Ecdysterone triacetate-induced molting in the American lobster (Homarus americanus)

Camp Bwchrm Phrsid.. Vol. 64A. pp 125 to I31 Q Pergamon PressLtd 1979.Pnntsd in Great Bntain

ECDYSTERONE TRIACETATE-INDUCED IN THE AMERICAN LOBSTER (HOMARUS AMERICANUS)

MOLTING

M. W. GILGAN and B. G. BURNS Department of Fisheries and the Environment, Maritimes Region, Technology Branch, P.O. Box 550, Halifax, N.S. B3J 2S7, Canada (Receiaed 18 December 1978) Abstract-l. Ecdysterone-2,3,22_triacetate (EAc,), the principal component of the crude ecdysterone acetate (EAc) preparations which successfully induced molting in the lobster, was responsible for the

bulk of the activity of the mixture. 2. EAc, treatment exhibits a dose-response threshold similar to that of ecdysterone (E). 3. EAc, on a weight basis is about one-fourth as active at premolt development induction as E. 4. Ethanolic solutions of EAc, were as effective as the original, successful EAc oil emulsions, 5. EAc, was about equally successful with male and female adult lobster both in the winter and summer. 6. Lobsters were more sensitive to EAc3 in the summer than the winter despite equivalent initial intermolt stages for both sets of experiments and identical physical experimentaf conditons.

INTRODUCTION Several studies by this laboratory (Gilgan & Zinck, 1975; Gilgan et al., 1977; Gilgan & Burns, 1977b; Gilgan & Farquharson, 1977) and others (Flint, 1972; Rao et ai., 1973) have been made with mixed success with ecdysterone injections in attempts to induce accelerated premolt development in the lobster (Homarus americanus) with normal survival after molting. The results were most successful with eyestalkless adult lobsters (Flint, 1972) which presumably would have molted precociously in any event, or with rapidly molting juvenile lobsters (Rao et al., 1973). Only modest success was achieved with intact adult lobster after repeated ecdysterone injections (Gilgan & Zinck, 1975) and under special circumstances after combined cc-ecdysone and ecdysterone treatments (Gilgan et al., 1977). Both procedures seemed to allow more prolonged exposure to the ecdysteroids. In attempts to further prolong the exposure, we subsequently discovered that treatment with an emulsion of the crude acetate mixture, produced by the acetylation of ecdysterone with acetic anhydride in pyridine, could induce premolt development followed by successful molting and subsequent survival (Gilgan & Burns, 1976). Comparable emulsions of equivalent amounts of free ecdysterone induced premolt development but the lobsters did not survive. We therefore wished to determine which part of the successful procedure was necessary for its success; the emulsion or major or minor components of the crude acetate mixture. Since previous studies had shown a pronounced difference in the response of lobsters captured and treated in the spring and summer as compared to those in the fall and winter (Gilgan et at., 1977) we wished to extend the previous ecdysterone acetate treatment studies using winter lobsters (Gilgan & Burns, 1976) to equivalent studies using summer lobsters. We wished also to determine if male and female lobsters differed, since female crustaceans

might be expected to behave differently because egg maturation and laying might complicate their endocrinology (Passano, 1960). Within our limited facility and availability of equivalent lobsters, we wished to determine sensitivity to the treatment by graded dose regimes. The following is a detailed report of our procedures and results following our preliminary report (Gilgan & Burns, 1977).

MATERIALS AND METHODS Test animnl conditions

Small adult lobsters were obtained from the commercial fishery and normally transported to the laboratory within 10 h of capture. At the laboratory they were maintained in unrecirculated, filtered seawater at 15°C in a room with controlled lighting (16LSD) as described previously (Gilgan & Burns, 1977). Only animais in stages C-D, or D,, determined as described previously (Gilgan & Zinck, 1975). were accepted as experimental animals. Ecdrsteroids

Standard ecdysterone (E,&eedysone, 20-hydroxyecdysone, crustecdysone; Rohto Pharmaceutical Co., Japan) solutions were normally prepared in ethanol (95%) and the concentration confirmed by measuring the optical density at 243 nm as before (Gilgan & Zinck, 1975). Oil emulsions were prepared from Freund’s incomplete adjuvent, ethanol and water (2:l: 1) as before (Gilgan & Burns, 1976). The crude ecdysterone acetate (EAc) preparation was made by reacting E with acetic anhydride in anhydrous pyridine as before (Gilgan & Burns, 1976). The major component could be purified by chromatography on a short alumina column (2 cm i.d. x 3 cm), after the residual pyridine and pyridinium salts were removed by repeated coevaporation with isobutanol in a rotary vacuum evaporator. The sample was applied to the column in ethyl acetate, the column washed thoroughly with that solvent and the EAc eluted with a small volume of redistilled acetone. The 125

