- Email: [email protected]
Cuticular sclerotization in larval and adult locusts, Schistocerca gregaria
J. Insect Physiol., 1974, Vet. 20, pp. 1537 to 1552. Pergamon Press. Printed in Great Britain
CUTICULAR SCLEROTIZATION IN LARVAL AND ADULT LOCUSTS, SCHISTOCERCA GREGARIA SVEND
OLAV
ANDERSEN
Zoophysiological Laboratory C, August Krogh Institute, The University of Copenhagen, 13, Universitetsparken, Copenhagen, Denmark (Received 18 January
1974)
Abstract-The sclerotization of both larval and adult cuticle from the desert locust, Schistocerca gregaria, has been studied by measuring the incorporation of radioactive dopamine and N-acetyldopamine into the cuticle. The results are compared with the degree of sclerotization of the cuticle and the amount of sclerotizing enzyme present. The various parts of the cuticle differ considerably with respect to the degree of sclerotization: in adult locusts the mandibles and the dorsal mesothoracic cuticle contain about twenty times as much cross-linking material per mg cuticle than is present in the abdominal tergites and sclerites. The degree of sclerotization in the various types of cuticle is apparently not determined by the amounts of sclerotizing enzyme present, and the rate at which radioactive dopamine or N-acetyldopamine is incorporated into the cuticle appears also to be unrelated to the amount of enzyme. The degree of sclerotization of the various parts of the cuticle from fifth instar larvae corresponds with the amounts of labelled dopamine which are incorporated during the first day after ecdysis, whereas there is no correlation between sclerotization and the amounts of labelled dopamine which are incorporated in older larvae. The degree of sclerotization of adult cuticle after 1 day corresponds to the incorporation of dopamine during the first day. When older animals are compared only little correlation is observed. The relative rates of sclerotization in the various parts of the cuticle must therefore change as the adult insect grows older. The changes in the incorporation pattern during the development of the locust are discussed in relation to the physiological control of the sclerotization process.
INTRODUCTION
ALTHOUGHthe sclerotization of insect cuticle has been studied for many years (review by HACKMAN,1971), we are still rather ignorant of many aspects of the process. We have recently investigated some of the chemical aspects of the sclerotization of the cuticle of the desert locust. We have studied the structure of the cross-links (ANDERSEN,1971; ANDERSENand BARRETT, 1971) and the properties of the cuticular enzyme which is involved in the formation of the crosslinks (ANDERSEN,1972). During these investigations it was found that sclerotization of adult cuticle continues uninterrupted for weeks with the result that both exo- and endocuticle become sclerotized, whereas in fifth instar larvae sclerotization 1537
1538
%TCND
OLAV~DERSEN
only occurs during the day after emergence and the cuticle deposited thereafter does not become sclerotized (ANDERSEN,1973). Since the activity of the crosslinking enzyme in the cuticle does not change in the course of an instar (ANDERSEN, 1972, and unpublished) the enzyme cannot be the factor which is responsible for bringing the sclerotization process to a halt, and the cause must be sought somewhere else. The mechanical properties of the various parts of locust cuticle appear to differ considerably, ranging from the soft and pliable cuticle in the intersegmental membranes to the very hard cuticle of the hindlegs and mandibles, and although these differences may partly be due to differences in composition and molecular architecture they may also be due to differences in the degree of sclerotization. The purpose of the present publication is to determine the degree of sclerotization of various types of locust cuticle, and to investigate whether the degree of sclerotization is determined mainly by the amount of enzyme present in the cuticle or whether the regulating factor is located somewhere else. The ability of the cuticle to form cross-links at various stages of development has been measured by injecting locusts with radioactive precursors of the cross-links and determining the degree of incorporation into the cuticle. The results indicate that the control of the degree of sclerotization is located in the epidermal cells.
MATERIALS AND METHODS Desert locusts, Schistocerca gregaria, were reared in cages in the laboratory under the conditions described earlier (ANDERSEN,1973). They were kept under regular observation, and immediately after emergence as either fifth instar larvae or as adults they were marked with coloured spots to indicate the time of emergence, so that their exact age at the time of injection would be known. Dopamine, generally labelled with tritium, was obtained from NEN-Radiochemicals, Germany. Before injection it was diluted 1 : 10 with distilled water and 5 ~1 of this dilution was injected into each animal by means of an Agla micro burette. The injection needle was introduced through the intersegmental membrane between the first and second abdominal sclerite and was pushed about 1 cm into the animal. Occasionally there was slight bleeding after withdrawal of the needle and the blood was then carefully washed away from the surface of the animal. Tritium-labelled N-acetyldopamine was synthesized from generally labelled dopamine by treatment with acetic anhydride in 10% potassium tetraborate, and the separation of the reaction products on BioGel P-2 was performed as described earlier (ANDER~EN,1971). After injection the animals were left in a cage under standard conditions for 24 hr and were then killed by freezing at - 18°C at which temperature they were stored until analysis. To prepare the cuticle for analysis an animal was divided into the appropriate parts, the internal organs were removed, and the pieces of cuticle were soaked in This treatment softens the remaining muscles and 1y0 potassium tetraborate.
CUTICULAR SCLEROTIZATIONIN
SCHISTOCERCA
1539
epidermis and thereby makes it easier to remove them. It also removes all watersoluble low molecular weight compounds, whereas both the sclerotized and nonsclerotized proteins in the cuticle are unaffected (ANDERSEN, unpublished). As soon as the tissues had softened enough to be easily removed, the pieces of cuticle were carefully cleaned by means of fine forceps and scalpels until no traces of cell remnants could be seen in the microscope. The cuticle was then washed in distilled water and dried to constant weight in an oven at 100°C. Twelve types of cuticle were used for the standard analyses : tibia and femur from the hindlegs, mandibles, head capsule with the cuticle of the compound eyes and the mouth-parts removed, pronotum, forewings, hindwings (it was not possible to remove the cells and haemolymph from the veins of the adult wings), dorsal, ventral, and lateral plates of the mesothorax, and dorsal and ventral abdominal cuticle (including the intersegmental membranes). The amount of tritium which had become incorporated into the various samples of cuticle was measured after burning the samples in a Packard Tri-Carb Sample Oxidizer, where the tritiated water which is formed during the combustion is collected in scintillation vials and mixed with scintillation liquid. The total amounts of ketocatechols which could be released from unlabelled cuticular samples were determined by hydrolysing the samples by reflux for 3 hr in 100 ml 1 N hydrochloric acid, whereafter the hydrolysates were taken to dryness, redissolved in 3 ml 0.2 M acetic acid, and fractionated on a column of BioGel P-2 (1.2 x 90 cm). Elution was performed with O-2 M acetic acid at a rate of 18 ml/hr. The extinction at 280 nm was measured for the fractions which contained the neutral ketocatechols, and the amount of ketocatechols present was calculated by means of the previously determined molar extinction coefficient of 8200 (ANDERSEN and BARRETT, 1971). To determine the activity of the cross-linking enzyme samples of cuticle were incubated together with N-acetyldopamine which was labelled with tritium at the Bcarbon atom of the side-chain. The amount of tritium which 1 mg cuticle could release from the substrate during 16 hr was used as a measure for the relative enzyme activity (ANDE~EN, 1972, 1973). RESULTS
Degree of sclerotixation
We have never obtained evidence for the presence of other types of cross-links in locust cuticle other than those which on acid hydrolysis give rise to ketocatechols, and it appears reasonable to use the amount of ketocatechols as a measure of the degree of cuticular sclerotization. Table 1 gives the amounts of ketocatechols which were obtained from various types of cuticle from locusts at different stages of development. Ketocatechols could be isolated from all types of cuticle which were investigated, but the amounts which were obtained varied considerably and depended both upon the age of the animal and the part of the body from which the sample was taken. Even cornea from the compound eyes and intersegmental
1540
SVEND OLAVANDERSEN
membranes from the abdomen of adult locusts gave significant amounts of ketocatechols although both types of cuticle are generally considered to be nonsclerotized. The only type of cuticle which we have found to give no ketocatechols on hydrolysis is soft, pre-ecdysial cuticle obtained from locusts caught immediately before or during emergence from the exuviae. TABLE I-AMOUNTS
OF KETOCATECHOLS OBTAINED FROM VARIOUS TYPES OF LOCUST CUTICLE Fifth instar larvae, between 5 and 6 days
Adults Less than 1 day
lo-12
days
Tibia Femur Head capsule Mandibles Pronotum Dorsal mesothorax Lateral mesothorax Ventral mesothorax Dorsal abdomen Ventral abdomen Forewings Hindwings Cornea from compound eyes Abdominal intersegmental membranes Abdominal sclerites Ribs from mesothorax
1.71 0.99 0.81 3.74 0.39 0.75 o-49 0.37 0.24 0.49 1.29 1.21 n.d. n.d. n.d. n.d.
