Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria

J. Insect .Physiol., 1973,

Vol. 19,pp. 1603to 1614. Pergamon Press.

Printed in Great Britain

COMPARISON BETWEEN THE SCLEROTIZATION OF ADULT AND LARVAL CUTICLE IN SCHISTOCERCA GREGARIA SVEND OLAV ANDERSEN Zoophysiological Laboratory C, August Krogh University of Copenhagen, Denmark (Received

9 February

Institute,

1973)

Abstract-Femur cuticle from fifth instar larvae of the desert locust, Schistoccrca gregaria, has been characterized with respect to composition, rate of deposition, and rate of sclerotization. The results are compared with those from adult cuticle of the same species. The protein compositions of the two types of cuticle are very similar, but the rates of deposition of both protein and chitin are different. The main difference is, however, that sclerotization is restricted tcg the first day after ecdysis in larval cuticle, whereas in adult cuticle sclerotizatic>n continues for at least a couple of weeks. The result is that the endocuticle remains untanned in the larvae, whereas in the adults the whole cuticle becomes tanned. INTRODUCTION

years I have been investigating the sclerotization of insect cuticle, and I have mainly used adults of the desert locust, Schistocercagregaria, as experimental animal:;. It was observed that the sclerotization of the cuticle, which starts soon after ecdysis and transforms the soft, pre-ecdysial cuticle into hard exocuticle, goes on for at least 2 weeks, so that also the cuticle deposited after ecdysis (endocuticle) becomes sclerotized and insoluble (ANDERSENand BARRETT, 1971). Both layers Iof the cuticle are thus chemically stabilized, and apparently the same chemical reaction is involved in both. A characteristic difference between adult cuticle and larval cuticle is that the former is not dissolved and reutilized later but lasts for the remaining part of the animal’s life, whereas larval cuticle is a temporary structure: it is formed to last for a short while, as it is partly redissolved and partly discarded at the next ecdysis. Therefore, it appeared possible that the results obtained for the stabilization of adult cuticle need not be valid for larval cuticle. An investigation of the development of cuticle during the fifth larval instar was therefore begun, and the results are reported below together with a discussion of the differences and similarities between the two types of cuticle. FOR SOME

MATERIALS

AND METHODS

Locusts were reared in the laboratory, and during their growth they were exposed to a diurnal rhythm of 14 hr night condition (darkness and room 1603

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SVENDOLAVANDERSEN

temperature, cu. 23°C) alternating with 10 hr day conditions where the cages were heated by a 60 W light-bulb, giving a temperature of 35°C in the centre of the cages. When an animal was preparing for ecdysis from the fourth to the fifth larval stage, it was kept under continuous observation, the exact time of emergence from the old cuticle was noted, and the animal was marked with coloured spots of paint time intervals on the pronotum to make it recognizable later. At predetermined the marked animals were killed by freezing and stored at - 18°C for later analysis. All analyses were performed on femur cuticle, because this cuticle is easy to clean for cells and adhering tissue and most of the determinations on adult cuticle The samples were cleaned manually in 1% have been performed on femurs. potassium tetraborate solutions (ANDERSEN, 1971a). They were then washed briefly in distilled water and dried at 70°C to constant weight. The procedures for extracting the cuticle and for determining the amount of cross-links have been described earlier (ANDERSEN and BARRETT, 1971). Determination of the activity of the cross-linking enzyme was performed as described by ANDERSEN (1972) with a single modification : N-acetyldopamine labelled with tritium only in the /3-position was used as substrate. It was synthesized by treating 3,4-dihydroxyphenylethyl-2-sH-amine, obtained from New England Nuclear, with acetic anhydride as described earlier (ANDERSEN, 1972). Proteins were obtained from unsclerotized femurs by extraction with 1% sodium dodecylsulphate (SDS) overnight at room temperature, and separated by means of analytical polyacrylamide electrophoresis in SDS- solution according to the method of WEBER and OSBORN (1969) and stained with Coomassie blue. For amino acid analysis cleaned femurs were hydrolysed in sealed, evacuated tubes for 20 hr at 110°C. The analyses of adult cuticle were performed in the Department of Zoology, Cambridge, on a Technicon Auto-Analyzer, and the analyses of larval cuticle were performed in this laboratory on a BioCal BC 201 amino acid analyser. RESULTS Under the conditions we use for rearing locusts, the fifth larval instar lasts about 10 days. Cuticle from animals of well-defined age was obtained for the first 9 days of the instar, and exuviae were used as a sample for day 10. Growth of cuticle Figure 1 shows how the femur cuticle increases in weight during the first 5 days of the instar due to deposition of endocuticle and how, after a short period of constant weight (days 5 to 7), it decreases in weight (days 7 to 10) due to dissolution of the cuticle by the moulting fluid during the preparation for the next apolysis. The amounts of protein and chitin present in the cuticle were determined by weighing the residue left after heating the samples in 2 N NaOH at 100°C for 2 hr and assuming that this treatment will dissolve all protein and leave the

