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A possible role of ecdysteroids in pre-ecdysial tanning in larvae of Sarcophaga bullata (Diptera: Sarcophagidae)
J. fr~,ec~rPl~.vsio/.,Vol. 28. No. 2. pp. 123-127, 1982.
0022-1910/82;020123-u5~03.00/0 <(‘:1982 Pergmot1 Pm.\ Lrd.
Printed in Great Brimin.
A POSSIBLE ROLE OF ECDYSTEROIDS IN PRE-ECDYSIAL TANNING IN LARVAE OF SARCOPHAGA BULLATA (DIPTERA: SARCOPHAGIDAE) BRIAN ROBERTS*, MARZ BAKER, MANDY KOTZMAN and SH~;RRIEL. W~NTWORTH Zoology Department, Monash University. Clayton, Victoria. Austraha
AbstractThe levels of ecdysteroids in Surcophuga hullata were determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) to after the 2nd ecdysis and from late larval to pupal development. Two distinct peaks of ecdysteroid activity were recorded mid-way through the first and second stadia (14 and 34 hr) and two smaller peaks occurred a few hours prior to each ecdysis. A large release of ecdysteroids occurred from 8 hr before and up to 18 hr after formation of the white prepupa. This peak initiated the formation of the prepupa. the tanning of the puparium, larval/pupal apolysis and secretion of the pupal cuticle. Assays for the cuticle tanning hormone, bursicon. in pre-ecdysial larvae were not positive and a possible role for ecdysone in pre-ecdysial tanning of larval cuticular structures is proposed. Kc,! Word Ind~~s: Ecdlsteroid titres. bursicon. Srrrco$qqcl
INTRODUCTION
MATERIALS
A (‘HARAC’TERISTIC feature of insects is their tough exoskeleton which is secreted by the epidermis. Moulting is indispensable for the growth of insects and other Arthropods and is controlled by hormones: the ecdysteroids playing a central role. It is now well documented that during the moulting cycle the ecdysteroids are responsible for apolysis. cell division, differentiation of tissues, and deposition of the new cuticle. There are many reviews of these topics, several ofwhichare WYATT(I~~~),GILBERT~~~ K1~~(1973), and RIDDIFORII and TRUMAN (1978). In the vast majority of insects the newly deposited cuticle is tanned shortly after the moult; the process being mediated by the polypeptide hormone bursicon (FRAEYKEL and HSIAO, 1965). However, in the higher Diptera. several cases of pre-ecydsial tanning have been recorded. The tanning of the pulvilli of the developing adult (WHITTEN, 1969), the tanning of the anterior and posterior spiracles (ROBERTS, 198 1) and the mandibles of the cephalopharyngeal apparatus (SMITH, 1933: ROBERTS,1976; KOTZMAN, unpublished) prior to larval ecdyses are several examples. Also, the tanning of the last larval cuticle to form the puparium prior to larval/pupal apolysis appears to be unique. It is now well documented that this process, in these flies, is initiated and controlled by ecdysteroids (SHAAYA and KARLSON, 1965). In an attempt to elucidate the nature of pre-ecdysial tanning of the spiracles and in the flesh fly Suucophuga hul/uru, we have monitored ecdysteroid titres during larval and prepupal development and have also assayed for bursicon at selected times.
* Present address: Department of Zoology. University North Carolina at Chapel Hill. NC 27514, U.S.A.
of
AND
METHODS
(I) Livirzg nznfrrial
Breeding stocks of the ovoviviparous flesh ily S. bullatu were reared and maintained in a constanttemperature room at 25 f 0.5’C with 40-SO”,, r.h. and 16 hr of daily illumination. Larvae and adults were maintained and aged according to methods previously described (ROBERTS and WARREN, 1975: ROBERTS, 1976; WENTWORTH PI ul., 1981). (2) Radioimtttuttoussa~~ Suitable numbers of larvae were removed from liver at specified times after larviposition, thoroughi) washed in water, dried and immediately weighed. For the first two larval stages at least 5 determinations were made for each point. During the third instar and the puparial stages, at least 5 animals were individuallj sampled for each time point. In all cases, prepupae were removed from the puparia before being homogenized. Organisms were homogenized with a mortar and and the homogenates pestle in IOO”,, methanol centrifuged at 2500 rpm for 3 min at 2O~‘C. When necessary, samples were covered and stored at 4 C. The assay was performed as previously described (BORST and O’CONNOR. 1974: WENTWORTH 6’1 ul.. 1981). (3) Bursicon hioussq Homogenates of whole 9 hr and 21 hr tirst instar larvae and tanning adults. sampled 30 min after eclosion, were prepared according to the methods of VINCENT (1972). Standard saline was added to the homogenates at the rate of 14.5 &mg fresh weight. Highly concentrated samples of 9 hr and 21 hr first instar larvae were prepared in the same manner but saline added at the rate of 0.09 pl:mg fresh weight.
123
BRIANROBERTS er al.
124
In accordance with the methods of FRAENKEL and HSIAO (1965) and VINCENT (1972), adult S. bulluta were prepared and used for the bursicon bioassay. Eclosion was immediately succeeded by cervical ligation and after 1 hr, 5 ~1 of test homogenate was injected with a microsyringe. The results were scored in the manner of Vincent (VINCENT, 1972 and see Fig. 3).
