in amounts as development proceeded, and became undetectable by the adult stage (Figs 2-5). Proteins 22, 24, 31 and 34 appeared by late third instar larvae and lasted only up to the brown pupal stage (Figs 3-5). Decreased amounts of protein 17 were found in late third instar larval cuticle compared to that of early third instar, in which large amounts were present (Figs 3 and 4). A protein 23 appeared by late third instar and persisted up to the adult stage.
Tsetse cuticle proteins
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Fig. 1. Electrophoresis of first instar cuticle proteins. Protein samples were separated in the first dimension by IEF gel electrophoresis with a 4: I mixture of ampholines, pH 5-7 and 3.5-10, respectively, and the second dimension by SDS-PAGE as described under Materials and Methods. The gel was stained for protein using silver.
Seven proteins (4, 17, 18-21, 34) were only found during the larval stages. Figures 6-8 showed proteins from different pupal stages. Proteins 36-38 ( M r s 3 1 ) and 40
(Mr ~ 45 kDa) appeared by day 5 of the pupal stage. The protein profiles during the pupal stage remained relatively constant. By the fifteenth day of the pupal stage, the number of cuticle proteins present was very
Fig. 2. Electrophoresis of second instar cuticle protein. Separation of the proteins was as described in Fig. 1.
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Fig. 3. Electrophoresis of third instar cuticle protein (aborted 2 days before larviposition). Separation of the proteins was as described in Fig. 1.
small. Five proteins (37-41) were found to be specific to the pupal stages. A series of proteins, relatively a b u n d a n t in early pharate adult cuticle extracts, were found in dimin-
ished amounts in eclosed adult cuticle extracts. A cluster of proteins 47-50 (M r ~ 37 kDa) and a protein 52 (M r ~ 43 kDa) relatively prominent, appeared in the cuticle of newly emerged adults (Fig. 9).
Fig. 4. Electrophoresis of third instar cuticle protein (larviposited). Separation of the proteins was as described in Fig. 1.
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Fig. 5. Electrophoresis of brown pupal cuticle protein. Separation of the proteins was as described in Fig. 1. DISCUSSION The results presented in this paper that distinct changes occur in the types amounts of protein synthesized by G. epidermis during the larval-to-adult
clearly show as well as the rn. morsitans
development.
The proteins could be classified into four groups: those specific to the larval, pupal and adult cuticles, and those that were c o m m o n to all the stages. The major larval cuticle proteins from G. m. morsitans were in the molecular weight range of Mr ~ 20-70 kDa. The range is close to the one
Fig. 6. Electrophoresis of 5-day-old pupal cuticle protein. Separation of the proteins was as described in Fig. 1. CBPB 105/2--H
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Fig. 7. Electrophoresis of 10-day-old pupal cuticle protein. Separation of the proteins was as described in Fig. 1. observed in the boll weevil larval cuticle proteins with the molecular weights ranging from 17 to 66 kDa (Stiles and Leopold, 1990). These proteins had molecular weights higher than M ~ ~ 3 0 k D a unlike L. cuprina and D. melanogaster in which the larval cuticle proteins had molecular weight range of
13-30 kDa (Skelly and Howells, 1988; Chihara et al., 1982). The pI range (4-6) of the G. m. rnorsitans cuticle proteins was comparable to the values (pI 3-6) reported for various other insects, including Hyalophora cecropia (Cox and Willis, 1985), Locusta
Fig. 8. Electrophoresis of 15-day-old pupal cuticle proteins. Separation of the proteins was as described in Fig. 1.
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Fig. 9. Electrophoresis of abdominal cuticle proteins from adult. Separation of the proteins was as described in Fig. 1.