OF CUTICLE IN SCHISTOCERCA

SCLEROTIZATION

GREGARIA

1605

chitin. Figure 1 shows that both protein and chitin gradually increase in amount during the first 5 days and that they decrease during the last 3 days of the instar. The ratio between protein and chitin is not constant during the period; the protein content constitutes 76 per cent of the dry weight of the cuticle of newly emerged animals, drops to about 58 per cent in the mid-instar period, and increases again to nearly 70 per cent in the exuviae.

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FIG. 1. Changes in dry weight and the amounts of protein and chitin during development of femur cuticle in fifth instar larvae of S. gregaria. O0, Dry x , mg protein per femur; 0 -0, mg chitin per femur ; weight of cuticle; x + -+ , percentage protein in cuticle. Day 0, Moment of ecdysis from fourth to fifth instar. Day 10, Exuviae from next ecdysis, which occurred between day 9 and day 10. The values are average values based on four measurements from each age group. Protein solubility The fraction of cuticular protein which can be extracted into dilute acetic acid changes also during the instar (Fig. 2). There is a drastic decrease in solubility during the first few hours after ecdysis, from 87 per cent immediately after ecdysis to 32 per cent 8 hr later, then an increase in solubility to about 50 per cent during the first: 5 days, which is followed by another decrease during the period of dissolution of the cuticle. Degree of cross-linking The protein solubility must depend upon the degree of sclerotization, which can be determined from the amount of ketocatechols formed by acid hydrolysis under s#tandard conditions (ANDERSEN and BARRETT, 1971). The results of the analyses are shown in Fig. 3. When the amounts of ketocatechols, which can be obtained per mg cuticle, are plotted against the age of the cuticle, a rapid increase

SVENDOLAVANDERSEN

1606

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FIG. 2. Solubility of cuticular proteins during development of fifth instar larvae 0, adult cuticle. Larval cuticle; Oand adults of S. gregaria. 0 -0, Cleaned femur cuticles were extracted overnight in 5 ml N acetic acid at room temperature and the weight loss was determined. The material removed was assumed to be protein, and from the original dry weight of the sample and the total content of protein the percentage of protein brought into solution was calculated.

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FIG. 3. Amounts of neutral ketocatechols which can be obtained from fifth instar

X-X, pmole femur cuticle on hydrolysis in 1 N HCI for 3 hr at 100°C. 0, pmole ketocatechol from cuticle of one ketocatechol per mg cuticle; Ofemur.