1600
-
1400-
RESULTS (1) Changes development
in ecdysteroid
levels during
early
larval
At larviposition (0 hr), the level of ecdysteroids was approx. 10 pg 20-hydroxyecdysone equivalents/mg fresh weight, but by 14 hr the level had reached a maximum of about 60 pg 20-hydroxyecdysone equivalentslmg fresh weight. The level of ecdysteroids then decreased and were low during the 18-21 hr period. A small, but significant peak (Studentized range test, P < 0.0 1, SOKAL and ROHLF, 1969) of 15 pg 20-hydroxyecdysone equivalents/mg fresh weight was recorded at 22 hr, some 2 hr prior to the first ecdysis. Ecdysteroid levels showed similar trends during the second instar with a maximum of 70 pg 20-hydroxyecdysone equivalents/mg fresh weight noted mid-way through the instar at 34 hr, and a small, but significant peak (Studentized range test, P < 0.01) of approx. 25 pg 20-hydroxyecdysone equivalents/mg fresh weight at about 40 hr of development. The ecdysteroid levels then decreased rapidly and remained below 5 pg 20-hydroxyecdysone equivalents /mg fresh weight at
____
90
-___m
I
I
60
60
n >n 'i ; : p
5o
I
3 :
40 :: pr := :. E 30 ; : -Z ,
',
20
:: 10
0 1,,,,1,,,,,,,1 0 4 8 12 16
20 Age
24
26
32
36
40
44
40
52
(h)
Fig. 1. Levels of ecdysteroids during early larval deof S. bulluta. The arrows indicate larval ecdyses and the dashed line the periods of pre-ecdysial tanning. Bars represent mean + standard error; where bars are not shown velopment
the standard
error was too small to be included.
OI
I
I
I
I
-20 -16-12 -6 -4
I
I
I
0
4
6
Age
I
I
I
I
12 16 20 24
I I 26 32
(h)
Fig. 2. Levels ofecdysteroids during late larval and prepupal development of S. bullma. The arrows represent the red spiracular stage ( -4 hr) and the white prepupal stage (0 -hr) and the dashed line the period of puparial tanning. Bars represent mean i standard error; where bars are not shown the standard error was too small to be included.
the time of the ecdysis (48 hr) and into the third instar. Figure 1 illustrates the results; the dashed line indicates the periods of pre-ecdysial tanning shown by the anterior and posterior spiracles and the mouth hooks of the cephalopharyngeal apparatus. (2) Changes in ecdvsteroid prepupal development
70 * ; *
zoo-
levels during late larval and
The formation of the immobile white prepupa occurred at 144 hr after larviposition. This developmental event provided an ideal time for synchronization of the population and was designated as 0 hr. All prepupal development was timed from this event. Figure 2 summarises the results. At 124 hr of larval development ( -20 hr) the level of ecdysteroids was low (IO pg 20-hydroxyecdysone equivalents/mg fresh weight), but gradually increased to about 90 pg 20-hydroxyecdysone equivalents/mg fresh weight at 136 hr ( - 8 hr). At 140 hr ( -4 hr) when larvae showed red sclerotized posterior spiracles, the ecdysteroid level has risen to approx. 250 pg 20-hydroxyecdysone equivalents/mg fresh weight. Some 4 hr later, at the formation of the immobile white prepupa (0 hr) the level was 300 pg 20-hydroxyecdysone equivalentslmg fresh weight. During the period of puparial tanning, the ecdysteroid levels continued to increase reaching a maximum of approx. 1.4 ng 20-hydroxyecdysone equivalents/mg fresh weight, 18 hr after the formation of the white prepupa. By 24 hr ecdysteroid levels were again relatively low and the tanning of the puparium was completed. (3) The bursicon bioassall Initially, bioassays were performed
with a control of
Pre-ecdysial
in larvae of
tanning
60
Sarcophaga
bullara
125
a
1 60.--
%
60
60
b0
40
80
e
60
d
60
60
% b0
40
20
20
0
0 0
1
2
2%
3
3 8'.
0
1
2
2 1% 3
3
Score
Score
Fig. 3. Relative distribution of tanning scores (expressed as a I’,,1recorded in the bursicon bioassay. Each 11) was injected with 5 ~(1of one of the following: (a) standard saline (11= 21): (b) pre-ecdysial larval homogenate (21 hr) (14.5 &mg: ,I = 12): (c) tanning fly homogenate (14.5 plimg: n = 20); (d) concentrated ‘young’ larval homogenate (9 hr) (0.09 &mg: ,I = IL)): (e) concentrated pre-ecdysial larval homogenate (21 hr) (0.09 &mg: II = 20). The tanning scores were assigned according to VINCENT (1972). 0. no darkening; I. slight darkening, < 30”” dorsal surface of abdomen: 2. darkening in patches on thorax or abdomen, > 30”,, of dorsal surface of abdomen: 21. abdomen entirely dark. thorax not dark or with very little darkening: 3. abdomen and thorax both dark or very near11 so: 3;. interference colours present at the tip to the abdomen and/or the scutellum: 4 (not observed in the bioassay). full4 darkened--i.e. with the ventral abdominal arthodial membrane also dark.
standard saline and dilute homogenates of preecdysial larvae and tanning flies. Pre-ecdysial larvae (21 hr) showed mandibles tanned at stages 1-2 as defined by ROBERTS (1976). Saline injected flies (Fig. 3a) achieved only 11.3”,,total darkening and injections of pre-ecdysial larval homogenate (Fig. 3b) yielded similar results (14.6’!, total darkening). In distinct contrast, injections of tanning fly homogenate (Fig. 3c) produced a total darkening of 67.5”,. These differences were tested with the KolmogorovSmirnov two-sample test (SIEGEL, 1956). The tanning fly homogenate was significantly different from both
the control and the pre-ecdysial larval homogenate (D,a, = 0.90 and 0.83 respectively; P < 0.001). while no significant difference was recorded between the control and the pre-ecdysial larval homogenate. In addition, highly concentrated homogenates of young (9 hr) (Fig. 3d) and pre-ecdysial(21 hr) (Fig. 3e) larvae were assayed (CU. 160 x more concentrated than previously used) yielding scores of only 23 and respectively. Both of these 30”” total darkening homogenates
were
significantly
different
from
the
control (Dn,J, = 0.35 and 0.41 respectively: P < 0.01) and from the tanning fly (D,,,, = 0.84 and 0.85
126
BRIANROBERTS rf al.