migratoria adults (Andersen and Hojrup, 1987), M. sexta larvae (Wolfgang and Riddiford, 1986), D. melanogaster (Chihara et al., 1982) and L. cuprina
(Hackman, 1980). Two-dimensional gel electrophoresis has been previously used to analyse cuticle proteins in many insects. For example, temporal changes in the proteins have been reported during larval-to-pupal transformation in the tobacco hornworm, Manduca sexta (Kiely and Riddiford, 1985a,b). Similarly, in Drosophila, different cuticular proteins have been shown to be present in each of four different stages, the first two larval instars, the third, the pupa and the adult (Chihara et al., 1982). Stage-specific cuticular protein banding patterns have been reported for the three larval stages in H. cecropia (Willis et al., 1981). In G. m. morsitans, the cuticle proteins from the first and second instar larvae were almost identical except for a few minor differences. Each subsequent stage (third instar larvae, pupa and adult) had a unique set of cuticle proteins. The cuticle proteins showed major differential deposition patterns during the third larval instar. For example, proteins 16-21 were deposited in relatively large amounts in the early third instar. Stage-specific proteins have also been observed in many other insects including Anthraea polyphemus (Sridhara, 1983), M. sexta (Kiely and Riddiford, 1985a,b), T. molitor (Lemoine and Delachambre, 1986), Locusta migratoria (Andersen and Hojrup, 1987) and L. cuprina (SkeUy and Howells, 1988). The differences in the cuticle proteins are likely to be related to the different functions of the various cuticles during insect development. A major
function of the cuticle is to protect the insect from the external environment. In G. m. morsitans, both the first and second instar larval stages are spent within the uterus of the mother. Consequently, the cuticle proteins from these instars were found to be almost identical since they were in a moist environment. Similarly, the first two days of third instar are spent in the same environment before larviposition occurs on third day. The most striking change in the electrophoretic profile of larval cuticle proteins occurs during the third instar. The larvae leave the moist environment within the uterus and enter a more dessicating environment. Coincident with this is a dramatic increase in the levels of a series of proteins (Mr ~ 30 kDa) within the cuticle. These proteins are likely to have a direct protective role for the larvae in the new environment and formation of the puparium. Within a short time after larviposition, the larva sclerotizes to form the puparium. This process involves reorientation of the cuticle components, the addition of cross-links between the components, and a decrease in their solubility (Lipke et al., 1983). There were no major differences observed between the cuticle proteins from late third instar larvae and brown pupa. By the brown pupal stage, apolysis has not yet occurred and still there is a very high chance of larval cuticle being present. By the fifth day pupal stage, there is a very dramatic decrease in the low molecular weight proteins (Mr ~ 30kDa). By this stage, apolysis has taken place and it is possible to dissect out pupal cuticle. The disappearance of the low molecular weight proteins by the fifth day pupal stage further supports the likelihood of the proteins
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being involved in protection and formation of the puparium. By this stage, the puparium is already formed and gives protection to the developing pupa. The pupal cuticle does not provide protection as it is itself enclosed at all times within the protective puparium. However, the cuticle must be sufficiently strong to withstand the hydrostatic pressure created by muscular contraction during head eversion (Fristrom, 1965). In dipterans, the pupal cuticle remains unsclerotized. Analysis of the cuticle proteins of L. cuprina (Skelly and Howells, 1988) and D. melanogaster (Chihara et al., 1982) showed that the patterns remained relatively constant during pupal development. Similarly, in G. m. morsitans, the patterns of proteins extracted during pupal development was the same. However, in insects where the pupal cuticle becomes sclerotized, for example, T. molitor and Galleria mellonella, the patterns of proteins change markedly during development (Srivastava, 1970; Roberts and Willis, 1980a,b). In G. m. morsitans, the third instar larvae undergo a behavioural change from a feeding to a wandering phase, a process which coincides with the appearance of specific and prominent new proteins in the cuticle. One may speculate that the mechanisms controlling both events are related, for example through the release or activation of hormones. Further work will be needed to examine the roles played by individual proteins during development and to establish whether the proteins are the products of one or several gene families. Acknowledgements--This work was supported by Funds
from the University of Nairobi. The authors also thank the Director of the ICIPE for constant encouragement during the course of this work.
