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Bursicon-mediated control of tanning in melanizing and non-melanizing first instar larvae of Schistocerca gregaria
J. Insect Physiol., 1976, Vol. 22, pp. 1447 to 1452. Pergamon Press. Printed in Great Britain.
BURSICON-MEDIATED CONTROL OF TANNING IN MELANIZING AND NON-MELANIZING FIRST INSTAR LARVAE OF SCHISTOCERCA GREGARIA D. E. PADGHAM Centre for Overseas Pest Research, College House, Wrights Lane, London W8 5SJ, England (Received 15 April 1976)
Abstract-Both melanization and sclerotization in first instar Schistocerca larvae are shown to have a common hormonal control mechanism. This hormone is shown to be analogous to bursicon. the tanning hormone of other insects, by the standard blowfly bioassay. The hormone is identical in both melanizing and non-melanizing first instar larvae of Schistocerca.
INTRODUCTION BURSICON,the tanning
hormone,
was first described
by FRAENKELand HSIAO (1962, 1963, 1965) in the blowfly Sarcophaga bullata and subsequently this and other blowflies have been used as a bioassay for the hormone. FRAJBKFLand HSIAO (1962, 1963) showed that blood from Periplaneta, Tenebrio, Phormia and Sarcophaga induced tanning when injected into neckligatured Calliphora. MILLS et al. (1965) used neckligatured Musca to bioassay the hormone from Periplaneta, whereas VINCENT(1972) used a Phormia bioassay for the blood of adult Locusta and Schistocerca. In addition to these bioassays the hormone was also assayed biochemically. POST(1972) and POST and DE JONG (1973) used its ability to initiate tyrosine metabolism as a measure of activity. In the present paper the relation between the melanization hormone described by PAKXXAM(1976) in first instar larvae of Schistocerca and bursicon is investigated using the standard blowfly bioassay. Although melanization and sclerotization are biochemically distinct (DENNELL,1958), they are considered as having the same humoral control by the hormone bursicon (FOGJZLand FRAENKEL,1969). This relationship between sclerotization and melanization has been investigated by a comparison between the sclerotizing and melanizing potential of blood from newly ecdysed melanizing, green and albino first instar larvae of Schistocerca gregaria. MATERIALS AND METHODS
advantages. Firstly, it provides a check on the tightness of the ligature since a loose ligature will allow the ptilinum to collapse back into the body. Secondly, an expanded ptilinum reduces the blood volume of the thorax and abdomen behind the ligature, thus making the test more sensitive. Finally, the reduced blood volume in the abdomen almost eliminates bleeding after injection. The ligatured blowflies were allowed to stand for 2 hr before being used for the bioassay and any flies showing darkening at this stage were rejected. The flies were then injected with 2 ~1 of blood using glass micropipettes drawn out with an SRI glass electrode puller. They were incubated for three hours at room temperature and then scored on a 0 to 4 scale for darkening analogous to that of VINCENT(1972):(O)No darkening. (1) Slight darkening; 30% of the dorsal surface of the abdomen. (2) Darkening on patches of the thorax or abdomen; 30% of the dorsal surface of the abdomen. (2.5) Abdomen entirely dark, thorax less so. (3) Abdomen and thorax almost totally dark. (3.5) Interference colours present at the tip of the abdomen. (4) Fully dark. The vermijkm larva bioassa) The use of vermiform larvae of Schistocerca for the bioassay of melanizing activity in haemolymph samples has been described previously (PADGHAM, 1976). Larvae used in this assay were also examined histologically for sclerotization using Mallory’s triple stain.
The blowfy bioassay
Cuticle digestion
The method adopted was that of VINCENT(1972). A culture of the blowfly Calliphora erythrocephala was
Schistocerca larvae at various stages before and after
reared from the late larval stage to the pupal stage at room temperature. Adults were ligatured with hair around the neck immediately they had emerged from the puparium. At this stage the ptilinum is still expanded and, as Vincent points out, this has three
the embryonic ecdysis were digested in weak alkali. The amount of protein going into solution gives a measure of the extent to which the cuticle is digested and is thus inversely proportional to the extent of tanning in the cuticle (Karlson et al., 1969).