respectively; P < 0.001). In spite of the differences in development, there was no significant difference between the tanning produced by these larval homogenates. DISCUSSION Of the many separate processes affected by the ecdysteroids, the moulting cycle is perhaps the best documented. The first visible event in arthropod moulting directly associated with ecdysteroid release is apolysis; the separation of the epidermis from the old cuticle. Previous work (ROBERTS et al., 1980; WENTWORTH et al., 1981) and our present data show clear cut releases of ecdysteroids approximately half way through the first two larval instars. Using first instar larvae of S. bullata, one of us (ROBERTS, 1981) has shown that apolysis of the tracheal epithelium takes place at 16 hr; some 2 hr after the first peak of ecdysteroids. The major biochemical process in the moulting cycle is deposition of new cuticle for which the epidermis synthesizes cuticular proteins, chitin and lipids. Synthesis of RNA and cuticular components has been induced by the addition of ecdysteroids to epidermal structures in vifro (MANDARON, 1970; RYERSE and LOCKE, 1977), which demonstrated the direct action of the hormone on these tissues. In first instar larvae of Sarcophaga, deposition of cuticle by the tracheal epithelium commenced at 18 hr (ROBERTS, 1981), some 4 hr after the maximum titre of 20hydroxyecdysone was recorded. It appears that the major peaks of ecdysteroids mid-way through the first and second instars are directly responsible for apolysis, differentiation of the tissues and the secretion of new larval cuticular structures. The formation of the anterior spiracle (ROBERTS, 1981), the posterior spiracles and the cepholopharyngeal apparatus (ROBERTS, 1976) support this statement. The role of the two smaller peaks of ecdysteroids, just prior to the larval ecdyses, will be discussed below. The tanning of the third instar larval cuticle to form the puparium is among the most investigated ecdysteroid-induced processes. From previous reports (SHAAYA and KARLSON, 1965; BARRI~T and BIRT, 1970; OH~AKI and TAKAHASHI, 1972; HODGETTSet al., 1977; ROBERTSet al., 1980; WENTWORTH et al., 1981), it has been clearly shown that the whole process of pupariation is triggered by the increasing levels of ecdysteroids at this stage of the life cycle. Our report showed that ecdysteroid levels commenced to increase some 8 hr prior to white prepuparium formation, to peak some 18 hr after this event, and to fall again to moderate levels by 32 hr of development. During the period of puparial tanning ecdysteroids activate the gene for decarboxylase which alters the metabolic fate of tyrosine and results in a tanned cuticle (KARLSON and SEKERIS, 1976). We consider the increasing ecdysteroid activity is not only required for the initiation of pupariation and tanning of the puparium but is also necessary for the sequential developmental events of larval/pupal apolysis (22-24 hr) and secretion of the pupal cuticle (25-28 hr) (WENTWORTH ef al., 1981), which occur some few hours after this high titre of ecdysteroids. Newly deposited cuticle is usually tanned just after
ecdysis and consequently it is considered that the ecdysone induced tanning of the dipteran puparium (KARLSON and SEKERIS, 1976) before the pupal moult is a special case in insect ontogeny (WYATT. 1972). Investigations to date have shown that post-ecdysial tanning in insects is controlled by the polypeptide hormone bursicon (FRAENKEL and HSIAO, 1965). Bursicon is believed to act by changing the metabolic fate of tyrosine, as ecdysone does in pupariating flies (FOGAL and FRAENKEL, 1969a; KARLSON and SEKERIS. 1976; RIDDIFORD and TRUMAN, 1978) from phydroxyphenyl propionic acid to dopamine (NEVILLE, 1975). Unlike ecdysone, bursicon is believed to achieve this by altering the permeability of haemocytes and epidermal cells to tyrosine (NEVILLE, 1975; RIDDIFORD and TRUMAN, 1978) and does not involve de now protein synthesis (FOGAL and FRAENKEL, 1969b). Our bursicon assay, scored in the manner of VINCENT(1972). showed that injections of pre-ecdysial larval homogenate yielded similar results to the control saline injected flies. In contrast, injections of tanning fly homogenate produced significant positive tanning. These findings suggest that pre-ecdysial larval tanning of the mouth hooks and spiracles is mediated by some agent other than bursicon. Results of the bioassay using highly concentrated homogenates of young larvae and pre-ecdysial larvae support this suggestion indicating that concentration alone was not responsible for the initial results. The relationship between the homogenate concentration and bursicon activity in S. bullata and Locusta (VINCENT, 1972) can be compared. In Locusta an increase from 14 to 30”, total darkening was produced by only a 2 times increase in concentration. while in Sarcopfmgn the same change in darkening was only achieved by a 160 times increase in concentration. It is possible that the bioassay tanning noted in concentrated homogenates was caused by a type of injury reaction. WIGGLESWORTH(1937) observed that when damaged cells were injected into an insect, they clustered against the recipient’s integument and initiated limited injury repair reactions. SALT (1970) described mechanisms with which insects combat invading organisms, documented the phenomenon of melanin encapsulation. Perhaps the larval homogenate injections resulted in damaged cells adhering to the integument of the bioassay flies and initiating melanin deposition, similar to normal tanning processes. This report showed for the first time that two significant releases of 20-hydroxyecdysone occurred some few hours prior to each of the larval ecdyses. The single peak late in the first instar is clear cut, that of the second instar is of higher values but is less clear due to normal variability in ageing larvae. These releases were not detected in previous investigations (ROBERTS et al., 1980: WENTWORTHet al., 1981) presumably due to greater time gaps between samples. During and after the times of these ecdysteroid releases it has been documented that specific cuticular structures, in S. bullata, undergo pre-ecdysial tanning. The anterior and posterior spiracles and the mouth hooks of the cephalopharyngeal apparatus (ROBERTS, 1976, 198 1) commence their tanning some 3-4 hr prior to ecdysis. Indeed these structures are almost completely tanned at the time of ecdysis; only the
Pre-ecdysial tanning in larvae of Surcophaga hullata articulation of the mandibular hook to the hypostomal sclerite remains to be fully sclerotised (ROBERTS. 1976). Consequently, with respect to the negative result of the bursicon bioassay, the accuracy of the RIA and to our ageing technique of the larvae, it is possible that in S. bulluta and indeed in the higher Diptera that ecdysteroids mediate pre-ecdysial tanning associated with both larval/larval moults in a manner similar to that of puparial tanning prior to the larval/pupal transformation. This conclusion implies that a specific gene set is used for tanning prior to metamorphosis and that a different system may be utilized for the completion of the tanning of the adult cuticle for which bursicon is the reported controlling hormone (FRAENKEL and HSIAO, 1965). We have not ruled out the possibility that the tanning process may be a consequence of the high ecdysteroid titres recorded midway through the instar. If this were the case the genes for tanning would be considered late developmental genes (ASHBURNERand RKHARDS, 1976) and would be functional only after the cuticular structures of the subsequent instar were fully formed. If this were so, the elevated titres just prior to the moult may be required to complete the sclerotization processes. We wish to thank Dr. J. D. ~r~noI~~il~~i~c~t~lrtrl. O‘CONNOK for the kind gift of H’ r-ecdysone and antiserum (Horn l-2).