REFERENCES
Andersen S. O. and Hojrup P. (1987) Extractable proteins from abdominal cuticle of sexually mature locusts, Locusta migratoria. Insect Biochem. 17, 45-51. Andersen S. O. (1979) Biochemistryof insect cuticle. A. Rev. Ent. 24, 29-61. Buxton P. A. (1955) The Natural History of Tsetse Flies. London School of Hygiene and Tropical Medicine, Memoir No. 10, H. K. Lewis, London. Chihara C. J., Silvert D. J. and Fristrom J. W. (1982) The cuticle protein of Drosophila melanogaster: stage specificity. Devl Biol. 89, 379-388. Cox D. L. and Willis J. H. (1985) The cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages. Insect Biochem. 15, 349-362. Fristrom J. W. (1965) Development of the morphological mutant cryptocephal of Drosophila melanogaster. Genetics 52, 299-318. Hackman R. H. and Goldberg M. (1971) Studies on the
hardening and darkening of insect cuticles. J. Insect Physiol. 17, 335-347.
Hackman R. H. (1980) Biochemicalmethods (proteins) In Cuticle Techniques in Arthropods (Edited by Miller T. A.), Chapt. 4, p. 151. Springer, New York. Jones P. P. (1980) Analysis of radiolabeled lymphocyte proteins by one and two-dimensional polyacrylamide gel electrophoresis. Selected Methods in Cellular Immunology (Edited by Mishell B. B. and Shiigi S. M.), pp. 398-440. W. H. Freeman, San Francisco. Kiely M. L. and Riddiford L. M. (1985a) Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation of Manduca sexta. Wilhelm Roux's Archs Devl Biol. 194, 325-335. Kiely M. L. and Riddiford L. M. (1985b)Temporal patterns of protein synthesis in Manduca epidermis during the change to pupal commitment in vitro: their modulation by 20-hydroxyecdysone and juvenile hormone. Wilhelm Roux's Archs Devl Biol. 194, 336-343. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-683. Lemoine A. and Delachambre J. (1986) A water soluble protein specific to the adult cuticle in Tenebrio. Insect Biochem. 16, 483-489. Lipke H., Sugumaran M. and Menzel W. (1983) Mechanism of sclerotization in dipterans. Adv. Insect Physiol. 17, 1-84.
O'Farrell P. Z., Goodman H. M. and O'Farrell P. H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1142. Richards A. G. (1978) The chemistry of insect cuticle. In Biochemistry o f Insects (Edited by Rockstein M.), pp. 205-232. Academic Press, New York. Roberts P. E. and Willis J. H. (1980a) The cuticular proteins of Tenebrio molitor. I. Electrophoretic banding patterns during post-embryonic development. Devl Biol. 75, 59-69. Roberts P. E. and WillisJ. H. (1980b)The cuticular proteins of Tenebrio molitor. 2. Patterns of synthesis during postembryonic development. Devl Biol. 75, 70-77. Silvert D. J. (1985) Cuticular proteins during postembryonic development. In Comprehensive Insect Physiology Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. L.), Vol. 2, pp. 239-254. Pergamon Press, New York. Skelly P. J. and Howells A. J. (1988) The cuticular proteins of Lucilia cuprina: stage specificity and immunological relatedness. Insect Biochem. 18, 237-247. Sridhara S. (1983) Cuticular proteins of the silkmoth, Antheraea polyphemus. Insect Biochem. 13, 665-675. Srivastava R. P. (1970) Electrophoretic behaviour of cuticular proteins of different developmental stages of Galleria mellonella. J. Insect Physiol. 16, 2345-2351. Stiles B. and Leopold R. A. (1990) Cuticle proteins from the Anthonomus grandis abdomen: stage specificity and immunological relatedness. Insect Biochem. 20, 113-125. Willis J. H., Regier J. C. and Debrunner B. A. (1981) The metamorphosis of arthropodin. In Current Topics in Insect Endocrinology and Nutrition (Edited by Bhaskaran G., Friedman S. and Rodriguez J.), pp. 27-46. Plenum Press, New York. Willis J. H. (1987) Cuticular proteins: the neglected components. Archs Insect Biochem. Physiol. 6, 203-215. Wolfgang W. J. and Riddiford L. M. (1986) Larval cuticular morphogenesisin the tobacco hornworm, Manduca sexta, and its hormonal regulation. Devl Biol. 113, 305-316.