I.P. 22/11-B
As a measure of cuticular tanning, the cuticles of
1447
1442(
D.E. PADGHAM
After removal of the alimentary canal the larvae were homogenized in water; the homogenate was left standing for 24 hr to extract water-soluble material. Since the epidermis cannot easily be separated from the cuticle, this initial extraction in water removes much of the more soluble epidermal protein so that cuticular protein is the principal component remaining. The suspension was centrifuged at 3000 rev/min and the supernatant discarded. The residue was washed three times in water. centrifuging and discarding the supernatant each time. The washed residue was freeze dried. 28 mg of residue was suspended in 25cm3 of 0.16”,, Na2S04 to digest for 16 hr. The suspension was centrifuged at 3000 rev/min and the supernatant collected and dialysed for 24 hr. The dialysed solution was then reduced from approximately 30 cm3 to exactly 5 cm3 by evaporation in an air current. The solution was then estimated for protein by the Folin and Ciocalteau method (LOWRY et al., 1951). Extracts were prepared from batches of 80 larvae from each of the following six times: (1) 14 hr before eclosion; (2) vermiform larvae (0 time): (3) 0.5 hr after eclosion; (4) 1 hr after eclosion; (5) 2 hr after eclosion: (6) 4 hr after eclosion. Non-rnelanizing lawur
On emergence from the embryonic cuticle most gregarious first instar larvae of Schistocerca are pale yellowish-green in colour except for the eyes, mandibles and the semi-lunar crescent of the metathoracic femoro-tibia1 joint. These larvae progressively darken over the next 2 to 3 hr until they are almost completely black. However, in a typical egg pod of gregarious Schistocerca a small number of the eggs at the bottom of the pod produce larvae which do not darken but remain green. These are slightly smaller than typical gregarious larvae and their initial coloration is more of a whitish-green. Frequently the eyes and mandibles are less pigmented and the semilunar crescent may be completely colourless. The green larvae change colour only slightly and are finally pale green. The characteristics of such larvae are not. however. totally reliable. Thus. when such larvae were used as blood donors, they were always
Table I. Melanization induced in neck-ligatured blowfly adults and vermiform Schistocerca larvae when injected with haemolymph from newly ecdysed first instar Schistocerca larvae or from 20min post-emergence adult Calliphoru. The Table gives the number of insects giving each particular score Bioassay recipient
Bioassay donor
Neck-ligatured blowflies Neck-ligatured blowflies Vermiform locusts Vermiform locusts
First instar locusts Adult blowflies First instar locusts Adult blowflies
Melanization score in recipient 2 2.5 3 4 10
12
3
-
2
13
6
-
3
-
12
19
~
8
13
enclosed first instar larvae of Schistocerca or from 20 min postemergence adult Calliphoru. The injection of locust haemolymph into vermiform larvae and of blowfly haemolymph into ligatured blowflies served as controls. After 3 hr incubation the neck-ligatured blowfly adult and vermiform larvae recipients were scored for melanization on their respective scales. The results are given in Table 1. There is no significant difference in the degree of melanization produced by either blowfly or locust haemolymph when injected into either bioassay insect. Cuticle digestion The digestibility of larval cuticle was used as a measure of its degree of sclerotization. Larvae at six stages on either side of eclosion were assayed. The quantity of protein going into solution was measured spectrophotometrically. Mean optical density readings for each of the six stages are given in Fig. 1. It is evident that there is a slight decrease in the amount of extractable protein per unit weight during the 14 hr before eclosion. After eclosion the quantity of extractable protein decreases markedly over a period of about an hour, after which the decline is less pronounced. In terms of cuticular sclerotization these results suggest a slow increase during the terminal stages of development but a very sharp increase immediately after eclosion.
kept and the presence or absence of melanization recorded as a check against the results in the recipient larvae. Albino Schistocr~u. kept as a stock culture at COPR. are morphologically identical to normal gregarious Schistocurca except for the absence of black pigmentation. They show a Mendelian pattern of inheritance in which albinism is recessive (HUNTER- Cuticular histology It is noteworthy that both the onset of rapid scleroJONES,1957). tization and the appearance of melanizing activity in the haemolymph are coincident with eclosion. The RESULTS association between melanization and sclerotization Melanization hioassa!’ can be demonstrated further in ligatured larvae which were scored for melanization and then stained histoBoth neck-ligatured blowflies and vermiform larvae logically for changes in sclerotization. Such a ligaof Schistocerca were used as bioassay recipients. Into these were injected haemolymph from either newly tured larva is shown in Fig. 2, the ligature being
-, .