REFERENCES ASIIBLIKN~.KM. and RICHARDS G. (1976) The role ofecdysone in the control of gene activity in the polytene chromosomes of Dro.so~/&ilu. S>,nry. R. Ent. Sot. 8, 203-225. BAKKIT L. C. and BIKE L. M. (1970) Prothoracic gland hormone in the sheep blowfly Luciliu cuprina. J. Insect PhJsiol. 16, 671-677. BOKST D. W. and O’CONNOR J. D. (1974) Trace analysis of ecdysone by gas liquid chromatography. radioimmunoassay and bioassay. Steroids 24, 637-656. FOGAL W. H. and FKAENEEL G. (1969a) The role of bursicon in melanization and endocuticle formation in the adult tlesh fly Sarcophaga hullata. J. Insect Physiol. E(7), 1235-1248. FOC;AL W. H. and FRAENKEL G. (1969b) Melanin in the puparium and adult integument of the flesh fly Sarcophaga hullata. J. Insect Physiol. 15(S), 1437-1448. FKA~NKEL G. and HASIO C. (1965) Bursicon, a hormone which mediates tanning of the cuticle in the adult fly and other insects. J. Insect Physiol. 11(5), 513-556. GILBERT L. I. and KING D. (1973) Physiology of growth and development. In Physiology oflnsecta (Ed. by ROCKSTEIN M.) 2nd ed.. pp. 249-370. Academic Press. New York. HUDGETTS R. B.. SAC~BB. and O’CONNOR J. D. (1977) Ecdqsone titre during post-embryonic development of Drosophila melanogaster. Devl. Biol. 60, 310-317.
117
KARLS~N P. and SEKER~SC. E. (1976) Control of tyrosine metabolism and cuticle sclerotization by ecdysone. In The Insect Integument (Ed. by HEPBURN H. R.) p. 571. E.lsevier. Amsterdam. MANDARON P. (1970) Development in vitro de disques imaginaux de la Drosophile. Aspects morphologiques et histologiques. Devl. Biol. 22, 298-320. NEVILLE A. C. (1975) Biology of the Arthropod Cuticle. Springer, New York. OHTAKI T. and TAKAHASHI M. (1972) Induction and termination of pupal diapause in relation to the change of ecdysone titre in the flesh fly SarcophaguprrcJ,qrina Jap. J. med. Sri. Biol. 25, 369-376. RIDDIFORD L. M. and TRUMAN J. W. (1978) Biochemistry of insect hormones and insect growth regulators. In Biochemistry of Insects (Ed. by ROCKSTEIN M.) pp. 308-357. Academic Press. New York. ROBERTS B. (1976) Larval development in the Australian flesh fly Tricholioproctia impatiens. .4nn. cwt. Sot. .4tn. 69(2). 158-164. ROBERTS B. ( I98 1) Development of the prothordcic spiracles in larvae of Sarcophuga buliata (Sarcophagidae:Diptera). Aust. J. Zool. 29, 199-204. ROBERTS B. and WARREN M. A. (1975) Diapause in the Australian flesh fly Tricholioproctiu intpatiwts. .4ust. J. Zool. 23, 563-567. ROBERTS B., WENTWORTH S. L. and O’CONNORJ. D. (1980) Ecdysone titres during post-embryonic development in Sarcophaga @dlata (Sarcophagidae:Diptera). XVI ht. Congr. Enr. (Kyoto, Japan) 16, 172. RYEKSE J. S. and LOCKE M. (1977) Ecdysterone-mediated cuticle deposition and the control of growth in insect tracheae. J. Insect Ph.vsiol. 24, 541-550. SALT G. (1970) The Cellular Defense Reuctions oj Inarcts. pp. 118. Cambridge University Press. Cambridge. SHAAYA E. and KARL~CINP. (1965) Der Ecdysontiter wahrend der Insektenent wicklung-II. Die postembryonale Entwicklung der Schmeissfliege Calliphoru er~throcephala Merg. J. Insect Physiol. 11, 6549. SIEGEL S. (1956) Nonparametric Statisticsjbr the Brhcn’ioural Sciences, p. 312, McGraw-Hill, London. SOKAL K. K. and ROHLF J. F. (1973). Introdut~tion to Biostatistics W. H. Freeman, San Francisco. SMITH C. N. (1933) Notes on the life history and moulting process of Sarcophaga securijrra (Villeneuve). Pro<,. enr. Sot. Wash. 35(7), 159-164. VINCENT J. F. V. (1972) The dynamics of release and the possible identity of bursicon in Locusta mrgratoria migratorioide.\. J. Insect Physiol. 25, 653-658. WENTWORTH S. L.. ROBERTS B. and O’CONNOR J. D. (1981) Ecdysteroid titres during post-embryonic development ol Surcophaga builata (Sarcophagidae: Diptera). J. Insect Physiol. 27, 435440. WHITTEN J. M. (1969) Coordinated development in the fly foot: sequential cuticle secretion. J. Morph. 1271 I ). 73-104. WIGGLESWORTH V. B. (1937) Wound healing in an insect (Rhodnius prolixus: Hemiptera). 1. e.xp. BioI. 14, 364-38 I. WYATT G. R. (1972) Insect hormones. In Biochm~ic~ol Acriom 01 Hormones (Ed. by LITWACK G.) Vol. 2. pp. 38-5-490. Academic Press, New York.