0.61 0.61 0.32 0.77 0.09 1.83 0.67 0.07 0.08 0.03 0.38 0.29 n.d. n.d. n.d. n.d.
2.41 2.65 3.03 3.36 2.46 5.25 2.66 1.51 0.33 0.27 1.97 2.02 0.22 0.20 0.38 4.47
n.d., No determinations. The results are expressed as a percentage of the dry weight of the samples.
The types of cuticle which appear to be most heavily sclerotized are mandibles from both larvae and adults and dorsal thorax and ribs from the lateral thoracic wall from adults. This indicates that the parts of the cuticle where mechanical strength is most needed become most sclerotized. The various parts of the cuticle from young adults are, as expected, much less sclerotized than the corresponding parts from more mature adults, and most of the samples from mid-instar larvae significance are sclerotized to an intermediate degree. This may be of functional as the larval cuticle will only last for a few days before most of it is redissolved in preparation for the next ecdysis. Amounts of enzyme In adult locusts the amount of cross-linking material per mg cuticle is about twenty times higher in the dorsal thorax than in the abdominal cuticle, and this difference could be due to a corresponding difference in the amount of cross-linking enzyme in the cuticle. However, the results shown in Fig. 1 demonstrate that this is not the case. The amount of enzyme present per mg cuticle varies between the
CUTICULAR
SCLEROTIZATION
IN SCHISTOCERCA
1541
different types of cuticle but not nearly as much as the variation in the degree of cross-linking, and it is not the cuticle richest in enzyme which becomes most heavily sclerotized. This indicates that the amount of enzyme present in the cuticle is not an important factor in determining the degree of sclerotization and that the control must be located elsewhere.
H
M
OT
LT
VT
OA
VA
P
FW
HW
FIG. 1. Activity of cross-linking enzyme in samples of cuticle from fifth instar larvae (open bars) and adults (cross-hatched bars). The samples were obtained from animals which had nearly finished ecdysis. After it had been cleaned the cuticle was incubated for 16 hr at room temperature in 2.5 ml O-2 M sodium acetate buffer, pH 5.3, containing N-acetyldopamine-/3-8H (14 x lo6 counts/min). The amounts of tritium which were released from the substrate during the incubation period were determined and used as a measure for the enzyme activity. T, Tibia; F, femur; H, head capsule; M, mandibles; DT, dorsal thorax; LT, lateral thorax; VT, ventral thorax; DA, dorsal abdomen; VA, ventral abdomen; P, pronotum; FW, forewings; HW, hindwings. The results are the average of two determinations.
Incorporation of tritiated dopamine into adults Experiments were then performed where animals of various ages were given a single injection of a radioactively labelled precursor for the cross-links whereafter the extent of incorporation into the various types of cuticle was determined. In the first series of experiments adult locusts, either quite young (2-6 hr after ecdysis) or mature (14 days after ecdysis), were injected with tritiated dopamine and killed 24 hr later; the various samples of cuticle were then cleaned and the incorporated amounts of tritium were determined. Fig. 2 shows the amount of radioactivity which became incorporated per mg cuticle in the various cuticular samples in both age groups. In the young locusts the dorsal thorax and the mandibles contain the highest concentrations of radioactivity; and the abdominal
1542
SVEND
OLAV
DT
LT
iiNDERSEZN
i/T
DA
VA
,P
FW
HW
FIG. 2. Incorporation of labelled dopamine into selected samples of adult locust cuticle. The animals were injected with tritiated dopamine (about lo5 counts/min per animal) either 2 to 6 hr after ecdysis (open bars) or 14 days after ecdysis (crosshatched bars). The animals were killed 24 hr later and the concentration of label in the various parts of the cuticle was determined. The values are the average of three measurements. For explanation of the symbols see legend to Fig. 1.
cuticles contain the lowest concentration; and, according to Table 1, the dorsal thorax and mandibles are the samples richest in cross-links, while the abdominal cuticle is poorest. In Fig. 3 the amounts of radioactivity incorporated per mg cuticle in young adults are plotted against the amounts of cross-links present in the various types of cuticle in animals of the same age. It shows that there is quite good agreement between the amount of activity which the cuticles incorporate and the degree of sclerotization of the cuticles in untreated animals. The only sample which does not fit into the relationship is the mandibles, which incorporate much more activity than expected according to their degree of sclerotization. Fig. 2 shows that the pattern of incorporation changes as the animals mature ; some types of mature cuticle incorporate less activity and other types incorporate more activity than the corresponding samples from young adults. However, the results are complicated by the increase in weight and thickness of the cuticle during the maturation period, so the observed decreases in incorporation may not be real but could be due to an increase in weight of the cuticle. The fraction of the total amount of incorporated label which each cuticular sample takes up is shown in Fig. 4 for both age groups. This demonstrates how the competition between the various types of cuticle for the available dopamine changes with the age of the locusts. The wings incorporate mainly radioactivity during the early period, whereas in mature locusts the abdominal cuticle and parts of the thorax consume relatively more of the available dopamine than they did in the young adults. There is a change with age in the pattern of incorporation of dopamine into the cuticle,
CUTICULAR
1543
IN SCHISTOCERCA
SCLBROTIZATION
and it is mainly the least sclerotized parts of the cuticle which in the older animals have increased their incorporation of dopamine.
lP ,iDA ?VA 0
I 05
I I.0 1.. %
I I.5
1 2.0
ketocatechol
FIG. 3. The relationship between incorporation of tritiated dopamine into cuticle from adult locusts injected 2 to 6 hr after ecdysis and the degree of sclerotization of corresponding samples of cuticle from adults less than 24 hr old. For explanation of the symbols see legend to Fig. 1.
F
H
H
DT
.T
VT
DA
VA
P
FW
FIG. 4. Relative distribution of incorporated tritiated dopamine between samples of adult cuticle. Open bars, locusts 2 to 6 hr old when injected. Cross-hatched bars, locusts 14 days old when injected. The incorporation of activity into each sample is calculated as a percentage of the total incorporation. For explanation of the symbols see legend to Fig. 1. The values are the average of three determinations
Incorporation of tritiated dopamine into fifth instar larvae Fifth instar larvae are also able to incorporate labelled dopamine into their cuticle, and the pattern of incorporation during the first day of the instar corresponds closely to the degree of sclerotization of the various parts of the cuticle in 50
SVENDOLAV ANDERSEN
1544
mature larvae (Fig. 5). The pattern of incorporation of labelled dopamine into the cuticle of fifth instar larvae also changes during the growth of the larvae (Fig. 6). The most striking changes are observed for the mandibles where the relative incorporation decreases from about 30 to about 5 per cent in the course of the
%
ketocotechol
FIG. 5. The relationship between incorporation of tritiated dopamine into cuticle from fifth instar larvae injected 4 to 21 l-u after ecdysis and the degree of sclerotization of corresponding samples of cuticle from fifth instar larvae 5 to 6 days old. For explanation of the symbols see legend to Fig. 1.
F
H
M
DT
LT
VT
DA
VA
P
FW
HW
FIG. 6. Relative distribution of incorporated tritiated dopamine between samples of cuticle from fifth instar larvae. Open bars, larvae 2 to 24 hr old when injected. Cross-hatched bars, larvae 5 to 7 days old when injected. The incorporation of activity into each sample is calculated as a percentage of the total incorporation. The values are the average of four determinations. For explanation of the symbols see legend to Fig. 1.
CUTICULAR SCLZROTUATION
IN SCHISTOCERCA
1545
instar. The dorsal abdominal cuticle during the same period increases its share from about 3 to about 22 per cent. These results show the relative distribution of radioactivity between the various samples of cuticle, and since the total amount of the injected dopamine which becomes incorporated into the cuticle may change during the instar, the results from the two larval groups cannot show any absolute changes in incorporation. To determine in more detail how the incorporation of the precursor varies with the age of the larvae a number of fifth instar larvae of ages covering the whole instar were injected with the same amount of tritiated dopamine and the total amount of radioactivity incorporated into selected samples of cuticle was determined. The results which are shown in Fig. 7 confirm that the relative incorporation of dopamine into the various parts of the cuticle changes during the fifth instar. Most of the selected samples show a gradual decrease in their ability to incorporate the label, but the abdominal cuticle and the pronotum show no significant decrease in incorporation during the instar. For some of the samples two periods of maximal
Age,
hr
FIG. 7. The relationship between age and incorporation of trititaed dopamine into six cuticular samples from fifth instar larvae. The ages are measured from the moment of ecdysis from fourth to fifth instar. The animals were injected with 5 x lo6 counts/min of tritiated dopamine and were killed 24 hr later. The total amount of radioactivity incorporated into each of the samples was determined. The results are the average of two determinations. The straight lines are calculated according to the least squares method. W, Wings; M, mandibles ; A, abdomen; P, pronotum; F, femur; T, tibia.