SCLEROTIZATION OF CUTICLEIN SCHISZ’OCERCA

GREGARIA

1607

is seen to occur during the first day, followed by a gradual decrease extending over most ef the instar, and immediately before ecdysis a considerable increase occurs again. If, however, the amount of ketocatechols, which can be isolated from a single femur, is plotted against age, the results appear simpler: an increase takes place during the first day, and thereafter the amount of ketocatechols stays nearly constant for the rest of the instar (Fig. 3). There is no significant difference between the amounts of ketocatechols which can be isolated from a femur taken from the exuvium and from a femur from an animal in mid-instar. Amounf of enzyme The initial increase in the amount of cross-links is significantly more rapid in larval than in adult cuticle: O-043 pmole ketocatechol is formed per mg larval cuticle during the first 3 hr as compared to 0=005 pmole per mg adult cuticle during the same period (ANDERSEN and BARRETT, 1971). This difference can be due either to a difference in the amount of cross-linking enzyme in the two types of cuticle or to a difference in the availability of precursors for the cross-links. The activity of the enzyme in both cuticles was therefore determined by following the release of tritium from N-acetyldopamine labelled with tritium in the /Iposition, when this compound was incubated with samples of cuticle. The results are given in Table 1. When the amount of enzyme is calculated per mg cuticle, TABLE 1-ENZYME ACTIVITYIN UNHARDENED FEMURCUTICLEFROMLARVAEANDADULTSOF S. gregaria Fifth instar larva Dry weight of cuticbe Area of external surface Radioactivity released per mg cuticle Radioactivity released per mm2 cuticle

Adult

0.871 + 0.052 mg

1.957 f 0.037 mg

90.4 + 5.1 mm2

142.3 + 2-6 mm2

5830 + 218 counts/min per mg

3259 + 237 counts/mm per mg

55.7 + 2-O counts/min per mm2

44.5 + 1.8 counts/mm per mm2

Cleaned femur cuticle from 0 hr old animals was incubated at room temperature for 6 hr in 2.2 ml O-2 M sodium acetate buffer, pH 5.3, containing N-acetyldoFlamine-2-3H (about lo5 counts/n& per ml). The supernatant was then pas.sed through a small column (0.5 cm2 x 1 cm) of activated charcoal, and the amount of tritium which was not retained by the column was measured (~DERSEN, 1972). Five samples of both types of cuticle were measured, and the results are given as the average + S.D.

the larval cuticle contains nearly twice as much enzyme as that from the adults, but since adult cuticle is thicker than larval, there is only little difference in the amount of enzyme present per unit surface area of cuticle.

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SVEND

OLAVANDERSEN

Electrophoresis The main part of the proteins present in unhardened cuticle can be brought into solution by extraction with neutral solutions of SDS, and this makes an electrophoretic separation of the proteins possible. When SDS extracts of unhardened femur cuticle from either fifth instar larvae or from adults were separated by polyacrylamide electrophoresis, a separation as shown in Fig. 4 was obtained. Both extracts contain a number of different proteins (at least 14 bands can be counted), and there are only minor differences between the two cuticles. All the major bands are present in both samples, although they do not appear to have the same relative strength when compared visually. Only among the slowly migrating bands, which correspond to the proteins with the largest molecular weights, are some qualitative differences to be seen. Amino acid analysis Table 2 gives the amino acid composition of both larval and adult cuticle. Again it is the similarities which are noticeable and the small differences are hardly significant. TABLE

~-AMINO

ACID

COMPOSITION AMINO

OF FEMUR

CUTICLE

FROM

Fifth instar larva Age : Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine The

,!i'. gregaria.

(RESIDUES

OF

ACIDS PER 100 RESIDUES).

0 hr 4.03 2.63 4.80 3.53 10.55 8.29 32.78 8.18 Nil Nil 3.20 4.59 9.31 Trace l-57 3.07 3.48

values are average values

5 days 3.80 2.79 4.98 3.75 9.99 8.16 32.26 8.48 Nil Nil 2.92 5.13 10.30 Trace 1.40 3.30 2.76

of four analyses

Adult 0 hr 3.98 2.37 4.67 3-23 11.10 7.99 35.05 8.55 Nil Nil 3.83 4.57 6.28 0.87 1.65 2.15 3.72

of each type of cuticle.