0022-1910/82;020123-u5~03.00/0 <(‘:1982 Pergmot1 Pm.\ Lrd.
Printed in Great Brimin.
A POSSIBLE ROLE OF ECDYSTEROIDS IN PRE-ECDYSIAL TANNING IN LARVAE OF SARCOPHAGA BULLATA (DIPTERA: SARCOPHAGIDAE) BRIAN ROBERTS*, MARZ BAKER, MANDY KOTZMAN and SH~;RRIEL. W~NTWORTH Zoology Department, Monash University. Clayton, Victoria. Austraha
AbstractThe levels of ecdysteroids in Surcophuga hullata were determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) to after the 2nd ecdysis and from late larval to pupal development. Two distinct peaks of ecdysteroid activity were recorded mid-way through the first and second stadia (14 and 34 hr) and two smaller peaks occurred a few hours prior to each ecdysis. A large release of ecdysteroids occurred from 8 hr before and up to 18 hr after formation of the white prepupa. This peak initiated the formation of the prepupa. the tanning of the puparium, larval/pupal apolysis and secretion of the pupal cuticle. Assays for the cuticle tanning hormone, bursicon. in pre-ecdysial larvae were not positive and a possible role for ecdysone in pre-ecdysial tanning of larval cuticular structures is proposed. Kc,! Word Ind~~s: Ecdlsteroid titres. bursicon. Srrrco$qqcl
INTRODUCTION
MATERIALS
A (‘HARAC’TERISTIC feature of insects is their tough exoskeleton which is secreted by the epidermis. Moulting is indispensable for the growth of insects and other Arthropods and is controlled by hormones: the ecdysteroids playing a central role. It is now well documented that during the moulting cycle the ecdysteroids are responsible for apolysis. cell division, differentiation of tissues, and deposition of the new cuticle. There are many reviews of these topics, several ofwhichare WYATT(I~~~),GILBERT~~~ K1~~(1973), and RIDDIFORII and TRUMAN (1978). In the vast majority of insects the newly deposited cuticle is tanned shortly after the moult; the process being mediated by the polypeptide hormone bursicon (FRAEYKEL and HSIAO, 1965). However, in the higher Diptera. several cases of pre-ecydsial tanning have been recorded. The tanning of the pulvilli of the developing adult (WHITTEN, 1969), the tanning of the anterior and posterior spiracles (ROBERTS, 198 1) and the mandibles of the cephalopharyngeal apparatus (SMITH, 1933: ROBERTS,1976; KOTZMAN, unpublished) prior to larval ecdyses are several examples. Also, the tanning of the last larval cuticle to form the puparium prior to larval/pupal apolysis appears to be unique. It is now well documented that this process, in these flies, is initiated and controlled by ecdysteroids (SHAAYA and KARLSON, 1965). In an attempt to elucidate the nature of pre-ecdysial tanning of the spiracles and in the flesh fly Suucophuga hul/uru, we have monitored ecdysteroid titres during larval and prepupal development and have also assayed for bursicon at selected times.
* Present address: Department of Zoology. University North Carolina at Chapel Hill. NC 27514, U.S.A.
of
AND
METHODS
(I) Livirzg nznfrrial
Breeding stocks of the ovoviviparous flesh ily S. bullatu were reared and maintained in a constanttemperature room at 25 f 0.5’C with 40-SO”,, r.h. and 16 hr of daily illumination. Larvae and adults were maintained and aged according to methods previously described (ROBERTS and WARREN, 1975: ROBERTS, 1976; WENTWORTH PI ul., 1981). (2) Radioimtttuttoussa~~ Suitable numbers of larvae were removed from liver at specified times after larviposition, thoroughi) washed in water, dried and immediately weighed. For the first two larval stages at least 5 determinations were made for each point. During the third instar and the puparial stages, at least 5 animals were individuallj sampled for each time point. In all cases, prepupae were removed from the puparia before being homogenized. Organisms were homogenized with a mortar and and the homogenates pestle in IOO”,, methanol centrifuged at 2500 rpm for 3 min at 2O~‘C. When necessary, samples were covered and stored at 4 C. The assay was performed as previously described (BORST and O’CONNOR. 1974: WENTWORTH 6’1 ul.. 1981). (3) Bursicon hioussq Homogenates of whole 9 hr and 21 hr tirst instar larvae and tanning adults. sampled 30 min after eclosion, were prepared according to the methods of VINCENT (1972). Standard saline was added to the homogenates at the rate of 14.5 &mg fresh weight. Highly concentrated samples of 9 hr and 21 hr first instar larvae were prepared in the same manner but saline added at the rate of 0.09 pl:mg fresh weight.
123
BRIANROBERTS er al.
124
In accordance with the methods of FRAENKEL and HSIAO (1965) and VINCENT (1972), adult S. bulluta were prepared and used for the bursicon bioassay. Eclosion was immediately succeeded by cervical ligation and after 1 hr, 5 ~1 of test homogenate was injected with a microsyringe. The results were scored in the manner of Vincent (VINCENT, 1972 and see Fig. 3).
1600
-
1400-
RESULTS (1) Changes development
in ecdysteroid
levels during
early
larval
At larviposition (0 hr), the level of ecdysteroids was approx. 10 pg 20-hydroxyecdysone equivalents/mg fresh weight, but by 14 hr the level had reached a maximum of about 60 pg 20-hydroxyecdysone equivalentslmg fresh weight. The level of ecdysteroids then decreased and were low during the 18-21 hr period. A small, but significant peak (Studentized range test, P < 0.0 1, SOKAL and ROHLF, 1969) of 15 pg 20-hydroxyecdysone equivalents/mg fresh weight was recorded at 22 hr, some 2 hr prior to the first ecdysis. Ecdysteroid levels showed similar trends during the second instar with a maximum of 70 pg 20-hydroxyecdysone equivalents/mg fresh weight noted mid-way through the instar at 34 hr, and a small, but significant peak (Studentized range test, P < 0.01) of approx. 25 pg 20-hydroxyecdysone equivalents/mg fresh weight at about 40 hr of development. The ecdysteroid levels then decreased rapidly and remained below 5 pg 20-hydroxyecdysone equivalents /mg fresh weight at
____
90
-___m
I
I
60
60
n >n 'i ; : p
5o
I
3 :
40 :: pr := :. E 30 ; : -Z ,
',
20
:: 10
0 1,,,,1,,,,,,,1 0 4 8 12 16
20 Age
24
26
32
36
40
44
40
52
(h)
Fig. 1. Levels of ecdysteroids during early larval deof S. bulluta. The arrows indicate larval ecdyses and the dashed line the periods of pre-ecdysial tanning. Bars represent mean + standard error; where bars are not shown velopment
the standard
error was too small to be included.