0305-0491/93 $6.00+ 0.00 © 1993Pergamon Press Ltd
Printed in Great Britain
TEMPORAL SYNTHESIS OF CUTICLE PROTEINS D U R I N G LARVAL DEVELOPMENT IN G L O S S I N A M O R S I T A N S VINCENT O. OCHIENG,* ELLIE O. OSIR,~':~JAMESO. OCHANDA* and NORAH K. OLEMBO* *Department of Biochemistry, University of Nairobi, P.O. Box 30197, Nairobi, Kenya (Fax 803-360); and tThe International Centre of Insect Physiology and Ecology (ICIPE), P. O. Box 30772, Nairobi, Kenya (Received 21 October 1992; accepted 27 November 1992)
Abstract--l. Larval development in Glossina species occurs in utero with the mature third instar larva being deposited after a developmental period of 7 days. 2. In this study, the patterns of cuticular protein synthesis during larval development were analysed by two-dimensional gel electrophoresis. 3. From the results, four types of cuticle proteins were identified: those specific to larval, pupal and adult cuticles, and others common to all the stages. 4. Few cutieular proteins were synthesized between the first and second larval instars. By the third larval instar (two days before larviposition), a large number of proteins (Mr ~<30 kDa) were induced. These proteins persisted up to the brown pupal stage and showed a rapid decline thereafter. Most of the proteins with molecular weights Mr ~<30 kDa were undeteetable at apolysis (5 days after larviposition). 5. By day 15 of the pupal stage, the number of cuticle proteins was very small. The protein profile during the pupal stages remained relatively constant. This was probably due to the fact that the pupal cuticle does not provide any protection since it is itself enclosed at all times within the protective puparium.
INTRODUCTION The insect cuticle functions both as an exoskeleton and a barrier between the living tissue and the environment. Furthermore, the cuticle determines the shape and appearance of insects. Proteins are a major component of the cuticle constituting approximately 50% of the dry weight (Richards, 1978). The main factors that contribute to the mechanical properties of cuticle are organization of chitin microfibrils, length of chitin microfibrils, chitin-protein interactions, protein conformation and protein-protein interactions (Andersen, 1979). The properties of the various highly specialized cuticles are due mainly to protein-protein interactions, which can be influenced by subtle changes in the surrounding medium (Andersen, 1979). Proteins provide a great deal of diversity to the cuticle composition. It seems likely that they define much of the specialized nature of the cuticle both in different anatomical regions and at different metamorphic stages (Silvert, 1985; Willis, 1987). Following ecdysis, electrophoretic banding patterns from cuticular extracts change, both quantitatively and qualitatively, due to sclerotization and the synthesis and secretion of new proteins (Roberts and Willis, 1980a,b; Chihara et al., 1982) Proteins extracted from the cuticles of holometabolous insects at different developmental stages ~To whom correspondence should be addressed. Abbreviations--pI: isoelectric point; IEF: isoelectric focus-
ing; SDS: sodium dodecyl sulphate; PAGE: polyacrylamide gel electrophoresis.
usually differ, as determined electrophoretically or by amino acid composition analysis (Srivastava, 1970; Roberts and Willis, 1980a,b; Chihara et al., 1982; Sridhara, 1983). The tsetse fly and other viviparous insects have a unique mode of reproduction. The larva completes its entire development within the uterus of the female and is then larviposited as a mature third instar larva. Each pregnancy cycle, lasting from 9 to 10 days, culminates in the production of a single fully grown third instar larva which burrows into the soil and forms a puparium within a few hours of larviposition (Buxton, 1955). In this study cuticle proteins of different developmental stages of G. m. morsitans are examined by two-dimensional gel electrophoresis.