1546
SVEND0LAvANDERsEN
incorporation can be seen, the first occurs at 80 to 90 hr after ecdysis and the other at about 200 hr, that is about 2 days before the final ecdysis. Relative weight changes of cuticular samples during the fifth
instar
Fig. 8 shows how the relative weights of the cuticular samples which were used in the above-mentioned experiment changed during the instar. It can be seen that during the whole instar the weight of the femur cuticle accounts for a nearly constant fraction of the total weight of all the samples from one animal. The sum of the samples does not represent the complete cuticle although they will account for a considerable part of it, and the weight of the total cuticle can therefore be assumed to change during the instar in the same manner as the weight of the femur cuticle. However, all the various samples of cuticle do not change in parallel: the relative weights of the mandibles decrease gradually from about 14 to about 10 per cent, and the weight of the abdominal cuticle increases from about 22 to 28 per cent. This indicates that the changes in the rate of deposition of endocuticle and the digestion by the moulting fluid are not completely synchronized in all parts of the animal. Radioactivity retained in exuviae
When larvae were injected with labelled dopamine during the last few days of the fifth instar, and were then allowed to live long enough to become adults, it was
L 0
I
I
40
80
I
I
120
I
I
160 200
hr FIG. 8. Relative weight of the cuticular samples used for incorporation
studies in Fig. 7. The weights are calculated as a percentage of the sum of the six cuticular samples from each animal. The straight lines are calculated according to the least squares method. For explanation of the symbols see legend to Fig. 7.
CUTICULAR
SCLRROTIZATION
IN
1547
SCHIS'TOCERCA
observed that part of the radioactivity had become incorporated into the exuviae while another part had become incorporated into the adult cuticle during its sclerotization. This agrees with the findings of KARLSON and SCHLOSSBERGERRAECKE (1962) and ANDEFWN and BARRETT(1971) that when fifth instar locust larvae are injected with radioactive tyrosine or DOPA a few days before ecdysis a significant part of the radioactivity can be recovered from the exuviae. Fig. 9 shows the amount of radioactivity found in the exuviae from locusts which had been injected with tritiated dopamine at different times before the last ecdysis. The incorporation decreases considerably during the last day before ecdysis, and a maximum of incorporation is observed 2 to 3 days before ecdysis. When labelled exuviae are divided into their various parts, and the distribution of label is determined, it is found that the distribution corresponds to that found in the cuticle from old fifth instar larvae which were not allowed to go through ecdysis, whereas the distribution of label in the cuticle of the adults which had been injected as larvae corresponded closely to the distribution in adults which were injected a few hours after ecdysis.
_ -- -__ I
\
,
25-
.
, \
0’
,
20 -
._ _ _
_*’
\
.’
e’--
\ 1 I
%
I
15-
. I
. IO-
5:.
0
I 100
t 80
I 60
I 40
I_ 20
-b--
I 0
hr before ecdysis
FIG. 9. Determination of the amounts of radioactivity retained in the exuviae from animals injected with tritiated dopamine at various times before the ecdysis from fifth instar larvae to adults. The horizontal lines drawn through some of the values indicate the uncertainty in the determination of the moment of ecdysis.
Incorporation of tritiated N-acetyldopamine Experiments have also been performed where both fifth instar larvae and adults were injected with tritiated N-acetyldopamine, whereafter the pattern of incorporation into the cuticle was determined. It was found that the incorporation of N-acetyldopamine follows the same pattern as the incorporation of dopamine: during the fifth instar there is a pronounced drop in the amount of activity which is
1.548
SEND
OLAVANDERSEN
incorporated into mandibles and a corresponding increase in the incorporation into abdominal cuticle (Fig. lo), and in the young adult it is the mandibles, the dorsal and lateral thorax, and the wings which incorporate the main part of the activity.
20
% IO
0 :
T
F
H
M
DT
LT
VT
DA
VA
P
FW
HW
FIG. 10. Relative distribution of labelled N-acetyldoparnine between samples of cuticle from fifth instar larvae. Open bars, larvae injected when 0.5 to 3.5 hr old. Cross-hatched bars, larvae 8 days old. The incorporation of activity into each sample is calculated as a percentage of the total incorporation. The results are the average of two determinations.- For explanation of the symbols see legend to Fig. 1.
DISCUSSION
Degree of sclerotixation When the amounts of ketocatechols formed on acid hydrolysis of cuticle are used as a measure for the amounts of cross-links present (ANDERSEN and BARRETT, 1971), it is found that all parts of the locust cuticle become more or less sclerotized. Even such samples as intersegmental membranes and cornea give small amounts of ketocatechols on hydrolysis although they are generally considered to be nonsclerotized. It has not yet been established where in these soft cuticles the crosslinks are present, but presumably they are located in the epicuticle or maybe in the exocuticle. In contrast to most other types of hard cuticle in locusts the abdominal sclerites and tergites are rather poor in cross-links. It is characteristic that the parts of the cuticle which during the normal life of the animal become most exposed to mechanical forces are also the most heavily sclerotized. The mandibles of both larvae and adults are thus very rich in crosslinks and the cuticle from the hindlegs, which is exposed to strong forces when the animal jumps, is also rich in cross-links in both larvae and adults. Parts of the exoskeleton which are involved in the flight of the adults (wings, dorsal thorax, and parts of the lateral thorax) are highly sclerotized in adult locusts, whereas the corresponding parts of larvae are much less sclerotized.