OI
I
I
I
I
-20 -16-12 -6 -4
I
I
I
0
4
6
Age
I
I
I
I
12 16 20 24
I I 26 32
(h)
Fig. 2. Levels ofecdysteroids during late larval and prepupal development of S. bullma. The arrows represent the red spiracular stage ( -4 hr) and the white prepupal stage (0 -hr) and the dashed line the period of puparial tanning. Bars represent mean i standard error; where bars are not shown the standard error was too small to be included.
the time of the ecdysis (48 hr) and into the third instar. Figure 1 illustrates the results; the dashed line indicates the periods of pre-ecdysial tanning shown by the anterior and posterior spiracles and the mouth hooks of the cephalopharyngeal apparatus. (2) Changes in ecdvsteroid prepupal development
70 * ; *
zoo-
levels during late larval and
The formation of the immobile white prepupa occurred at 144 hr after larviposition. This developmental event provided an ideal time for synchronization of the population and was designated as 0 hr. All prepupal development was timed from this event. Figure 2 summarises the results. At 124 hr of larval development ( -20 hr) the level of ecdysteroids was low (IO pg 20-hydroxyecdysone equivalents/mg fresh weight), but gradually increased to about 90 pg 20-hydroxyecdysone equivalents/mg fresh weight at 136 hr ( - 8 hr). At 140 hr ( -4 hr) when larvae showed red sclerotized posterior spiracles, the ecdysteroid level has risen to approx. 250 pg 20-hydroxyecdysone equivalents/mg fresh weight. Some 4 hr later, at the formation of the immobile white prepupa (0 hr) the level was 300 pg 20-hydroxyecdysone equivalentslmg fresh weight. During the period of puparial tanning, the ecdysteroid levels continued to increase reaching a maximum of approx. 1.4 ng 20-hydroxyecdysone equivalents/mg fresh weight, 18 hr after the formation of the white prepupa. By 24 hr ecdysteroid levels were again relatively low and the tanning of the puparium was completed. (3) The bursicon bioassall Initially, bioassays were performed
with a control of
Pre-ecdysial
in larvae of
tanning
60
Sarcophaga
bullara
125
a
1 60.--
%
60
60
b0
40
80
e
60
d
60
60
% b0
40
20
20
0
0 0
1
2
2%
3
3 8'.
0
1
2
2 1% 3
3
Score
Score
Fig. 3. Relative distribution of tanning scores (expressed as a I’,,1recorded in the bursicon bioassay. Each 11) was injected with 5 ~(1of one of the following: (a) standard saline (11= 21): (b) pre-ecdysial larval homogenate (21 hr) (14.5 &mg: ,I = 12): (c) tanning fly homogenate (14.5 plimg: n = 20); (d) concentrated ‘young’ larval homogenate (9 hr) (0.09 &mg: ,I = IL)): (e) concentrated pre-ecdysial larval homogenate (21 hr) (0.09 &mg: II = 20). The tanning scores were assigned according to VINCENT (1972). 0. no darkening; I. slight darkening, < 30”” dorsal surface of abdomen: 2. darkening in patches on thorax or abdomen, > 30”,, of dorsal surface of abdomen: 21. abdomen entirely dark. thorax not dark or with very little darkening: 3. abdomen and thorax both dark or very near11 so: 3;. interference colours present at the tip to the abdomen and/or the scutellum: 4 (not observed in the bioassay). full4 darkened--i.e. with the ventral abdominal arthodial membrane also dark.
standard saline and dilute homogenates of preecdysial larvae and tanning flies. Pre-ecdysial larvae (21 hr) showed mandibles tanned at stages 1-2 as defined by ROBERTS (1976). Saline injected flies (Fig. 3a) achieved only 11.3”,,total darkening and injections of pre-ecdysial larval homogenate (Fig. 3b) yielded similar results (14.6’!, total darkening). In distinct contrast, injections of tanning fly homogenate (Fig. 3c) produced a total darkening of 67.5”,. These differences were tested with the KolmogorovSmirnov two-sample test (SIEGEL, 1956). The tanning fly homogenate was significantly different from both
the control and the pre-ecdysial larval homogenate (D,a, = 0.90 and 0.83 respectively; P < 0.001). while no significant difference was recorded between the control and the pre-ecdysial larval homogenate. In addition, highly concentrated homogenates of young (9 hr) (Fig. 3d) and pre-ecdysial(21 hr) (Fig. 3e) larvae were assayed (CU. 160 x more concentrated than previously used) yielding scores of only 23 and respectively. Both of these 30”” total darkening homogenates
were
significantly
different
from
the
control (Dn,J, = 0.35 and 0.41 respectively: P < 0.01) and from the tanning fly (D,,,, = 0.84 and 0.85
126
BRIANROBERTS rf al.