MATERIALSAND METHODS Experimental insects
Tsetse flies, Glossina morsitans morsitans Westwood, were supplied by the Insect and Animal Breeding Unit of the International Centre of Insect Physiology and Ecology. They were reared on a 12 light: 12 dark photoperiod at 70-80% relative humidity and 25°C. The insects were fed daily on rabbit ears. Pregnant flies were dissected to obtain first and second instar larvae. Third instar larvae (2 days before larviposition) were forcibly expelled from the uterus by applying slight pressure on the female abdomen. Some flies were left to larviposit normally
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and the pupae selected at different stages. The rest of the pupae were kept until adult emergence. Preparation o f cuticles
Whole cuticles were dissected from larval, pupal and adult stages in tsetse Ringer solution (Kiely and Riddiford, 1985a,b). Larvae were anaesthetized in water and placed on a microscope slide. After removing the polyneustic lobes from the anterior region using a scalpel, the larvae were opened and the viscera removed. The integument was then scraped in tsetse Ringer solution from the body wall musculature. The pupal cuticle separates from puparium 5 days after white puparium formation. For adults, only the abdominal cuticles were used. In this case, cuticles were freed of contaminating tissue under a dissecting microscope using a previously described procedure (Hackman and Goldberg, 1971). The dissected cuticles were rinsed three times in tsetse Ringer solution, blotted dry on a filter paper and stored at - 7 0 ° C until analysis. Extraction o f cuticle proteins
The extraction of cuticle proteins was carried out as previously described (Kiely and Riddiford, 1985 a,b). Briefly, samples were homogenized at 4°C using 100 pl/insect of 6.25 mM Tris-HC1, pH 6.8, 1% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co., St Louis, MO). The protein content of extracts was determined using the bicinchoninic acid assay (BCA; Pierce, Rockford, IL). Bovine serum albumin (Fraction V; Calbiochem, San Diego, CA) was used as the protein standard. Gel electrophoresis
Two-dimensional gel electrophoresis was carried out according to O'Farrell et al. (1977) as modified by Jones (1980). Protein samples were separated by isoelectric focusing (IEF) gels in the first dimension using a 4:1 ratio of pH 5-7 and pH 3-10 ampholines (LKB, Pharmacia, Bromma, Sweden). The gels (11 cm) were cast in Pyrex tubes (140 x 30 mm, i.d.). The samples were prepared in IEF sample buffer (9.5 M urea, 2% (w/v) NP-40, 2% ampholines composed of 1.6%, pH range 5-7, and 0.4%, pH range 3-10, and 5% fl-mercaptoethanol). The cathode and anode solutions were 10mM NaOH and 20mM H3PO4, respectively. The gels were prefocused according to the following schedule for each gel: (a) 20 V for 25 min; (b) 30 V for 30 min; (c) 40 V for 30 min. The extruded gels were placed in 1 ml of equilibrium soaking solution (O'Farrell, 1977). After shaking gently for 30 min at room temperature, the gels were kept at - 70°C until used. For the second dimension, the SDS-polyacrylamide gel electrophoresis (SDSPAGE) was used (Laemmli, 1970). The running gel consisted of 8.5% polyacrylamide overlaid with 3% stacking gel. Individual proteins were resolved as discrete spots that were visualized after silver staining (O'Farrell et al., 1977).
RESULTS In order to ensure that the cuticles used in this study were free of contaminating tissue and haemolymph, the cuticles were thoroughly cleaned and then rinsed several times. Examination under a dissecting microscope was carried out to ascertain that the cuticles were clean. Furthermore, the haemolymph protein bands were different from those of the cuticle proteins. Initial attempts to separate the cuticular proteins were carried out by the nonequilibrium pH gradient gel (NEPHGE) system which involved the use of pH 3.5-10 ampholines in the first dimension. This system gave a poor separation since approximately two-thirds of the protein spots could not be seen. However, the use of isoelectric focusing, instead of NEPHGE in the first dimension, gave a good resolution of the proteins. Using this system, very striking changes were revealed in the patterns of cuticle proteins during larval development. The most interesting of these was the disappearance of some polypeptides as well as the appearance of others as the larva developed into an adult. From these results, four types of cuticle proteins were identified, namely, those specific to larval, pupal and adult cuticles, and others common to the stages. The first and second instar larvae exhibited the same protein profiles except for proteins 4, 15 and 16, which appeared by day 1 (second instar) and disappeared by day 5 (pupal stage) (Figs 1 and 2). By the third larval instar stage (2 days before larviposition), a large number of proteins appeared (Fig. 3). For example, a group of acidic proteins (16-20 and 26) with molecular weights of less than Mr ~ 30 kDa appeared in large amounts, persisted up to the brown pupal stage and thereafter showed a rapid decline, becoming undetectable at apolysis (5 days after larviposition) (Fig. 6). Protein 18, present in early third instar larvae, was not visible by the late third instar. Similarly, protein 19 which, in early third instar, was present in large amounts, was found only in trace amounts in late third instar, and became undetectable by the brown pupal stage. Proteins 5-6 and 9 appeared by early third instar larvae and persisted up to the adult stage. A number of proteins (20-30 and 42) with molecular weights of M r ~ 4 5 - 6 6 k D a , appeared by early third instar. These proteins increased in amounts by late third instar larvae (larviposition) and attained the highest amounts by the brown pupal stage. The proteins then decreased in amounts as development proceeded, and became undetectable by the adult stage (Figs 2-5). Proteins 22, 24, 31 and 34 appeared by late third instar larvae and lasted only up to the brown pupal stage (Figs 3-5). Decreased amounts of protein 17 were found in late third instar larval cuticle compared to that of early third instar, in which large amounts were present (Figs 3 and 4). A protein 23 appeared by late third instar and persisted up to the adult stage.