CUTICULAR
SCLEROTIZATION
IN
SCHISTOCERCA
1549
I know of only a few measurements of the mechanical properties of hard cuticle from locusts, and none of them can be used in an attempt to correlate mechanical strength and degree of sclerotization between the various parts of the locust exoskeleton. That the mechanical strength of locust femur is dependent upon the degree of sclerotization has recently been demonstrated by HEPBURN and JOFFE (1974). They find that the force necessary to produce a given elongation of femur cuticle from Locusta migratoria increases in parallel with the increase in amounts of cross-links. In larval femur cuticle cross-links are formed only during the first day after ecdysis, thereafter the total amount of cross-links per femur stays constant for the rest of the instar although the cuticle grows in thickness for several days and later becomes partly redissolved (ANDERSEN,1973). In adult femur cuticle both the thickness of the cuticle and amount of cross-links per femur increase for at least 12 days after ecdysis (ANDERSN and BARRETT,1971). These earlier investigations were concerned only with femur cuticle and the results were not considered valid for the whole cuticle, but the results reported in this paper indicate that the difference between larval and adult femur cuticle is also valid for the other hard parts of the exoskeleton. The correlation between incorporation of labelled dopamine into the cuticle early in the fifth instar and the total degree of sclerotization of the cuticle immediately before it starts being dissolved by the moulting fluid confirms that cross-links are formed only during the early period of the instar. In the adults there is a reasonably good agreement between early incorporation of labelled dopamine and the degree of sclerotization in very young animals, whereas there is little agreement between incorporation of dopamine and the degree of sclerotization in more mature animals, indicating both that sclerotization continues during the period of endocuticle formation and that it proceeds at different rates in the various parts of the cuticle. The larval endocuticle, in contrast to adult endocuticle, has to be digested by the enzymes in the moulting fluid in preparation for the next ecdysis, and it may be essential for proper digestion of the endocuticle that it does not become sclerotized. In adults the endocuticle is a permanent structure and they can therefore allow themselves to strengthen it by sclerotization. Amount of enzyme The differences in the degree of sclerotization of the various parts of the cuticle could be due to corresponding differences in the amounts of the cross-linking enzyme in the cuticle, or it could be the rate at which the substrate (N-acetyldopamine) gains access to the cross-linking enzyme which determines how fast and how much the cuticle becomes sclerotized. Determinations of the enzyme activity in the various cuticular samples show that there is little, if any, correlation between the amount of enzyme present and the degree of sclerotization which takes place. Neither is there any correlation between the amount of enzyme and the rate of incorporation of labelled dopamine. The mandibles have the lowest content of enzyme activity of all the samples, and they have a very high incorporation
1550
SVENDOLAVANDERSEN
rate in recently ecdysed animals and in mature animals they have a high degree of sclerotization. This indicates that the other cuticular samples must contain a surplus of enzyme activity, and that the concentration of enzyme cannot be the ratelimiting factor. The control of both the rate and degree of sclerotization in the cuticle can therefore be assumed to be located in that part of the system which regulates the access of substrate to the enzyme, and it is presumably the epidermal cells which possess this control. It has not yet been established where the biosynthesis of the substrate, N-acetyldopamine, takes place. MILLS and WHITEHEAD (1970) have shown that in cockroaches the transformation of tyrosine via DOPA (or tyramine) to dopamine occurs in the haemocytes, and they mention that the acetylation of dopamine to N-acetyldopamine could take place either in the haemocytes or in the epidermal cells. Incorporation of labelled dopamine and N-acetyldopamine
According to this scheme, two cell types, haemocytes and epidermal cells, are involved in the sclerotization. The haemocytes transform tyrosine to dopamine and maybe to N-acetyldopamine, and the epidermal cells will take up dopamine (or N-acetyldopamine) from the haemolymph and secrete N-acetyldopamine into the cuticle, where it comes into contact with the sclerotizing enzyme. The uptake of tyrosine into the haemocytes is controlled by the hormone bursicon (WHITEHEAD, 1969; MILLS and WHITEHEAD,1970; POST, 1972). By introducing labelled dopamine or N-acetyldopamine into locusts and following the incorporation of label into the cuticle it should be possible to circumvent part of the biosynthetic pathway and obtain some information on the importance of the local control systems in determining the degree of sclerotization. Both dopamine and N-acetyldopamine will when injected into adult locusts give rise to cuticular cross-links which cannot be distinguished from the natural cross-links (ANDERSEN,1971), and similar results have been obtained for fifth instar larvae. It has been possible by means of limited acid hydrolysis to isolate labelled N-acetylarterenone from larval cuticle into which labelled dopamine had been incorporated, indicating that also in the larvae dopamine becomes N-acetylated before incorporation into the cuticle. Single injections of labelled dopamine into fifth instar larvae show that dopamine can become incorporated into the cuticle during the whole instar, although sclerotization normally occurs only during the first day of the instar. Dopamine can thus be introduced into the metabolic pathway leading to cuticular cross-links at any time during the fifth instar, although the pathway is only available for tyrosine early in the instar. The pattern of incorporation of labelled dopamine during the first day of the instar closely follows the pattern of sclerotization in untreated larvae, whereas incorporation of dopamine during the rest of the instar gradually changes to another pattern, which bears little relationship to the observed sclerotization in untreated animals. Changes in the incorporation of labelled dopamine into the cuticle of adults during their maturation have not been investigated in as much detail as
CUTICULAR
SCLEROTIZATION
IN
SCHISTOCERCA
1551
incorporation into larval cuticle, but the results available indicate that the changes occurring during the two stages are similar. In the adults these changes can be of physiological importance since normal sclerotization ccntinues for a prolonged period with the result that certain parts of the cuticle (such as the mandibles and the wings) become hardened very fast whereas other parts, such as the abdomen and pronotum, only later obtain a significant degree of sclerotization. In the larvae these changes occur during a period where sclerotization does not normally occur, presumably due to the fact that the uptake of tyrosine into the haemocytes is blocked, and the changes in the ability of the cuticle to form cross-links can therefore hardly be of any physiological importance to the larvae. The changing pattern of incorporation of injected dopamine into the larval cuticle is presumably due to some change in the activity of the epidermal cells, and it appears improbable that the change is centrally controlled, since some cuticular parts (wings and mandibles) show a steady decline in their ability to incorporate dopamine, while other parts (pronotal shield and abdomen) show two periods of increased incorporation during the instar. To demonstrate that the changes are locally determined, it will be necessary to perform incorporation experiments on isolated parts of cuticle with intact epidermis. The experiments on whole animals reported here can best be explained by assuming that the programme for the timedependent changes in sclerotization is present in the epidermal cells and that it is the availability of dopamine or N-acetyldopamine which determines whether this programme shall be put into action or not. Changes in cuticular weight It has been shown (ANDERSEN, 1973) that the femur cuticle during the first 5 days of the fifth instar increases its weight to about four times that at ecdysis, and this period is followed by a few days of nearly constant weight, followed by 3 days where the weight decreases due to dissolution of the endocuticle. From the measurements reported in this paper it appears that the various parts of the cuticle do not change their weight completely in parallel : during the instar the weight of the femur cuticle constitutes a constant fraction of the sum of the weights of the cuticle from the tibiae, femurs, wings, abdomen, pronotal shield, and mandibles, and the weight changes of the femur cuticle can therefore presumably be considered as typical for the total cuticle. However, some parts of the cuticle (such as the wings) show a relative decrease in their weight while other parts (abdomen) increase in relative weight. This could indicate that the deposition of endocuticle does not stop simultaneously all over the animal, and that the dissolution of the cuticle does not start at the same time all over the animal. The cuticular sampIes which during the instar decrease in relative weight (mandibles and wings) also show a pronounced decrease in their ability to incorporate labelled dopamine. Similarly, the samples which increase in relative weight during the instar also tend to increase their share of incorporated dopamine. However, so little is known about the metabolism of the epidermal cells that it is impossible to suggest any correlations between the various activities.
1552
S~FZND OLAVANDERSEN CONCLUSIONS
The main result of this study is the demonstration that the sclerotization of locust cuticle must be controlled at two different levels. There is a central control which regulates the access of tyrosine to the biosynthetic pathway leading to Nacetyldopamine, the sclerotization compound. This control is hormonal: soon after ecdysis the hormone bursicon is released from the central nervous system and affects the haemocytes in such a way that their cell membrane becomes permeable to tyrosine, which is then transformed to N-acetyldopamine. Bursicon can in this way determine how much material is available for the sclerotization of the cuticle, but the control of the sclerotization of the various parts of the cuticle must be located in the epidermal cells. Presumably the epidermal cells in the various parts of the body compete with each other and with alternative metabolic pathways for the available N-acetyldopamine (or dopamine), and in some regions the cells will be much more effective in this competition than cells from other regions. During the maturation of the animals the epidermal cells change their ability to transport N-acetyldopamine into the cuticle with the result that in adult locusts, where Nacetyldopamine is being formed continuously, the relative degree of sclerotization of the various parts of the cuticle changes gradually. A change in sclerotization pattern is not observed in larval cuticle, although larval epidermal cells change their incorporation pattern in the same way as adult cells, because N-acetyldopamine is not formed in the larvae after the first day of the instar. Acknowledgements--I thank mag. scient. SIGNE NEDERGAARD for allowing us to use the Packard Tri-Carb Sample Oxidizer and Mrs. KARENKROGHfor skilful technical assistance. REFERENCES ANDERSENS. 0. (1971) Phenolic compounds isolated from insect hard cuticle and their relationship to the sclerotization process. Insect Biochem. 1, 157-170. ANDERSIZN S. 0. (1972) An enzyme from locust cuticle involved in the formation of crosslinks from N-acetyldopamine. J. Insect Physiol. 18, 527-540. ANDERSENS. 0. (1973) Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria. J. Insect Physiol. 19, 1603-1614. ANDER~ENS. 0. and BARREN F. M. (1971) The isolation of ketocatechols from insect cuticle and their possible rBle in sclerotization. J. Insect Physiol. 17, 6943. HACKMAN R. H. (1971) The integument of arthropoda. In ChemicalZoology (Ed. by FLORKIN M. and SCHEERB. T.) 6, l-62. Academic Press, New York. HEPBURN H. R. and JOFFE I. (1974) Hardening of locust sclerites. J. Insect Physiol. 20, 631-635. KARLSONP. and SCHLOSSBERGER-RAECKE I. (1962) Zum Tyrosinstoffwechsel der InsektenVIII. Der Sklerotisierung der Cuticula bei der Wildform und der Albinomutante von Schistocerca gregaria Forsk. J. Insect Physiol. 8, 441-452. MILLS R. R. and WHITEHEADD. L. (1970) H ormonal control of tanning in the American cockroach: changes in blood cell permeability during ecdysis. J. Insect Physiol. 16, 331-340. POST L. C. (1972) Bursicon: its effect on tyrosine permeation into insect haemocytes. Biochim. boiphys. Acta 290, 424-428. WHITEHEADD. L. (1969) New evidence for the control mechanism of sclerotization in insects. Nature, Lond. 224,721-723.