;I , 1
.i
:
Fig. 2. First-instar larva of Schistocerca ligatured between the prothoracic and mesothoracic legs just as the embryonic ecdysis was beginning. Melanization is limited to that part of the body posterior to the ligature. Fig. 3. Transverse section of the prothoracic femur (right-hand side) and the mesothoracic femur (left-hand side) of the ligatured larva shown above. The prothoracic femur shows the thick, strongly acid fuchsin positive cuticle characteristic of an unsclerotized vermiform larva. Conversely the mesothoracic femur shows the thinner, more extended cuticle, which is refractory to acid fuchsin, characteristic of a sclerotized first instar larva.
Tanning in melanizing and non-melanizing larvae of S. gregaria
1451
the release site, melanization and sclerotization contined to the anterior end of the body.
are
Interchanges of haemolymph between melanizing, nonmelanizing green, and non-melanizing albino larvae of
Schistocerca
Time on either
side
of ecdysis,
hours,
Fig. 1, Changes in the amount of cuticular protein soluble in weak alkali in the period from 14hr before eclosion to 4 hr after eclosion. Vertical bars represent one standard deviation on either side of the mean.
between the prothoracic described
previously
and mesothoracic legs. As (PALIGHAM,1976), the release site
of the melanization hormone is immediately posterior to the metathoracic ganglion and thus the ligatured larva shows melanization posterior to the ligature but none anterior. The cuticular histology of this same larva is shown in Fig. 3. The section cuts the prothoracic and mesothoracic tibiae. The prothoracic tibia, which shows no melanization, also exhibits the typically thick bright red staining cuticle of unsclerotized vermiform larvae. In contrast, the melanized mesothoracic tibia is both more refractory to the acid fuchsin staining and shows the typically thin extended cuticle described by BERNAYS (1972) for newly sclerotized 4 hr post-eclosion Schistocerca larvae. Similarly, when a larva is ligatured posterior to
Haemolymph from newly ecdysed larvae of each of the three locust forms was injected into vermiform larvae of a different form. Thus, haemolymph from green first instar larvae was injected into potentially melanizing vermiform larvae and vice-versa. Haemolymph from albino larvae was similarly interchanged. Melanization was recorded by observations of darkening, and sclerotization by the change in the histological appearance of sections to Mallory’s triple stain from strongly acid fuchsin positive cuticle to thin non-staining cuticle. The results of these reciprocal blood transfers, together with those for two control groups, are given in Table 2. Haemolymph from newly ecdysed melanizing, nonmelanizing green and non-melanizing albino larvae is capable of inducing sclerotization and melanization in vermiforrn gregarious larvae even though the latter two forms do not themselves melanize. Conversely haemolymph from a newly ecdysed gregarious donor, which induces melanization in the donor, does not do so in either the green non-melanizing or the albino vermiform recipients, although it does induce sclerotization in both. DISCUSSION Haemolymph containing the melanization hormone of first instar Schistocerca, described by PADGHAM (1976), has been shown to have almost identical
Table 2. The melanizing and sclerotizing potential of haemolymph from newly ecdysed melanizing, non-melanizing green and non-melanizing albino locust donors when injected into reciprocal recipients of these forms Number of larvae 16
9
28
25
10
10
Donor
Recipient
Melanization in recipient
Sclerotization in recipient
Non-melanizing first instar. just ecdysed Melanizing first instar, just ecdysed
Potentially melanizing vermiform larva
16
16
Potentially non-melanizing vermiform larva
0
9
Albino first instar, just ecdysed Melanizing first instar. just ecdysed Potentially melanizing vermiform larva
Potentially melanizing vermiform larva
21
21
Albino vermiform larva
0
25
Albino vermiform larva
0
0
Albino first instar 5 hr after ecdysis
Potentially melanizing vermiform larva
0
0
1457
D. E. PAoGHAM