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Fig. 1. Electrophoresis of first instar cuticle proteins. Protein samples were separated in the first dimension by IEF gel electrophoresis with a 4: I mixture of ampholines, pH 5-7 and 3.5-10, respectively, and the second dimension by SDS-PAGE as described under Materials and Methods. The gel was stained for protein using silver.
Seven proteins (4, 17, 18-21, 34) were only found during the larval stages. Figures 6-8 showed proteins from different pupal stages. Proteins 36-38 ( M r s 3 1 ) and 40
(Mr ~ 45 kDa) appeared by day 5 of the pupal stage. The protein profiles during the pupal stage remained relatively constant. By the fifteenth day of the pupal stage, the number of cuticle proteins present was very
Fig. 2. Electrophoresis of second instar cuticle protein. Separation of the proteins was as described in Fig. 1.
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Fig. 3. Electrophoresis of third instar cuticle protein (aborted 2 days before larviposition). Separation of the proteins was as described in Fig. 1.
small. Five proteins (37-41) were found to be specific to the pupal stages. A series of proteins, relatively a b u n d a n t in early pharate adult cuticle extracts, were found in dimin-
ished amounts in eclosed adult cuticle extracts. A cluster of proteins 47-50 (M r ~ 37 kDa) and a protein 52 (M r ~ 43 kDa) relatively prominent, appeared in the cuticle of newly emerged adults (Fig. 9).
Fig. 4. Electrophoresis of third instar cuticle protein (larviposited). Separation of the proteins was as described in Fig. 1.
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Fig. 5. Electrophoresis of brown pupal cuticle protein. Separation of the proteins was as described in Fig. 1. DISCUSSION The results presented in this paper that distinct changes occur in the types amounts of protein synthesized by G. epidermis during the larval-to-adult
clearly show as well as the rn. morsitans
development.
The proteins could be classified into four groups: those specific to the larval, pupal and adult cuticles, and those that were c o m m o n to all the stages. The major larval cuticle proteins from G. m. morsitans were in the molecular weight range of Mr ~ 20-70 kDa. The range is close to the one
Fig. 6. Electrophoresis of 5-day-old pupal cuticle protein. Separation of the proteins was as described in Fig. 1. CBPB 105/2--H
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Fig. 7. Electrophoresis of 10-day-old pupal cuticle protein. Separation of the proteins was as described in Fig. 1. observed in the boll weevil larval cuticle proteins with the molecular weights ranging from 17 to 66 kDa (Stiles and Leopold, 1990). These proteins had molecular weights higher than M ~ ~ 3 0 k D a unlike L. cuprina and D. melanogaster in which the larval cuticle proteins had molecular weight range of
13-30 kDa (Skelly and Howells, 1988; Chihara et al., 1982). The pI range (4-6) of the G. m. rnorsitans cuticle proteins was comparable to the values (pI 3-6) reported for various other insects, including Hyalophora cecropia (Cox and Willis, 1985), Locusta
Fig. 8. Electrophoresis of 15-day-old pupal cuticle proteins. Separation of the proteins was as described in Fig. 1.