CUTICULAR SCLEROTIZATION IN LARVAL AND ADULT LOCUSTS, SCHISTOCERCA GREGARIA SVEND
OLAV
ANDERSEN
Zoophysiological Laboratory C, August Krogh Institute, The University of Copenhagen, 13, Universitetsparken, Copenhagen, Denmark (Received 18 January
1974)
Abstract-The sclerotization of both larval and adult cuticle from the desert locust, Schistocerca gregaria, has been studied by measuring the incorporation of radioactive dopamine and N-acetyldopamine into the cuticle. The results are compared with the degree of sclerotization of the cuticle and the amount of sclerotizing enzyme present. The various parts of the cuticle differ considerably with respect to the degree of sclerotization: in adult locusts the mandibles and the dorsal mesothoracic cuticle contain about twenty times as much cross-linking material per mg cuticle than is present in the abdominal tergites and sclerites. The degree of sclerotization in the various types of cuticle is apparently not determined by the amounts of sclerotizing enzyme present, and the rate at which radioactive dopamine or N-acetyldopamine is incorporated into the cuticle appears also to be unrelated to the amount of enzyme. The degree of sclerotization of the various parts of the cuticle from fifth instar larvae corresponds with the amounts of labelled dopamine which are incorporated during the first day after ecdysis, whereas there is no correlation between sclerotization and the amounts of labelled dopamine which are incorporated in older larvae. The degree of sclerotization of adult cuticle after 1 day corresponds to the incorporation of dopamine during the first day. When older animals are compared only little correlation is observed. The relative rates of sclerotization in the various parts of the cuticle must therefore change as the adult insect grows older. The changes in the incorporation pattern during the development of the locust are discussed in relation to the physiological control of the sclerotization process.
INTRODUCTION
ALTHOUGHthe sclerotization of insect cuticle has been studied for many years (review by HACKMAN,1971), we are still rather ignorant of many aspects of the process. We have recently investigated some of the chemical aspects of the sclerotization of the cuticle of the desert locust. We have studied the structure of the cross-links (ANDERSEN,1971; ANDERSENand BARRETT, 1971) and the properties of the cuticular enzyme which is involved in the formation of the crosslinks (ANDERSEN,1972). During these investigations it was found that sclerotization of adult cuticle continues uninterrupted for weeks with the result that both exo- and endocuticle become sclerotized, whereas in fifth instar larvae sclerotization 1537
1538
%TCND
OLAV~DERSEN
only occurs during the day after emergence and the cuticle deposited thereafter does not become sclerotized (ANDERSEN,1973). Since the activity of the crosslinking enzyme in the cuticle does not change in the course of an instar (ANDERSEN, 1972, and unpublished) the enzyme cannot be the factor which is responsible for bringing the sclerotization process to a halt, and the cause must be sought somewhere else. The mechanical properties of the various parts of locust cuticle appear to differ considerably, ranging from the soft and pliable cuticle in the intersegmental membranes to the very hard cuticle of the hindlegs and mandibles, and although these differences may partly be due to differences in composition and molecular architecture they may also be due to differences in the degree of sclerotization. The purpose of the present publication is to determine the degree of sclerotization of various types of locust cuticle, and to investigate whether the degree of sclerotization is determined mainly by the amount of enzyme present in the cuticle or whether the regulating factor is located somewhere else. The ability of the cuticle to form cross-links at various stages of development has been measured by injecting locusts with radioactive precursors of the cross-links and determining the degree of incorporation into the cuticle. The results indicate that the control of the degree of sclerotization is located in the epidermal cells.
MATERIALS AND METHODS Desert locusts, Schistocerca gregaria, were reared in cages in the laboratory under the conditions described earlier (ANDERSEN,1973). They were kept under regular observation, and immediately after emergence as either fifth instar larvae or as adults they were marked with coloured spots to indicate the time of emergence, so that their exact age at the time of injection would be known. Dopamine, generally labelled with tritium, was obtained from NEN-Radiochemicals, Germany. Before injection it was diluted 1 : 10 with distilled water and 5 ~1 of this dilution was injected into each animal by means of an Agla micro burette. The injection needle was introduced through the intersegmental membrane between the first and second abdominal sclerite and was pushed about 1 cm into the animal. Occasionally there was slight bleeding after withdrawal of the needle and the blood was then carefully washed away from the surface of the animal. Tritium-labelled N-acetyldopamine was synthesized from generally labelled dopamine by treatment with acetic anhydride in 10% potassium tetraborate, and the separation of the reaction products on BioGel P-2 was performed as described earlier (ANDER~EN,1971). After injection the animals were left in a cage under standard conditions for 24 hr and were then killed by freezing at - 18°C at which temperature they were stored until analysis. To prepare the cuticle for analysis an animal was divided into the appropriate parts, the internal organs were removed, and the pieces of cuticle were soaked in This treatment softens the remaining muscles and 1y0 potassium tetraborate.
CUTICULAR SCLEROTIZATIONIN
SCHISTOCERCA
1539
epidermis and thereby makes it easier to remove them. It also removes all watersoluble low molecular weight compounds, whereas both the sclerotized and nonsclerotized proteins in the cuticle are unaffected (ANDERSEN, unpublished). As soon as the tissues had softened enough to be easily removed, the pieces of cuticle were carefully cleaned by means of fine forceps and scalpels until no traces of cell remnants could be seen in the microscope. The cuticle was then washed in distilled water and dried to constant weight in an oven at 100°C. Twelve types of cuticle were used for the standard analyses : tibia and femur from the hindlegs, mandibles, head capsule with the cuticle of the compound eyes and the mouth-parts removed, pronotum, forewings, hindwings (it was not possible to remove the cells and haemolymph from the veins of the adult wings), dorsal, ventral, and lateral plates of the mesothorax, and dorsal and ventral abdominal cuticle (including the intersegmental membranes). The amount of tritium which had become incorporated into the various samples of cuticle was measured after burning the samples in a Packard Tri-Carb Sample Oxidizer, where the tritiated water which is formed during the combustion is collected in scintillation vials and mixed with scintillation liquid. The total amounts of ketocatechols which could be released from unlabelled cuticular samples were determined by hydrolysing the samples by reflux for 3 hr in 100 ml 1 N hydrochloric acid, whereafter the hydrolysates were taken to dryness, redissolved in 3 ml 0.2 M acetic acid, and fractionated on a column of BioGel P-2 (1.2 x 90 cm). Elution was performed with O-2 M acetic acid at a rate of 18 ml/hr. The extinction at 280 nm was measured for the fractions which contained the neutral ketocatechols, and the amount of ketocatechols present was calculated by means of the previously determined molar extinction coefficient of 8200 (ANDERSEN and BARRETT, 1971). To determine the activity of the cross-linking enzyme samples of cuticle were incubated together with N-acetyldopamine which was labelled with tritium at the Bcarbon atom of the side-chain. The amount of tritium which 1 mg cuticle could release from the substrate during 16 hr was used as a measure for the relative enzyme activity (ANDE~EN, 1972, 1973). RESULTS
Degree of sclerotixation
We have never obtained evidence for the presence of other types of cross-links in locust cuticle other than those which on acid hydrolysis give rise to ketocatechols, and it appears reasonable to use the amount of ketocatechols as a measure of the degree of cuticular sclerotization. Table 1 gives the amounts of ketocatechols which were obtained from various types of cuticle from locusts at different stages of development. Ketocatechols could be isolated from all types of cuticle which were investigated, but the amounts which were obtained varied considerably and depended both upon the age of the animal and the part of the body from which the sample was taken. Even cornea from the compound eyes and intersegmental
1540
SVEND OLAVANDERSEN
membranes from the abdomen of adult locusts gave significant amounts of ketocatechols although both types of cuticle are generally considered to be nonsclerotized. The only type of cuticle which we have found to give no ketocatechols on hydrolysis is soft, pre-ecdysial cuticle obtained from locusts caught immediately before or during emergence from the exuviae. TABLE I-AMOUNTS
OF KETOCATECHOLS OBTAINED FROM VARIOUS TYPES OF LOCUST CUTICLE Fifth instar larvae, between 5 and 6 days
Adults Less than 1 day
lo-12
days
Tibia Femur Head capsule Mandibles Pronotum Dorsal mesothorax Lateral mesothorax Ventral mesothorax Dorsal abdomen Ventral abdomen Forewings Hindwings Cornea from compound eyes Abdominal intersegmental membranes Abdominal sclerites Ribs from mesothorax
1.71 0.99 0.81 3.74 0.39 0.75 o-49 0.37 0.24 0.49 1.29 1.21 n.d. n.d. n.d. n.d.