activity to blood containing bursicon. In the reciprocerca) or triggers and maintains (adult male Z_~custu) cal transfers of active haemolymph between vermi- this epidermal potentiality if it is present. form larvae of Schistocerca and ligatured adult Culliphora there was no significant difference in the capaAcknowlecigements~I would like to thank Dr R. F. city of undiluted blood from either insect to induce CHAPMANand Mr R. G. DAVIESfor their supervision durdarkening in the bioassay. That the Schistocerca ing this work and Dr D. W. EWEX for his critical reading melanization hormone is also a tanning hormone is of the manuscript. shown by the ligatured larvae which both melanize and sclerotize in the body half containing the release REFERENCES site but do neither in the other half. There is also ANDERSENS. 0. (1974) Evidence for two mechanisms of evidence for the coincident onset of both sclerotizasclerotization in insect cuticle. Nature, Lond. 251, tion and melanization. Melanization is initiated at the 507-508. time of eclosion (PADGHAM,1976). Similarly. as shown BERNAYSE. (1972) Changes in the first instar cuticle of in Fig. 1, the rapid onset of cuticular sclerotization Schistocerca gregaria before and associated with hatchbegins at eclosion. This is in agreement with the ing. J. Insect Physiol. 18, 897-912. observations of BERNAYS(1972) who also reports a DENNELLR. (1958) The hardening of insect cuticles. Biol. Reo. 33, 178-196. decrease in the more soluble fraction of Schistocerca F~GAL W. and FRAENKELG. (1969) The rBle of bursicon cuticle in the first hour after eclosion. in melanization and endocuticle formation in the adult The reactions of the melanizing, non-melanizing flesh fly Sarcophaga bullata. J. Insect Physiol. 15, green, and albino larvae to the various reciprocal in1235-1247. jections of ‘active’ blood suggest that the hormone FRAENKELG. and HSIAOC. (1962) Hormonal and nervous bursicon is acting as a trigger and that the determinacontrol of tanning in the fly. Science, Wash. 138, 27-29. tion of the type and degree of sclerotization and FRAENKELG. and Hsl~o C. (1963) Tanning in the adult melanization is an epidermal property. Within a grefly: A new function of neurosecretion in the brain. Science, Wash. 141, 1057-1058. garious first instar larva there is both dark brown and relatively colourless sclerotization as well as FRAENKELG. and Hsl~o C. (1965) Bursicon, a hormone which mediates tanning of the cuticle in the adult fly melanized and non-melanized areas of cuticle. and other insects. J. Insect Phvsiol. 11. 513-556. ANDER~EN (1974) suggests that these types of sclerotiHUN~R-JONES P, (1975) An aldino strain of the desert zation and the presence or absence of melanization locust. Nature, Lond. 180, 236237. are dependent upon specific epidermal enzymes. KARL~~NP., KALLIOPEE. S., und MARMARASV. I. (1969) MALEK(1975) also attributes the presence or absence Die Aminostiurezusammensetzung verschiedener Proof melanin in the cuticle to a differential distribution teinfraktionen aus der Cuticula von Calkphora erythroof phenolase enzymes in the outer layers of the exocucephala in verschiedenen Entwicklungsstadien. J. Insect Physiol. 15, 319-323. tide. The results given here confirm these suggestions N. J., FARR A. L., and RANin so far as they show that the blood capable of in- LOWRY0. H.. ROSENBOROUGH DALLR. J. (1951) Protein measurement with the folin ducing melanization in a melanizing larva does not phenol reagent. J. biol. Chem. 193, 265-275. have this capacity in a green or albino larva. ConMALEK S. R. A. (1957) Sclerotization and melanization: versely, blood which has the capacity to induce only , Two independent processes in the cuticle of the desert sclerotization in a green or albino larva will trigger locust. Nature, Lond. 180, 237. both sclerotization and melanization in a melanizing MILLS R. R., MATHURR. B., and GUERRAA. A. (1965) larva. Thus. the difference between melanizing, nonStudies on the hormonal control of tanning in the Amermelanizing green, and albino larvae resides in the epiican cockroach. I. Release site of an activation factor from the terminal abdominal ganglion. J. Insect Physiol. dermis rather than in the hormone. 