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Fig. 9. Electrophoresis of abdominal cuticle proteins from adult. Separation of the proteins was as described in Fig. 1.
migratoria adults (Andersen and Hojrup, 1987), M. sexta larvae (Wolfgang and Riddiford, 1986), D. melanogaster (Chihara et al., 1982) and L. cuprina
(Hackman, 1980). Two-dimensional gel electrophoresis has been previously used to analyse cuticle proteins in many insects. For example, temporal changes in the proteins have been reported during larval-to-pupal transformation in the tobacco hornworm, Manduca sexta (Kiely and Riddiford, 1985a,b). Similarly, in Drosophila, different cuticular proteins have been shown to be present in each of four different stages, the first two larval instars, the third, the pupa and the adult (Chihara et al., 1982). Stage-specific cuticular protein banding patterns have been reported for the three larval stages in H. cecropia (Willis et al., 1981). In G. m. morsitans, the cuticle proteins from the first and second instar larvae were almost identical except for a few minor differences. Each subsequent stage (third instar larvae, pupa and adult) had a unique set of cuticle proteins. The cuticle proteins showed major differential deposition patterns during the third larval instar. For example, proteins 16-21 were deposited in relatively large amounts in the early third instar. Stage-specific proteins have also been observed in many other insects including Anthraea polyphemus (Sridhara, 1983), M. sexta (Kiely and Riddiford, 1985a,b), T. molitor (Lemoine and Delachambre, 1986), Locusta migratoria (Andersen and Hojrup, 1987) and L. cuprina (SkeUy and Howells, 1988). The differences in the cuticle proteins are likely to be related to the different functions of the various cuticles during insect development. A major
function of the cuticle is to protect the insect from the external environment. In G. m. morsitans, both the first and second instar larval stages are spent within the uterus of the mother. Consequently, the cuticle proteins from these instars were found to be almost identical since they were in a moist environment. Similarly, the first two days of third instar are spent in the same environment before larviposition occurs on third day. The most striking change in the electrophoretic profile of larval cuticle proteins occurs during the third instar. The larvae leave the moist environment within the uterus and enter a more dessicating environment. Coincident with this is a dramatic increase in the levels of a series of proteins (Mr ~ 30 kDa) within the cuticle. These proteins are likely to have a direct protective role for the larvae in the new environment and formation of the puparium. Within a short time after larviposition, the larva sclerotizes to form the puparium. This process involves reorientation of the cuticle components, the addition of cross-links between the components, and a decrease in their solubility (Lipke et al., 1983). There were no major differences observed between the cuticle proteins from late third instar larvae and brown pupa. By the brown pupal stage, apolysis has not yet occurred and still there is a very high chance of larval cuticle being present. By the fifth day pupal stage, there is a very dramatic decrease in the low molecular weight proteins (Mr ~ 30kDa). By this stage, apolysis has taken place and it is possible to dissect out pupal cuticle. The disappearance of the low molecular weight proteins by the fifth day pupal stage further supports the likelihood of the proteins
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being involved in protection and formation of the puparium. By this stage, the puparium is already formed and gives protection to the developing pupa. The pupal cuticle does not provide protection as it is itself enclosed at all times within the protective puparium. However, the cuticle must be sufficiently strong to withstand the hydrostatic pressure created by muscular contraction during head eversion (Fristrom, 1965). In dipterans, the pupal cuticle remains unsclerotized. Analysis of the cuticle proteins of L. cuprina (Skelly and Howells, 1988) and D. melanogaster (Chihara et al., 1982) showed that the patterns remained relatively constant during pupal development. Similarly, in G. m. morsitans, the patterns of proteins extracted during pupal development was the same. However, in insects where the pupal cuticle becomes sclerotized, for example, T. molitor and Galleria mellonella, the patterns of proteins change markedly during development (Srivastava, 1970; Roberts and Willis, 1980a,b). In G. m. morsitans, the third instar larvae undergo a behavioural change from a feeding to a wandering phase, a process which coincides with the appearance of specific and prominent new proteins in the cuticle. One may speculate that the mechanisms controlling both events are related, for example through the release or activation of hormones. Further work will be needed to examine the roles played by individual proteins during development and to establish whether the proteins are the products of one or several gene families. Acknowledgements--This work was supported by Funds
from the University of Nairobi. The authors also thank the Director of the ICIPE for constant encouragement during the course of this work.
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