0.61 0.61 0.32 0.77 0.09 1.83 0.67 0.07 0.08 0.03 0.38 0.29 n.d. n.d. n.d. n.d.
2.41 2.65 3.03 3.36 2.46 5.25 2.66 1.51 0.33 0.27 1.97 2.02 0.22 0.20 0.38 4.47
n.d., No determinations. The results are expressed as a percentage of the dry weight of the samples.
The types of cuticle which appear to be most heavily sclerotized are mandibles from both larvae and adults and dorsal thorax and ribs from the lateral thoracic wall from adults. This indicates that the parts of the cuticle where mechanical strength is most needed become most sclerotized. The various parts of the cuticle from young adults are, as expected, much less sclerotized than the corresponding parts from more mature adults, and most of the samples from mid-instar larvae significance are sclerotized to an intermediate degree. This may be of functional as the larval cuticle will only last for a few days before most of it is redissolved in preparation for the next ecdysis. Amounts of enzyme In adult locusts the amount of cross-linking material per mg cuticle is about twenty times higher in the dorsal thorax than in the abdominal cuticle, and this difference could be due to a corresponding difference in the amount of cross-linking enzyme in the cuticle. However, the results shown in Fig. 1 demonstrate that this is not the case. The amount of enzyme present per mg cuticle varies between the
CUTICULAR
SCLEROTIZATION
IN SCHISTOCERCA
1541
different types of cuticle but not nearly as much as the variation in the degree of cross-linking, and it is not the cuticle richest in enzyme which becomes most heavily sclerotized. This indicates that the amount of enzyme present in the cuticle is not an important factor in determining the degree of sclerotization and that the control must be located elsewhere.
H
M
OT
LT
VT
OA
VA
P
FW
HW
FIG. 1. Activity of cross-linking enzyme in samples of cuticle from fifth instar larvae (open bars) and adults (cross-hatched bars). The samples were obtained from animals which had nearly finished ecdysis. After it had been cleaned the cuticle was incubated for 16 hr at room temperature in 2.5 ml O-2 M sodium acetate buffer, pH 5.3, containing N-acetyldopamine-/3-8H (14 x lo6 counts/min). The amounts of tritium which were released from the substrate during the incubation period were determined and used as a measure for the enzyme activity. T, Tibia; F, femur; H, head capsule; M, mandibles; DT, dorsal thorax; LT, lateral thorax; VT, ventral thorax; DA, dorsal abdomen; VA, ventral abdomen; P, pronotum; FW, forewings; HW, hindwings. The results are the average of two determinations.
Incorporation of tritiated dopamine into adults Experiments were then performed where animals of various ages were given a single injection of a radioactively labelled precursor for the cross-links whereafter the extent of incorporation into the various types of cuticle was determined. In the first series of experiments adult locusts, either quite young (2-6 hr after ecdysis) or mature (14 days after ecdysis), were injected with tritiated dopamine and killed 24 hr later; the various samples of cuticle were then cleaned and the incorporated amounts of tritium were determined. Fig. 2 shows the amount of radioactivity which became incorporated per mg cuticle in the various cuticular samples in both age groups. In the young locusts the dorsal thorax and the mandibles contain the highest concentrations of radioactivity; and the abdominal
1542
SVEND
OLAV
DT
LT
iiNDERSEZN
i/T
DA
VA
,P
FW
HW
FIG. 2. Incorporation of labelled dopamine into selected samples of adult locust cuticle. The animals were injected with tritiated dopamine (about lo5 counts/min per animal) either 2 to 6 hr after ecdysis (open bars) or 14 days after ecdysis (crosshatched bars). The animals were killed 24 hr later and the concentration of label in the various parts of the cuticle was determined. The values are the average of three measurements. For explanation of the symbols see legend to Fig. 1.
cuticles contain the lowest concentration; and, according to Table 1, the dorsal thorax and mandibles are the samples richest in cross-links, while the abdominal cuticle is poorest. In Fig. 3 the amounts of radioactivity incorporated per mg cuticle in young adults are plotted against the amounts of cross-links present in the various types of cuticle in animals of the same age. It shows that there is quite good agreement between the amount of activity which the cuticles incorporate and the degree of sclerotization of the cuticles in untreated animals. The only sample which does not fit into the relationship is the mandibles, which incorporate much more activity than expected according to their degree of sclerotization. Fig. 2 shows that the pattern of incorporation changes as the animals mature ; some types of mature cuticle incorporate less activity and other types incorporate more activity than the corresponding samples from young adults. However, the results are complicated by the increase in weight and thickness of the cuticle during the maturation period, so the observed decreases in incorporation may not be real but could be due to an increase in weight of the cuticle. The fraction of the total amount of incorporated label which each cuticular sample takes up is shown in Fig. 4 for both age groups. This demonstrates how the competition between the various types of cuticle for the available dopamine changes with the age of the locusts. The wings incorporate mainly radioactivity during the early period, whereas in mature locusts the abdominal cuticle and parts of the thorax consume relatively more of the available dopamine than they did in the young adults. There is a change with age in the pattern of incorporation of dopamine into the cuticle,
CUTICULAR
1543
IN SCHISTOCERCA
SCLBROTIZATION
and it is mainly the least sclerotized parts of the cuticle which in the older animals have increased their incorporation of dopamine.
lP ,iDA ?VA 0
I 05
I I.0 1.. %
I I.5
1 2.0
ketocatechol
FIG. 3. The relationship between incorporation of tritiated dopamine into cuticle from adult locusts injected 2 to 6 hr after ecdysis and the degree of sclerotization of corresponding samples of cuticle from adults less than 24 hr old. For explanation of the symbols see legend to Fig. 1.
F
H
H
DT
.T
VT
DA
VA
P
FW
FIG. 4. Relative distribution of incorporated tritiated dopamine between samples of adult cuticle. Open bars, locusts 2 to 6 hr old when injected. Cross-hatched bars, locusts 14 days old when injected. The incorporation of activity into each sample is calculated as a percentage of the total incorporation. For explanation of the symbols see legend to Fig. 1. The values are the average of three determinations
Incorporation of tritiated dopamine into fifth instar larvae Fifth instar larvae are also able to incorporate labelled dopamine into their cuticle, and the pattern of incorporation during the first day of the instar corresponds closely to the degree of sclerotization of the various parts of the cuticle in 50
SVENDOLAV ANDERSEN
1544
mature larvae (Fig. 5). The pattern of incorporation of labelled dopamine into the cuticle of fifth instar larvae also changes during the growth of the larvae (Fig. 6). The most striking changes are observed for the mandibles where the relative incorporation decreases from about 30 to about 5 per cent in the course of the
%
ketocotechol
FIG. 5. The relationship between incorporation of tritiated dopamine into cuticle from fifth instar larvae injected 4 to 21 l-u after ecdysis and the degree of sclerotization of corresponding samples of cuticle from fifth instar larvae 5 to 6 days old. For explanation of the symbols see legend to Fig. 1.
F
H
M
DT
LT
VT
DA
VA
P
FW
HW
FIG. 6. Relative distribution of incorporated tritiated dopamine between samples of cuticle from fifth instar larvae. Open bars, larvae 2 to 24 hr old when injected. Cross-hatched bars, larvae 5 to 7 days old when injected. The incorporation of activity into each sample is calculated as a percentage of the total incorporation. The values are the average of four determinations. For explanation of the symbols see legend to Fig. 1.
CUTICULAR SCLZROTUATION
IN SCHISTOCERCA
1545
instar. The dorsal abdominal cuticle during the same period increases its share from about 3 to about 22 per cent. These results show the relative distribution of radioactivity between the various samples of cuticle, and since the total amount of the injected dopamine which becomes incorporated into the cuticle may change during the instar, the results from the two larval groups cannot show any absolute changes in incorporation. To determine in more detail how the incorporation of the precursor varies with the age of the larvae a number of fifth instar larvae of ages covering the whole instar were injected with the same amount of tritiated dopamine and the total amount of radioactivity incorporated into selected samples of cuticle was determined. The results which are shown in Fig. 7 confirm that the relative incorporation of dopamine into the various parts of the cuticle changes during the fifth instar. Most of the selected samples show a gradual decrease in their ability to incorporate the label, but the abdominal cuticle and the pronotum show no significant decrease in incorporation during the instar. For some of the samples two periods of maximal
Age,
hr
FIG. 7. The relationship between age and incorporation of trititaed dopamine into six cuticular samples from fifth instar larvae. The ages are measured from the moment of ecdysis from fourth to fifth instar. The animals were injected with 5 x lo6 counts/min of tritiated dopamine and were killed 24 hr later. The total amount of radioactivity incorporated into each of the samples was determined. The results are the average of two determinations. The straight lines are calculated according to the least squares method. W, Wings; M, mandibles ; A, abdomen; P, pronotum; F, femur; T, tibia.