11, 1047-1053. In gregarious adult Locusta migrutoria migrutorPADCHAMD. E. (1976) Control of melanization in first inioides the development of the yellow mature male colstar Schistocerca gregaria. In press. oration is controlled by the corpora allata (PENERet PENERM. P. (1976) The differential effect of the corpora al., 1972). Implants of corpora allata into newly allata on yellow coloration in crowded and isolated ecdysed gregarious males will speed up this yellow Locusta migratoria migratorioides males. Acrida In press. colour development. However, corpora allata im- PENERM. P., GIRARDIEA., and JOLYP. (1972) Neurosecreplanted into solitarious Locusta males, which show tory and corpus allatum controlled effects on mating yellowing on their hind wings, does not induce any behaviour and colour change in adult Locusta migratoria migratorioides males. Gen. camp. Endocr. 19, 484508. further yellow coloration (PENER,in press). Thus, in the development of both the black coloration in first POST L. C. (1972) Bursicon: its effect on tyrosine permeation into haemocytes. Biochim. biophys. Acta 290, instar larvae of Schistocerca and the yellow coloration 424428. in adult male Locusta the hormonal factor which inPOST L. C. and DE JONG B. J. (1973) Bursicon and the itiates the process in the gregarious phase does not metabolism of tyrosine in the moulting cycle of Pieris do so in the solitarious phase. In both instances the larvae. J. Znsecf Physiol. 19, 1541-1546. cuticular or epidermal coloration depends on the VINCENTJ. F. V. (1972) The dynamics of release and the potentiality of the epidermal cell and not upon the possible identity of bursicon in Locusta migratoria migrahormone, which only triggers (first instar Schisrotorioides. J. Insect Physiol. 18, 757-780.
BURSICON-MEDIATED CONTROL OF TANNING IN MELANIZING AND NON-MELANIZING FIRST INSTAR LARVAE OF SCHISTOCERCA GREGARIA D. E. PADGHAM Centre for Overseas Pest Research, College House, Wrights Lane, London W8 5SJ, England (Received 15 April 1976)
Abstract-Both melanization and sclerotization in first instar Schistocerca larvae are shown to have a common hormonal control mechanism. This hormone is shown to be analogous to bursicon. the tanning hormone of other insects, by the standard blowfly bioassay. The hormone is identical in both melanizing and non-melanizing first instar larvae of Schistocerca.
INTRODUCTION BURSICON,the tanning
hormone,
was first described
by FRAENKELand HSIAO (1962, 1963, 1965) in the blowfly Sarcophaga bullata and subsequently this and other blowflies have been used as a bioassay for the hormone. FRAJBKFLand HSIAO (1962, 1963) showed that blood from Periplaneta, Tenebrio, Phormia and Sarcophaga induced tanning when injected into neckligatured Calliphora. MILLS et al. (1965) used neckligatured Musca to bioassay the hormone from Periplaneta, whereas VINCENT(1972) used a Phormia bioassay for the blood of adult Locusta and Schistocerca. In addition to these bioassays the hormone was also assayed biochemically. POST(1972) and POST and DE JONG (1973) used its ability to initiate tyrosine metabolism as a measure of activity. In the present paper the relation between the melanization hormone described by PAKXXAM(1976) in first instar larvae of Schistocerca and bursicon is investigated using the standard blowfly bioassay. Although melanization and sclerotization are biochemically distinct (DENNELL,1958), they are considered as having the same humoral control by the hormone bursicon (FOGJZLand FRAENKEL,1969). This relationship between sclerotization and melanization has been investigated by a comparison between the sclerotizing and melanizing potential of blood from newly ecdysed melanizing, green and albino first instar larvae of Schistocerca gregaria. MATERIALS AND METHODS