DISCUSSION

Hard cuticle obtained from fifth instar locusts appears to have many properties in common with adult hard cuticle, but some differences have been found, which

FIG 4. Pa llyacrylamide

electrophoresis of SDS-soluble proteins from uInhardened cuti tzle frc )rn femurs of S. gregaria. a, Adult cuticle; b, fifth instar IarT
SCLEROTIZATION

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will be discussed in the following section. It must be emphasized that all the measurements have been performed on femur cuticle and the results need neither be valid for cuticle from other parts of the same animals nor for cuticle from other insect species. Polyacrylamide gel electrophoresis shows that a number of proteins can be extracted with SDS from both larval and adult unhardened cuticle (the preecdysial cuticle, which when hardened forms the exocuticle) and that all the major proteins are common to both types of cuticle. There are a few differences among the high molecular weight proteins, but these proteins are minor components in the extracts. Amino acid analysis of whole, unextracted samples of larval and adult cuticle shows that the overall amino acid composition is nearly identical in the two types of cuticle and that the composition does not change during deposition of the endocuticle. This indicates that the proteins forming the endocuticle are of the epidermal cells the same as those deposited before ecdysis. The programme for syrrthesizing and depositing proteins remains thus virtually unchanged, both while the animals are changing from the larval part of their life cycle to the adult part, and while switching from the formation of exocuticle to the formation of endocuticle. This is in contrast to the results we have obtained from the beetle, Tenebrio molitor, where larval and pupal cuticle are of identical composition, but strikingly different from adult cuticle (ANDERSEN et al., 1973). Moreover, in the adult stage, in contrast to the two earlier stages, exo- and endocuticle of Tenebrio have d:.fferent amino acid composition. The relationship between amino acid composition and the properties of various insect cuticles is as yet poorly understood. A discussion of the relationship between amino acid composition and cuticular hardness in S. gregariu has been attempted (ANDERSEN, 1971b), but a more detailed investigation is needed. We can, however, conclude that differences in properties between larval and adult cuticle in S. gregaria cannot be explained by differences in protein composition. Adu.lt locust cuticle grows continuously in thickness by the regular deposition of chitin and protein, and the newly deposited proteins become gradually sclerotized and insoluble (ANDERSEN and BARRETT,1971). In the larval cuticle, which is a temporary structure, there is an initially rapid deposition of protein and chitin which gradually slows down and stops completely after 5 days, at which time the weight of the cuticle has been increased to four times that at ecdysis. After a few more days the weight starts to decrease due to the dissolving action of the moulting fluid. The percentage of chitin increases from 24 per cent at ecdysis to above 40 per cent in the mid-instar period, indicating that the relative amounts of protein and chitin secreted from the epidermal cells varies with the age of the animals, or, to say it in another way, the deposition of proteins in the cuticle slows down more rapidly than the deposition of chitin. From the results shown in Fig. 1 it can be calculated that the cuticle deposited from day 2 to day 6, which corresponds approximately to the inner half of the endocuticle, contains about 55% chitin, whereas the outer half of the endocuticle, which is deposited during the first day after ecdysis, contains 38% chitin.