1546
SVEND0LAvANDERsEN
incorporation can be seen, the first occurs at 80 to 90 hr after ecdysis and the other at about 200 hr, that is about 2 days before the final ecdysis. Relative weight changes of cuticular samples during the fifth
instar
Fig. 8 shows how the relative weights of the cuticular samples which were used in the above-mentioned experiment changed during the instar. It can be seen that during the whole instar the weight of the femur cuticle accounts for a nearly constant fraction of the total weight of all the samples from one animal. The sum of the samples does not represent the complete cuticle although they will account for a considerable part of it, and the weight of the total cuticle can therefore be assumed to change during the instar in the same manner as the weight of the femur cuticle. However, all the various samples of cuticle do not change in parallel: the relative weights of the mandibles decrease gradually from about 14 to about 10 per cent, and the weight of the abdominal cuticle increases from about 22 to 28 per cent. This indicates that the changes in the rate of deposition of endocuticle and the digestion by the moulting fluid are not completely synchronized in all parts of the animal. Radioactivity retained in exuviae
When larvae were injected with labelled dopamine during the last few days of the fifth instar, and were then allowed to live long enough to become adults, it was
L 0
I
I
40
80
I
I
120
I
I
160 200
hr FIG. 8. Relative weight of the cuticular samples used for incorporation
studies in Fig. 7. The weights are calculated as a percentage of the sum of the six cuticular samples from each animal. The straight lines are calculated according to the least squares method. For explanation of the symbols see legend to Fig. 7.
CUTICULAR
SCLRROTIZATION
IN
1547
SCHIS'TOCERCA
observed that part of the radioactivity had become incorporated into the exuviae while another part had become incorporated into the adult cuticle during its sclerotization. This agrees with the findings of KARLSON and SCHLOSSBERGERRAECKE (1962) and ANDEFWN and BARRETT(1971) that when fifth instar locust larvae are injected with radioactive tyrosine or DOPA a few days before ecdysis a significant part of the radioactivity can be recovered from the exuviae. Fig. 9 shows the amount of radioactivity found in the exuviae from locusts which had been injected with tritiated dopamine at different times before the last ecdysis. The incorporation decreases considerably during the last day before ecdysis, and a maximum of incorporation is observed 2 to 3 days before ecdysis. When labelled exuviae are divided into their various parts, and the distribution of label is determined, it is found that the distribution corresponds to that found in the cuticle from old fifth instar larvae which were not allowed to go through ecdysis, whereas the distribution of label in the cuticle of the adults which had been injected as larvae corresponded closely to the distribution in adults which were injected a few hours after ecdysis.
_ -- -__ I
\
,
25-
.
, \
0’
,
20 -
._ _ _
_*’
\
.’
e’--
\ 1 I
%
I
15-
. I
. IO-
5:.
0
I 100
t 80
I 60
I 40
I_ 20
-b--
I 0
hr before ecdysis
FIG. 9. Determination of the amounts of radioactivity retained in the exuviae from animals injected with tritiated dopamine at various times before the ecdysis from fifth instar larvae to adults. The horizontal lines drawn through some of the values indicate the uncertainty in the determination of the moment of ecdysis.
Incorporation of tritiated N-acetyldopamine Experiments have also been performed where both fifth instar larvae and adults were injected with tritiated N-acetyldopamine, whereafter the pattern of incorporation into the cuticle was determined. It was found that the incorporation of N-acetyldopamine follows the same pattern as the incorporation of dopamine: during the fifth instar there is a pronounced drop in the amount of activity which is
1.548
SEND
OLAVANDERSEN
incorporated into mandibles and a corresponding increase in the incorporation into abdominal cuticle (Fig. lo), and in the young adult it is the mandibles, the dorsal and lateral thorax, and the wings which incorporate the main part of the activity.
20
% IO
0 :
T
F
H
M
DT
LT
VT
DA
VA
P
FW
HW
FIG. 10. Relative distribution of labelled N-acetyldoparnine between samples of cuticle from fifth instar larvae. Open bars, larvae injected when 0.5 to 3.5 hr old. Cross-hatched bars, larvae 8 days old. The incorporation of activity into each sample is calculated as a percentage of the total incorporation. The results are the average of two determinations.- For explanation of the symbols see legend to Fig. 1.
DISCUSSION
Degree of sclerotixation When the amounts of ketocatechols formed on acid hydrolysis of cuticle are used as a measure for the amounts of cross-links present (ANDERSEN and BARRETT, 1971), it is found that all parts of the locust cuticle become more or less sclerotized. Even such samples as intersegmental membranes and cornea give small amounts of ketocatechols on hydrolysis although they are generally considered to be nonsclerotized. It has not yet been established where in these soft cuticles the crosslinks are present, but presumably they are located in the epicuticle or maybe in the exocuticle. In contrast to most other types of hard cuticle in locusts the abdominal sclerites and tergites are rather poor in cross-links. It is characteristic that the parts of the cuticle which during the normal life of the animal become most exposed to mechanical forces are also the most heavily sclerotized. The mandibles of both larvae and adults are thus very rich in crosslinks and the cuticle from the hindlegs, which is exposed to strong forces when the animal jumps, is also rich in cross-links in both larvae and adults. Parts of the exoskeleton which are involved in the flight of the adults (wings, dorsal thorax, and parts of the lateral thorax) are highly sclerotized in adult locusts, whereas the corresponding parts of larvae are much less sclerotized.
CUTICULAR
SCLEROTIZATION
IN
SCHISTOCERCA
1549
I know of only a few measurements of the mechanical properties of hard cuticle from locusts, and none of them can be used in an attempt to correlate mechanical strength and degree of sclerotization between the various parts of the locust exoskeleton. That the mechanical strength of locust femur is dependent upon the degree of sclerotization has recently been demonstrated by HEPBURN and JOFFE (1974). They find that the force necessary to produce a given elongation of femur cuticle from Locusta migratoria increases in parallel with the increase in amounts of cross-links. In larval femur cuticle cross-links are formed only during the first day after ecdysis, thereafter the total amount of cross-links per femur stays constant for the rest of the instar although the cuticle grows in thickness for several days and later becomes partly redissolved (ANDERSEN,1973). In adult femur cuticle both the thickness of the cuticle and amount of cross-links per femur increase for at least 12 days after ecdysis (ANDERSN and BARRETT,1971). These earlier investigations were concerned only with femur cuticle and the results were not considered valid for the whole cuticle, but the results reported in this paper indicate that the difference between larval and adult femur cuticle is also valid for the other hard parts of the exoskeleton. The correlation between incorporation of labelled dopamine into the cuticle early in the fifth instar and the total degree of sclerotization of the cuticle immediately before it starts being dissolved by the moulting fluid confirms that cross-links are formed only during the early period of the instar. In the adults there is a reasonably good agreement between early incorporation of labelled dopamine and the degree of sclerotization in very young animals, whereas there is little agreement between incorporation of dopamine and the degree of sclerotization in more mature animals, indicating both that sclerotization continues during the period of endocuticle formation and that it proceeds at different rates in the various parts of the cuticle. The larval endocuticle, in contrast to adult endocuticle, has to be digested by the enzymes in the moulting fluid in preparation for the next ecdysis, and it may be essential for proper digestion of the endocuticle that it does not become sclerotized. In adults the endocuticle is a permanent structure and they can therefore allow themselves to strengthen it by sclerotization. Amount of enzyme The differences in the degree of sclerotization of the various parts of the cuticle could be due to corresponding differences in the amounts of the cross-linking enzyme in the cuticle, or it could be the rate at which the substrate (N-acetyldopamine) gains access to the cross-linking enzyme which determines how fast and how much the cuticle becomes sclerotized. Determinations of the enzyme activity in the various cuticular samples show that there is little, if any, correlation between the amount of enzyme present and the degree of sclerotization which takes place. Neither is there any correlation between the amount of enzyme and the rate of incorporation of labelled dopamine. The mandibles have the lowest content of enzyme activity of all the samples, and they have a very high incorporation
1550
SVENDOLAVANDERSEN