advantages. Firstly, it provides a check on the tightness of the ligature since a loose ligature will allow the ptilinum to collapse back into the body. Secondly, an expanded ptilinum reduces the blood volume of the thorax and abdomen behind the ligature, thus making the test more sensitive. Finally, the reduced blood volume in the abdomen almost eliminates bleeding after injection. The ligatured blowflies were allowed to stand for 2 hr before being used for the bioassay and any flies showing darkening at this stage were rejected. The flies were then injected with 2 ~1 of blood using glass micropipettes drawn out with an SRI glass electrode puller. They were incubated for three hours at room temperature and then scored on a 0 to 4 scale for darkening analogous to that of VINCENT(1972):(O)No darkening. (1) Slight darkening; 30% of the dorsal surface of the abdomen. (2) Darkening on patches of the thorax or abdomen; 30% of the dorsal surface of the abdomen. (2.5) Abdomen entirely dark, thorax less so. (3) Abdomen and thorax almost totally dark. (3.5) Interference colours present at the tip of the abdomen. (4) Fully dark. The vermijkm larva bioassa) The use of vermiform larvae of Schistocerca for the bioassay of melanizing activity in haemolymph samples has been described previously (PADGHAM, 1976). Larvae used in this assay were also examined histologically for sclerotization using Mallory’s triple stain.
The blowfy bioassay
Cuticle digestion
The method adopted was that of VINCENT(1972). A culture of the blowfly Calliphora erythrocephala was
Schistocerca larvae at various stages before and after
reared from the late larval stage to the pupal stage at room temperature. Adults were ligatured with hair around the neck immediately they had emerged from the puparium. At this stage the ptilinum is still expanded and, as Vincent points out, this has three
the embryonic ecdysis were digested in weak alkali. The amount of protein going into solution gives a measure of the extent to which the cuticle is digested and is thus inversely proportional to the extent of tanning in the cuticle (Karlson et al., 1969).
I.P. 22/11-B
As a measure of cuticular tanning, the cuticles of
1447
1442(
D.E. PADGHAM
After removal of the alimentary canal the larvae were homogenized in water; the homogenate was left standing for 24 hr to extract water-soluble material. Since the epidermis cannot easily be separated from the cuticle, this initial extraction in water removes much of the more soluble epidermal protein so that cuticular protein is the principal component remaining. The suspension was centrifuged at 3000 rev/min and the supernatant discarded. The residue was washed three times in water. centrifuging and discarding the supernatant each time. The washed residue was freeze dried. 28 mg of residue was suspended in 25cm3 of 0.16”,, Na2S04 to digest for 16 hr. The suspension was centrifuged at 3000 rev/min and the supernatant collected and dialysed for 24 hr. The dialysed solution was then reduced from approximately 30 cm3 to exactly 5 cm3 by evaporation in an air current. The solution was then estimated for protein by the Folin and Ciocalteau method (LOWRY et al., 1951). Extracts were prepared from batches of 80 larvae from each of the following six times: (1) 14 hr before eclosion; (2) vermiform larvae (0 time): (3) 0.5 hr after eclosion; (4) 1 hr after eclosion; (5) 2 hr after eclosion: (6) 4 hr after eclosion. Non-rnelanizing lawur
On emergence from the embryonic cuticle most gregarious first instar larvae of Schistocerca are pale yellowish-green in colour except for the eyes, mandibles and the semi-lunar crescent of the metathoracic femoro-tibia1 joint. These larvae progressively darken over the next 2 to 3 hr until they are almost completely black. However, in a typical egg pod of gregarious Schistocerca a small number of the eggs at the bottom of the pod produce larvae which do not darken but remain green. These are slightly smaller than typical gregarious larvae and their initial coloration is more of a whitish-green. Frequently the eyes and mandibles are less pigmented and the semilunar crescent may be completely colourless. The green larvae change colour only slightly and are finally pale green. The characteristics of such larvae are not. however. totally reliable. Thus. when such larvae were used as blood donors, they were always