respectively; P < 0.001). In spite of the differences in development, there was no significant difference between the tanning produced by these larval homogenates. DISCUSSION Of the many separate processes affected by the ecdysteroids, the moulting cycle is perhaps the best documented. The first visible event in arthropod moulting directly associated with ecdysteroid release is apolysis; the separation of the epidermis from the old cuticle. Previous work (ROBERTS et al., 1980; WENTWORTH et al., 1981) and our present data show clear cut releases of ecdysteroids approximately half way through the first two larval instars. Using first instar larvae of S. bullata, one of us (ROBERTS, 1981) has shown that apolysis of the tracheal epithelium takes place at 16 hr; some 2 hr after the first peak of ecdysteroids. The major biochemical process in the moulting cycle is deposition of new cuticle for which the epidermis synthesizes cuticular proteins, chitin and lipids. Synthesis of RNA and cuticular components has been induced by the addition of ecdysteroids to epidermal structures in vifro (MANDARON, 1970; RYERSE and LOCKE, 1977), which demonstrated the direct action of the hormone on these tissues. In first instar larvae of Sarcophaga, deposition of cuticle by the tracheal epithelium commenced at 18 hr (ROBERTS, 1981), some 4 hr after the maximum titre of 20hydroxyecdysone was recorded. It appears that the major peaks of ecdysteroids mid-way through the first and second instars are directly responsible for apolysis, differentiation of the tissues and the secretion of new larval cuticular structures. The formation of the anterior spiracle (ROBERTS, 1981), the posterior spiracles and the cepholopharyngeal apparatus (ROBERTS, 1976) support this statement. The role of the two smaller peaks of ecdysteroids, just prior to the larval ecdyses, will be discussed below. The tanning of the third instar larval cuticle to form the puparium is among the most investigated ecdysteroid-induced processes. From previous reports (SHAAYA and KARLSON, 1965; BARRI~T and BIRT, 1970; OH~AKI and TAKAHASHI, 1972; HODGETTSet al., 1977; ROBERTSet al., 1980; WENTWORTH et al., 1981), it has been clearly shown that the whole process of pupariation is triggered by the increasing levels of ecdysteroids at this stage of the life cycle. Our report showed that ecdysteroid levels commenced to increase some 8 hr prior to white prepuparium formation, to peak some 18 hr after this event, and to fall again to moderate levels by 32 hr of development. During the period of puparial tanning ecdysteroids activate the gene for decarboxylase which alters the metabolic fate of tyrosine and results in a tanned cuticle (KARLSON and SEKERIS, 1976). We consider the increasing ecdysteroid activity is not only required for the initiation of pupariation and tanning of the puparium but is also necessary for the sequential developmental events of larval/pupal apolysis (22-24 hr) and secretion of the pupal cuticle (25-28 hr) (WENTWORTH ef al., 1981), which occur some few hours after this high titre of ecdysteroids. Newly deposited cuticle is usually tanned just after
ecdysis and consequently it is considered that the ecdysone induced tanning of the dipteran puparium (KARLSON and SEKERIS, 1976) before the pupal moult is a special case in insect ontogeny (WYATT. 1972). Investigations to date have shown that post-ecdysial tanning in insects is controlled by the polypeptide hormone bursicon (FRAENKEL and HSIAO, 1965). Bursicon is believed to act by changing the metabolic fate of tyrosine, as ecdysone does in pupariating flies (FOGAL and FRAENKEL, 1969a; KARLSON and SEKERIS. 1976; RIDDIFORD and TRUMAN, 1978) from phydroxyphenyl propionic acid to dopamine (NEVILLE, 1975). Unlike ecdysone, bursicon is believed to achieve this by altering the permeability of haemocytes and epidermal cells to tyrosine (NEVILLE, 1975; RIDDIFORD and TRUMAN, 1978) and does not involve de now protein synthesis (FOGAL and FRAENKEL, 1969b). Our bursicon assay, scored in the manner of VINCENT(1972). showed that injections of pre-ecdysial larval homogenate yielded similar results to the control saline injected flies. In contrast, injections of tanning fly homogenate produced significant positive tanning. These findings suggest that pre-ecdysial larval tanning of the mouth hooks and spiracles is mediated by some agent other than bursicon. Results of the bioassay using highly concentrated homogenates of young larvae and pre-ecdysial larvae support this suggestion indicating that concentration alone was not responsible for the initial results. The relationship between the homogenate concentration and bursicon activity in S. bullata and Locusta (VINCENT, 1972) can be compared. In Locusta an increase from 14 to 30”, total darkening was produced by only a 2 times increase in concentration. while in Sarcopfmgn the same change in darkening was only achieved by a 160 times increase in concentration. It is possible that the bioassay tanning noted in concentrated homogenates was caused by a type of injury reaction. WIGGLESWORTH(1937) observed that when damaged cells were injected into an insect, they clustered against the recipient’s integument and initiated limited injury repair reactions. SALT (1970) described mechanisms with which insects combat invading organisms, documented the phenomenon of melanin encapsulation. Perhaps the larval homogenate injections resulted in damaged cells adhering to the integument of the bioassay flies and initiating melanin deposition, similar to normal tanning processes. This report showed for the first time that two significant releases of 20-hydroxyecdysone occurred some few hours prior to each of the larval ecdyses. The single peak late in the first instar is clear cut, that of the second instar is of higher values but is less clear due to normal variability in ageing larvae. These releases were not detected in previous investigations (ROBERTS et al., 1980: WENTWORTHet al., 1981) presumably due to greater time gaps between samples. During and after the times of these ecdysteroid releases it has been documented that specific cuticular structures, in S. bullata, undergo pre-ecdysial tanning. The anterior and posterior spiracles and the mouth hooks of the cephalopharyngeal apparatus (ROBERTS, 1976, 198 1) commence their tanning some 3-4 hr prior to ecdysis. Indeed these structures are almost completely tanned at the time of ecdysis; only the
Pre-ecdysial tanning in larvae of Surcophaga hullata articulation of the mandibular hook to the hypostomal sclerite remains to be fully sclerotised (ROBERTS. 1976). Consequently, with respect to the negative result of the bursicon bioassay, the accuracy of the RIA and to our ageing technique of the larvae, it is possible that in S. bulluta and indeed in the higher Diptera that ecdysteroids mediate pre-ecdysial tanning associated with both larval/larval moults in a manner similar to that of puparial tanning prior to the larval/pupal transformation. This conclusion implies that a specific gene set is used for tanning prior to metamorphosis and that a different system may be utilized for the completion of the tanning of the adult cuticle for which bursicon is the reported controlling hormone (FRAENKEL and HSIAO, 1965). We have not ruled out the possibility that the tanning process may be a consequence of the high ecdysteroid titres recorded midway through the instar. If this were the case the genes for tanning would be considered late developmental genes (ASHBURNERand RKHARDS, 1976) and would be functional only after the cuticular structures of the subsequent instar were fully formed. If this were so, the elevated titres just prior to the moult may be required to complete the sclerotization processes. We wish to thank Dr. J. D. ~r~noI~~il~~i~c~t~lrtrl. O‘CONNOK for the kind gift of H’ r-ecdysone and antiserum (Horn l-2).