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SVENDOLAVANDER.SEN

MALEK (1958) reported that the innermost lamellae of the endocuticle of larvae of S. greguria give rise to the ecdysial membrane, which is formed before apolysis due to the resistance of this part of the endocuticle towards the enzymes in the moulting fluid. He also reported that the membrane is chitinous and impregnated with a lipoprotein complex which makes it resistant towards digestion. This membrane may correspond to the part of the endocuticle which is formed while some deposition of chitin still continues and protein deposition has nearly stopped. The dissolution of the cuticle during the last 3 days of the instar involves enzymatic removal of both protein and chitin, which is in agreement with investigations of the composition and action of the moulting fluid in other insect species (PASSONNEAUand WILLIAMS, 1953). The weight of the exuviae is close to the weight of the cuticle on day 0, indicating that nearly all endocuticle becomes digested by the moulting fluid. The changes observed in extractability of the cuticular proteins during development of the fifth instar larvae indicate that sclerotization of the cuticle takes place The initial phase, lasting for about 8 hr, during which the shortly after ecdysis. solubility of the proteins decreases drastically due to their sclerotization, is followed by a period lasting several days where the relative solubility increases again. This period corresponds to the interval where the main part of the endocuticle becomes deposited, and the increase in solubility can therefore be explained by the addition of fresh proteins, which are not (or only to a minor extent) rendered insoluble by sclerotization. During the period of dissolution of the cuticle (day 7 to day 10) the solubility of the proteins in the cuticle decreases again and reaches a very low level in the exuviae. The results agree with the generally accepted idea that it is the unsclerotized part of the cuticle which is dissolved by the moulting fluid, whereas the sclerotized exocuticle is resistant (LOCKE, 1964). This idea was confirmed by determination of the amount of cross-links present in the cuticular proteins at various stages of development of the fifth instar larvae. The determination is based on the assumption that the ketocatechols, which are liberated from the cuticle on acid hydrolysis, are derived from the cross-links formed during sclerotization (ANDERSEN and BARRETT, 1971). Cuticle obtained immediately after ecdysis did not give any ketocatechols at all, whereas cuticle only 1 hr old did give significant amounts. A maximum yield of ketocatechols was obtained from 1 day old cuticle, while during the following days a steady decrease was observed in the yields which could be obtained per mg cuticle. This decrease can be explained by the assumption that cross-links are only formed during the first day after ecdysis and that the continuing deposition of protein during endocuticle formation simply brings the relative amount of cross-links down. The increased yield observed in exuviae should therefore be due to the removal of the proteins which do not contain cross-links. The interpretation is supported by calculating the total amount of ketocatechols obtainable from one femur. This amount is roughly constant from day 1 to the shedding of the exuvium.

SCLEROTIZATION

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These results are in contrast to those obtained for cuticle from adult locusts where it was found that sclerotization goes on for at least 12 days after ecdysis (ANDERsEN and BARRETT, 1971). The initial rates of formation of cross-links are also different for the two types of cuticle: from 3 hr old larval cuticle about eight ti:mes as much ketocatechol can be obtained as from the same amount of 3 hr old adult cuticle. The difference can be due either to a difference in the amounts of cross-linking enzyme present (ANDERSEN, 1972) or to a difference in the avalability of N-acetyldopamine, the precursor for the cross-links (KARLSON and SEIZRIS, 1962; ANDERSEN, 1971a). So far it has not been possible to determine the rate at which N-acetyldopamine reaches the enzyme in the cuticle, but a determination of the amount of enzyme present in 0 hr old cuticle showed that larval cuticle contains slightly more enzyme per unit surface area than does adult cuticle, while there is nearly twice as much enzyme per mg larval cuticle as per mg adult cuticle. The difference in enzyme content in the two types of cuticle is presumably not enough to account for the difference in cross-linking rate, and it can be suggested that there must also be more substrate available in larval cuticle for the enzyme to act upon. The haemocytes play an essential role in the formation of N-acetyldopamine from tyrosine, and the rate-limiting step in the transformation, which is controlled by the .tanning hormone, bursicon, appears to be the uptake of tyrosine into the haemocytes (WHITEHEAD, 1969; MILLS and WHITEHEAD, 1970; POST, 1972). According to this scheme sclerotization can first start when bursicon is released from the central nervous system after ecdysis, as no N-acetyldopamine is formed before the uptake of tyrosine into the haemocytes is stimulated. Bursicon has been reported to influence the deposition of endocuticle in flies (FOGAL and FRAENKEL, 1969) and in the locust Locusta migratoriu (VINCENT, 1971). In L. migratmiu bursicon activity is rapidly removed from the haemolymph: 1 to 1.5 hr after ecdysis the hormone concentration has dropped to such a low level that its presence cannot be demonstrated any longer (VINCENT, 1972). If these results are also valid for S. gregaria, there is a discrepancy between the duration of the period in which bursicon can be demonstrated in the haemolymph and of the periods during which the cellular activities stimulated by bursicon continue. In the larvae the sclerotization goes on for about a day and endocuticle deposition goes on for 4 to 5 days, while in adults both processes last for a couple of weeks, at least. Does this mean that the cells can take up the hormone, so that it cannot be demonstrated in the haemolymph any longer, but is still available for the activation of the cells ? The gradual disappearance of the induced activities could then be due to metabolic degradation of the cell-bound hormone, a degradation which would have to be much slower in adults than in larvae. One observation, which it is difficult to reconcile with the assumption that the synthesis of N-acetyldopamine and therefore sclerotization is controlled by bursicon, is the finding that labelled DOPA, when it is injected into fifth stage larvae a few days before ecdysis, becomes incorporated into the old cuticle (KARLSON and SCHLOSSBERGER-RAEcKE, 1962), and after hydrolysis of the exuvium the