rate in recently ecdysed animals and in mature animals they have a high degree of sclerotization. This indicates that the other cuticular samples must contain a surplus of enzyme activity, and that the concentration of enzyme cannot be the ratelimiting factor. The control of both the rate and degree of sclerotization in the cuticle can therefore be assumed to be located in that part of the system which regulates the access of substrate to the enzyme, and it is presumably the epidermal cells which possess this control. It has not yet been established where the biosynthesis of the substrate, N-acetyldopamine, takes place. MILLS and WHITEHEAD (1970) have shown that in cockroaches the transformation of tyrosine via DOPA (or tyramine) to dopamine occurs in the haemocytes, and they mention that the acetylation of dopamine to N-acetyldopamine could take place either in the haemocytes or in the epidermal cells. Incorporation of labelled dopamine and N-acetyldopamine
According to this scheme, two cell types, haemocytes and epidermal cells, are involved in the sclerotization. The haemocytes transform tyrosine to dopamine and maybe to N-acetyldopamine, and the epidermal cells will take up dopamine (or N-acetyldopamine) from the haemolymph and secrete N-acetyldopamine into the cuticle, where it comes into contact with the sclerotizing enzyme. The uptake of tyrosine into the haemocytes is controlled by the hormone bursicon (WHITEHEAD, 1969; MILLS and WHITEHEAD,1970; POST, 1972). By introducing labelled dopamine or N-acetyldopamine into locusts and following the incorporation of label into the cuticle it should be possible to circumvent part of the biosynthetic pathway and obtain some information on the importance of the local control systems in determining the degree of sclerotization. Both dopamine and N-acetyldopamine will when injected into adult locusts give rise to cuticular cross-links which cannot be distinguished from the natural cross-links (ANDERSEN,1971), and similar results have been obtained for fifth instar larvae. It has been possible by means of limited acid hydrolysis to isolate labelled N-acetylarterenone from larval cuticle into which labelled dopamine had been incorporated, indicating that also in the larvae dopamine becomes N-acetylated before incorporation into the cuticle. Single injections of labelled dopamine into fifth instar larvae show that dopamine can become incorporated into the cuticle during the whole instar, although sclerotization normally occurs only during the first day of the instar. Dopamine can thus be introduced into the metabolic pathway leading to cuticular cross-links at any time during the fifth instar, although the pathway is only available for tyrosine early in the instar. The pattern of incorporation of labelled dopamine during the first day of the instar closely follows the pattern of sclerotization in untreated larvae, whereas incorporation of dopamine during the rest of the instar gradually changes to another pattern, which bears little relationship to the observed sclerotization in untreated animals. Changes in the incorporation of labelled dopamine into the cuticle of adults during their maturation have not been investigated in as much detail as
CUTICULAR
SCLEROTIZATION
IN
SCHISTOCERCA
1551
incorporation into larval cuticle, but the results available indicate that the changes occurring during the two stages are similar. In the adults these changes can be of physiological importance since normal sclerotization ccntinues for a prolonged period with the result that certain parts of the cuticle (such as the mandibles and the wings) become hardened very fast whereas other parts, such as the abdomen and pronotum, only later obtain a significant degree of sclerotization. In the larvae these changes occur during a period where sclerotization does not normally occur, presumably due to the fact that the uptake of tyrosine into the haemocytes is blocked, and the changes in the ability of the cuticle to form cross-links can therefore hardly be of any physiological importance to the larvae. The changing pattern of incorporation of injected dopamine into the larval cuticle is presumably due to some change in the activity of the epidermal cells, and it appears improbable that the change is centrally controlled, since some cuticular parts (wings and mandibles) show a steady decline in their ability to incorporate dopamine, while other parts (pronotal shield and abdomen) show two periods of increased incorporation during the instar. To demonstrate that the changes are locally determined, it will be necessary to perform incorporation experiments on isolated parts of cuticle with intact epidermis. The experiments on whole animals reported here can best be explained by assuming that the programme for the timedependent changes in sclerotization is present in the epidermal cells and that it is the availability of dopamine or N-acetyldopamine which determines whether this programme shall be put into action or not. Changes in cuticular weight It has been shown (ANDERSEN, 1973) that the femur cuticle during the first 5 days of the fifth instar increases its weight to about four times that at ecdysis, and this period is followed by a few days of nearly constant weight, followed by 3 days where the weight decreases due to dissolution of the endocuticle. From the measurements reported in this paper it appears that the various parts of the cuticle do not change their weight completely in parallel : during the instar the weight of the femur cuticle constitutes a constant fraction of the sum of the weights of the cuticle from the tibiae, femurs, wings, abdomen, pronotal shield, and mandibles, and the weight changes of the femur cuticle can therefore presumably be considered as typical for the total cuticle. However, some parts of the cuticle (such as the wings) show a relative decrease in their weight while other parts (abdomen) increase in relative weight. This could indicate that the deposition of endocuticle does not stop simultaneously all over the animal, and that the dissolution of the cuticle does not start at the same time all over the animal. The cuticular sampIes which during the instar decrease in relative weight (mandibles and wings) also show a pronounced decrease in their ability to incorporate labelled dopamine. Similarly, the samples which increase in relative weight during the instar also tend to increase their share of incorporated dopamine. However, so little is known about the metabolism of the epidermal cells that it is impossible to suggest any correlations between the various activities.
1552
S~FZND OLAVANDERSEN CONCLUSIONS
The main result of this study is the demonstration that the sclerotization of locust cuticle must be controlled at two different levels. There is a central control which regulates the access of tyrosine to the biosynthetic pathway leading to Nacetyldopamine, the sclerotization compound. This control is hormonal: soon after ecdysis the hormone bursicon is released from the central nervous system and affects the haemocytes in such a way that their cell membrane becomes permeable to tyrosine, which is then transformed to N-acetyldopamine. Bursicon can in this way determine how much material is available for the sclerotization of the cuticle, but the control of the sclerotization of the various parts of the cuticle must be located in the epidermal cells. Presumably the epidermal cells in the various parts of the body compete with each other and with alternative metabolic pathways for the available N-acetyldopamine (or dopamine), and in some regions the cells will be much more effective in this competition than cells from other regions. During the maturation of the animals the epidermal cells change their ability to transport N-acetyldopamine into the cuticle with the result that in adult locusts, where Nacetyldopamine is being formed continuously, the relative degree of sclerotization of the various parts of the cuticle changes gradually. A change in sclerotization pattern is not observed in larval cuticle, although larval epidermal cells change their incorporation pattern in the same way as adult cells, because N-acetyldopamine is not formed in the larvae after the first day of the instar. Acknowledgements--I thank mag. scient. SIGNE NEDERGAARD for allowing us to use the Packard Tri-Carb Sample Oxidizer and Mrs. KARENKROGHfor skilful technical assistance. REFERENCES ANDERSENS. 0. (1971) Phenolic compounds isolated from insect hard cuticle and their relationship to the sclerotization process. Insect Biochem. 1, 157-170. ANDERSIZN S. 0. (1972) An enzyme from locust cuticle involved in the formation of crosslinks from N-acetyldopamine. J. Insect Physiol. 18, 527-540. ANDERSENS. 0. (1973) Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria. J. Insect Physiol. 19, 1603-1614. ANDER~ENS. 0. and BARREN F. M. (1971) The isolation of ketocatechols from insect cuticle and their possible rBle in sclerotization. J. Insect Physiol. 17, 6943. HACKMAN R. H. (1971) The integument of arthropoda. In ChemicalZoology (Ed. by FLORKIN M. and SCHEERB. T.) 6, l-62. Academic Press, New York. HEPBURN H. R. and JOFFE I. (1974) Hardening of locust sclerites. J. Insect Physiol. 20, 631-635. KARLSONP. and SCHLOSSBERGER-RAECKE I. (1962) Zum Tyrosinstoffwechsel der InsektenVIII. Der Sklerotisierung der Cuticula bei der Wildform und der Albinomutante von Schistocerca gregaria Forsk. J. Insect Physiol. 8, 441-452. MILLS R. R. and WHITEHEADD. L. (1970) H ormonal control of tanning in the American cockroach: changes in blood cell permeability during ecdysis. J. Insect Physiol. 16, 331-340. POST L. C. (1972) Bursicon: its effect on tyrosine permeation into insect haemocytes. Biochim. boiphys. Acta 290, 424-428. WHITEHEADD. L. (1969) New evidence for the control mechanism of sclerotization in insects. Nature, Lond. 224,721-723.