REFERENCES ASIIBLIKN~.KM. and RICHARDS G. (1976) The role ofecdysone in the control of gene activity in the polytene chromosomes of Dro.so~/&ilu. S>,nry. R. Ent. Sot. 8, 203-225. BAKKIT L. C. and BIKE L. M. (1970) Prothoracic gland hormone in the sheep blowfly Luciliu cuprina. J. Insect PhJsiol. 16, 671-677. BOKST D. W. and O’CONNOR J. D. (1974) Trace analysis of ecdysone by gas liquid chromatography. radioimmunoassay and bioassay. Steroids 24, 637-656. FOGAL W. H. and FKAENEEL G. (1969a) The role of bursicon in melanization and endocuticle formation in the adult tlesh fly Sarcophaga hullata. J. Insect Physiol. E(7), 1235-1248. FOC;AL W. H. and FRAENKEL G. (1969b) Melanin in the puparium and adult integument of the flesh fly Sarcophaga hullata. J. Insect Physiol. 15(S), 1437-1448. FKA~NKEL G. and HASIO C. (1965) Bursicon, a hormone which mediates tanning of the cuticle in the adult fly and other insects. J. Insect Physiol. 11(5), 513-556. GILBERT L. I. and KING D. (1973) Physiology of growth and development. In Physiology oflnsecta (Ed. by ROCKSTEIN M.) 2nd ed.. pp. 249-370. Academic Press. New York. HUDGETTS R. B.. SAC~BB. and O’CONNOR J. D. (1977) Ecdqsone titre during post-embryonic development of Drosophila melanogaster. Devl. Biol. 60, 310-317.
117
KARLS~N P. and SEKER~SC. E. (1976) Control of tyrosine metabolism and cuticle sclerotization by ecdysone. In The Insect Integument (Ed. by HEPBURN H. R.) p. 571. E.lsevier. Amsterdam. MANDARON P. (1970) Development in vitro de disques imaginaux de la Drosophile. Aspects morphologiques et histologiques. Devl. Biol. 22, 298-320. NEVILLE A. C. (1975) Biology of the Arthropod Cuticle. Springer, New York. OHTAKI T. and TAKAHASHI M. (1972) Induction and termination of pupal diapause in relation to the change of ecdysone titre in the flesh fly SarcophaguprrcJ,qrina Jap. J. med. Sri. Biol. 25, 369-376. RIDDIFORD L. M. and TRUMAN J. W. (1978) Biochemistry of insect hormones and insect growth regulators. In Biochemistry of Insects (Ed. by ROCKSTEIN M.) pp. 308-357. Academic Press. New York. ROBERTS B. (1976) Larval development in the Australian flesh fly Tricholioproctia impatiens. .4nn. cwt. Sot. .4tn. 69(2). 158-164. ROBERTS B. ( I98 1) Development of the prothordcic spiracles in larvae of Sarcophuga buliata (Sarcophagidae:Diptera). Aust. J. Zool. 29, 199-204. ROBERTS B. and WARREN M. A. (1975) Diapause in the Australian flesh fly Tricholioproctiu intpatiwts. .4ust. J. Zool. 23, 563-567. ROBERTS B., WENTWORTH S. L. and O’CONNORJ. D. (1980) Ecdysone titres during post-embryonic development in Sarcophaga @dlata (Sarcophagidae:Diptera). XVI ht. Congr. Enr. (Kyoto, Japan) 16, 172. RYEKSE J. S. and LOCKE M. (1977) Ecdysterone-mediated cuticle deposition and the control of growth in insect tracheae. J. Insect Ph.vsiol. 24, 541-550. SALT G. (1970) The Cellular Defense Reuctions oj Inarcts. pp. 118. Cambridge University Press. Cambridge. SHAAYA E. and KARL~CINP. (1965) Der Ecdysontiter wahrend der Insektenent wicklung-II. Die postembryonale Entwicklung der Schmeissfliege Calliphoru er~throcephala Merg. J. Insect Physiol. 11, 6549. SIEGEL S. (1956) Nonparametric Statisticsjbr the Brhcn’ioural Sciences, p. 312, McGraw-Hill, London. SOKAL K. K. and ROHLF J. F. (1973). Introdut~tion to Biostatistics W. H. Freeman, San Francisco. SMITH C. N. (1933) Notes on the life history and moulting process of Sarcophaga securijrra (Villeneuve). Pro<,. enr. Sot. Wash. 35(7), 159-164. VINCENT J. F. V. (1972) The dynamics of release and the possible identity of bursicon in Locusta mrgratoria migratorioide.\. J. Insect Physiol. 25, 653-658. WENTWORTH S. L.. ROBERTS B. and O’CONNOR J. D. (1981) Ecdysteroid titres during post-embryonic development ol Surcophaga builata (Sarcophagidae: Diptera). J. Insect Physiol. 27, 435440. WHITTEN J. M. (1969) Coordinated development in the fly foot: sequential cuticle secretion. J. Morph. 1271 I ). 73-104. WIGGLESWORTH V. B. (1937) Wound healing in an insect (Rhodnius prolixus: Hemiptera). 1. e.xp. BioI. 14, 364-38 I. WYATT G. R. (1972) Insect hormones. In Biochm~ic~ol Acriom 01 Hormones (Ed. by LITWACK G.) Vol. 2. pp. 38-5-490. Academic Press, New York.