1614

activity can be recovered

SVENDOLAV ANDERSEN

in the neutral ketocatechols

(ANDERSEN and BARRETT,

1971). Before ecdysis there should be no bursicon present, and it is difficult explain why incorporation of the sclerotizing compound should take place.

to

REFERENCES ANDERSENS. 0. (197la) Phenolic compounds isolated from insect hard cuticle and their relationship to the sclerotization process. Insect Biochem. 1, 157-170. ANDERSEN S. 0. (1971b) Resilin. In Comprehensive Biochemistry (Ed. by FLORKINM. and STOTZE. H.) 26C, 633-657. Elsevier, Amsterdam. ANDERSEN S. 0. (1972) An enzyme from locust cuticle involved in the formation of crosslinks from N-acetyldopamine. r. Insect Physiol. 18, 527-540. ANDERSEN S. 0. and BARRETTF. M. (1971) The isolation of ketocatechols from insect cuticle and their possible role in sclerotization. J. Insect Physiol. 17, 69-83. ANDERSEN S. O., CHASEA. M., and WILLIS J. H. (1973) The amino acid composition of cuticles from Tenebrio molitor with special reference to the action of juvenile hormone. Insect Biochem. In press. FOGALW. and FRAENKEL G. (1969) The rBle of bursicon in melanization and endocuticle formation in the adult fleshfly, Sarcophaga bullata. J. Insect Physiol. 15, 1235-1247. KARLSONP. and SCHLOSSBERGER-RAECKE I. (1962) 2 urn Tyrosinstoffwechsel der InsektenVIII. Die Sklerotisierung der Cuticula bei der Wildform und der Albinomutante von Schistocerca gregaria Forsk. J. Insect Physiol. 8, 441-452. KARLSONP. and SEKERISC. E. (1962) N-acetyldopamine as the sclerotizing agent of the insect cuticle. Nature, Lond. 195, 183-I 84. LOCKEM. (1964) The structure and formation of the integument in insects. In The Physiology of Insects (Ed. by ROCKSTEINM.) 3, 379-470. Academic Press, New York. MALEK S. R. A. (1958) The origin and nature of the ecdysial membrane in Schistocerca gregaria (Forskll). J. Insect Physiol. 2, 298-312. MILLS R. R. and WHITEHEADD. L. (1970) Hormonal control of tanning in the American cockroach: changes in blood cell permeability during ecdysis. r. Insect Physiol. 16, 331-340. PASSONNEAU J. V. and WILLIAMSC. M. (1953) The moulting fluid of the Cecropia silkworm. r. exp. Biol. 30, 545-560. POST L. C. (1972) Bursicon: its effect on tyrosine permeation into insect haemocytes. Biochim. biophys. Acta 290, 424-428. VINCENTJ. F. V. (1971) Effects of but-&con on cuticular properties in Locusta migratoria migvatorioides. J. Insect Physiol. 17, 625-636. VINCENTJ. F. V. (1972) The dynamics of release and the possible identity of bursicon in Locusta migratoria migratorioides. J. Insect Physiol. 18, 757-780. WEBERE. and OSBORNM. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412. WHITEHEADD. L. (1969) New evidence for the control mechanism of sclerotization in insects. Nature, Lond. 224, 721-723.