Table I. Melanization induced in neck-ligatured blowfly adults and vermiform Schistocerca larvae when injected with haemolymph from newly ecdysed first instar Schistocerca larvae or from 20min post-emergence adult Calliphoru. The Table gives the number of insects giving each particular score Bioassay recipient
Bioassay donor
Neck-ligatured blowflies Neck-ligatured blowflies Vermiform locusts Vermiform locusts
First instar locusts Adult blowflies First instar locusts Adult blowflies
Melanization score in recipient 2 2.5 3 4 10
12
3
-
2
13
6
-
3
-
12
19
~
8
13
enclosed first instar larvae of Schistocerca or from 20 min postemergence adult Calliphoru. The injection of locust haemolymph into vermiform larvae and of blowfly haemolymph into ligatured blowflies served as controls. After 3 hr incubation the neck-ligatured blowfly adult and vermiform larvae recipients were scored for melanization on their respective scales. The results are given in Table 1. There is no significant difference in the degree of melanization produced by either blowfly or locust haemolymph when injected into either bioassay insect. Cuticle digestion The digestibility of larval cuticle was used as a measure of its degree of sclerotization. Larvae at six stages on either side of eclosion were assayed. The quantity of protein going into solution was measured spectrophotometrically. Mean optical density readings for each of the six stages are given in Fig. 1. It is evident that there is a slight decrease in the amount of extractable protein per unit weight during the 14 hr before eclosion. After eclosion the quantity of extractable protein decreases markedly over a period of about an hour, after which the decline is less pronounced. In terms of cuticular sclerotization these results suggest a slow increase during the terminal stages of development but a very sharp increase immediately after eclosion.
kept and the presence or absence of melanization recorded as a check against the results in the recipient larvae. Albino Schistocr~u. kept as a stock culture at COPR. are morphologically identical to normal gregarious Schistocurca except for the absence of black pigmentation. They show a Mendelian pattern of inheritance in which albinism is recessive (HUNTER- Cuticular histology It is noteworthy that both the onset of rapid scleroJONES,1957). tization and the appearance of melanizing activity in the haemolymph are coincident with eclosion. The RESULTS association between melanization and sclerotization Melanization hioassa!’ can be demonstrated further in ligatured larvae which were scored for melanization and then stained histoBoth neck-ligatured blowflies and vermiform larvae logically for changes in sclerotization. Such a ligaof Schistocerca were used as bioassay recipients. Into these were injected haemolymph from either newly tured larva is shown in Fig. 2, the ligature being
-, .
;I , 1
.i
:
Fig. 2. First-instar larva of Schistocerca ligatured between the prothoracic and mesothoracic legs just as the embryonic ecdysis was beginning. Melanization is limited to that part of the body posterior to the ligature. Fig. 3. Transverse section of the prothoracic femur (right-hand side) and the mesothoracic femur (left-hand side) of the ligatured larva shown above. The prothoracic femur shows the thick, strongly acid fuchsin positive cuticle characteristic of an unsclerotized vermiform larva. Conversely the mesothoracic femur shows the thinner, more extended cuticle, which is refractory to acid fuchsin, characteristic of a sclerotized first instar larva.
Tanning in melanizing and non-melanizing larvae of S. gregaria
1451
the release site, melanization and sclerotization contined to the anterior end of the body.
are
Interchanges of haemolymph between melanizing, nonmelanizing green, and non-melanizing albino larvae of
Schistocerca
Time on either
side
of ecdysis,
hours,
Fig. 1, Changes in the amount of cuticular protein soluble in weak alkali in the period from 14hr before eclosion to 4 hr after eclosion. Vertical bars represent one standard deviation on either side of the mean.
between the prothoracic described
previously
and mesothoracic legs. As (PALIGHAM,1976), the release site
of the melanization hormone is immediately posterior to the metathoracic ganglion and thus the ligatured larva shows melanization posterior to the ligature but none anterior. The cuticular histology of this same larva is shown in Fig. 3. The section cuts the prothoracic and mesothoracic tibiae. The prothoracic tibia, which shows no melanization, also exhibits the typically thick bright red staining cuticle of unsclerotized vermiform larvae. In contrast, the melanized mesothoracic tibia is both more refractory to the acid fuchsin staining and shows the typically thin extended cuticle described by BERNAYS (1972) for newly sclerotized 4 hr post-eclosion Schistocerca larvae. Similarly, when a larva is ligatured posterior to
Haemolymph from newly ecdysed larvae of each of the three locust forms was injected into vermiform larvae of a different form. Thus, haemolymph from green first instar larvae was injected into potentially melanizing vermiform larvae and vice-versa. Haemolymph from albino larvae was similarly interchanged. Melanization was recorded by observations of darkening, and sclerotization by the change in the histological appearance of sections to Mallory’s triple stain from strongly acid fuchsin positive cuticle to thin non-staining cuticle. The results of these reciprocal blood transfers, together with those for two control groups, are given in Table 2. Haemolymph from newly ecdysed melanizing, nonmelanizing green and non-melanizing albino larvae is capable of inducing sclerotization and melanization in vermiforrn gregarious larvae even though the latter two forms do not themselves melanize. Conversely haemolymph from a newly ecdysed gregarious donor, which induces melanization in the donor, does not do so in either the green non-melanizing or the albino vermiform recipients, although it does induce sclerotization in both. DISCUSSION Haemolymph containing the melanization hormone of first instar Schistocerca, described by PADGHAM (1976), has been shown to have almost identical
Table 2. The melanizing and sclerotizing potential of haemolymph from newly ecdysed melanizing, non-melanizing green and non-melanizing albino locust donors when injected into reciprocal recipients of these forms Number of larvae 16
9
28
25
10
10
Donor
Recipient
Melanization in recipient
Sclerotization in recipient
Non-melanizing first instar. just ecdysed Melanizing first instar, just ecdysed
Potentially melanizing vermiform larva
16
16
Potentially non-melanizing vermiform larva
0
9
Albino first instar, just ecdysed Melanizing first instar. just ecdysed Potentially melanizing vermiform larva
Potentially melanizing vermiform larva
21
21
Albino vermiform larva
0
25
Albino vermiform larva
0
0
Albino first instar 5 hr after ecdysis
Potentially melanizing vermiform larva
0
0
1457
D. E. PAoGHAM
activity to blood containing bursicon. In the reciprocerca) or triggers and maintains (adult male Z_~custu) cal transfers of active haemolymph between vermi- this epidermal potentiality if it is present. form larvae of Schistocerca and ligatured adult Culliphora there was no significant difference in the capaAcknowlecigements~I would like to thank Dr R. F. city of undiluted blood from either insect to induce CHAPMANand Mr R. G. DAVIESfor their supervision durdarkening in the bioassay. That the Schistocerca ing this work and Dr D. W. EWEX for his critical reading melanization hormone is also a tanning hormone is of the manuscript. shown by the ligatured larvae which both melanize and sclerotize in the body half containing the release REFERENCES site but do neither in the other half. There is also ANDERSENS. 0. (1974) Evidence for two mechanisms of evidence for the coincident onset of both sclerotizasclerotization in insect cuticle. Nature, Lond. 251, tion and melanization. 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(1962) Hormonal and nervous bursicon is acting as a trigger and that the determinacontrol of tanning in the fly. Science, Wash. 138, 27-29. tion of the type and degree of sclerotization and FRAENKELG. and Hsl~o C. (1963) Tanning in the adult melanization is an epidermal property. Within a grefly: A new function of neurosecretion in the brain. Science, Wash. 141, 1057-1058. garious first instar larva there is both dark brown and relatively colourless sclerotization as well as FRAENKELG. and Hsl~o C. (1965) Bursicon, a hormone which mediates tanning of the cuticle in the adult fly melanized and non-melanized areas of cuticle. and other insects. J. Insect Phvsiol. 11. 513-556. ANDER~EN (1974) suggests that these types of sclerotiHUN~R-JONES P, (1975) An aldino strain of the desert zation and the presence or absence of melanization locust. Nature, Lond. 180, 236237. are dependent upon specific epidermal enzymes. KARL~~NP., KALLIOPEE. S., und MARMARASV. I. 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