of the selected lobhter\ Ivr Experiment I h) random numbers. For the other experiment\ the lobsters were distributed among groups to allow uniform average weights and weight distribution. The lobster\ uere allowed to acclimatize to their small tank and the water temperature for at least one week before thrv were treated. fe\t solutions or cmulalons were m~ected into the tail muscIc at a constant dose of solution (? jtl:g) or emulsion (0.X 111F) irrespective of the ecdysteroid dose. The ecdystercold concentration wab therefore selected to buit the neccssar? doac bolumc. The trcatmcnt ratca for the ccdystcroids, \\erc as indicated in /q ecdysteroid,g lite weight. The prsmolt development of the lobsters before and dur~ng the experiment was monitored by examining the shell and \wm~merct tips as berore (Gilgan & Zmck. 197.5; Alhen. 1973). Also as before we comerted the premolt \tagc 01 Indiwdunl lobsters to corresponding number values. averaged the numbers for the treatment group and recon\crted the number\ to the aberage premolt stages ah hho*n in F1g5 I 4 depicting premolt development. This allowed ur to assess the effectiveness 01” the initial trcatment and dctcrmlnc when the indicated second treatment I+ 35 ncccw1r\ Test

groups

here assi_ened

The adult. male lobsters for this experiment were obtained from Three Fathom Harbour in May and the trial was started in June. The groups of five lobsters. wlccted and distributed as mentioned above. were treated with the doses indicated in Table I in 011 emulsions. The crystalline EAc, dose (2.7~1g;g) contained the same amount of hormone as the free ecdysterone dose (X)/cgi~).

tt 1/

CONTROL

,

E (2.OpgIg)

EAc,

(2.7pgIg)

i

I+

- -

I

-

I

EAc (3.8yg)

t

-~ *_

1

.-i. t

DAYS

FROM

FIRST

TREATMENT

Fig. I. Premolt development of male lobsters after treatment in the late sprmg wth <)il-emulslor of ecdysterone (E), crystalline ecdysterone triacetate (EAc,) or crude ecdysterone acetate (FAc) in 1111 emulsions. When used. the second treatment time is indicated (7) (m = molts. t .r deathsl.

127

Molting in the American lobster

-15

15

0

30

DAYS

FROM

45

60

FIRST

75

90

105

120

135

TREATMENT

Fig. 2. Premolt development of adult, male lobsters after ~eatment with graded ecdysterone triacetate doses in oil-emulsions. The second treatment time is indicated (7) ( x , control; 0, 0.6; Q 1.3; A, 2.5; 0, 4.0 and 8, 5.0 pg EAc,/g live weight).

Nuclear Magnetic Resonance (NMR) spectrum compound in perdeuteropyridine which showed the three distinct methyl proton peaks area where acetate methyl proton absorption

1 c -15

0

15

of the clearly in the would

occur. Since the remainder of the NMR spectrum was essentially that of E and the ultra-violet spectrum was also unchanged from that of E we concluded that the crystalline material was in fact ecdysterone-2,3,22-

30 45 60 75 90 105 DAYS FROM FIRST TREATMENT

120

135

Fig. 3. Premolt development of adult, male lobsters after treatment with graded ecdysterone triacetate doses in ethanol. The second treatment time is indicated (7) ( x, control; 0, 0.6; q, 1.3; h, 2.5pg EAc,/g live weight).

DAYS

FROM

FIRST

TREATMENT

Fig. 4: Premolt development of adult, female lobsters after treatment with graded doses of ecdysterone triacetate in oil-emulsions, The second treatment time is indicated (7) (x, control: 0. 0.6: F. 1.3: iI,, 2.5; l , 4.0 and V. 5.Opg EAc,/g live weight).

triacetate the principal product of our acetylation procedure and hence the principal component of our injected crude EAc. Experiment

1

The treatment procedures of this experiment were intention~ly not varied from those used previously (Giigan & Burns, 1976). We wished to compare the effectiveness of the material with lobsters which would be expected to soon molt naturally (during summer or early fall, that we refer to as summer lobsters), to those used previously which would not have molted naturally for about 6 months from the time of first treatment (Gilgan 2%.Burns, 1976). The

Table

I. Treatment

Treatment* (K&f Control E 2.0 EAc 25 EAc

3.8

EAc 5.0 EAc, 2.1

of groups

Average weight (g + S.D.1

of five male

only significant modification was to include treatment utilizing crystalline EAc,. It is apparent from the results of Table 1 that E. while it induced rapid premolt development as usual (Fig. l), was lethal at the dose given. The crude EAc dose, containing an equivalent quantity of ecdysterone, on the other hand induced less rapid premolt development (Fig. 1) but allowed a high degree of successful molting. With the “summer” lobsters. it second dose was not required at 3.8pg;g and above. The pure EAcs was approximately as effective at inducing premolt development and at allowing subsequent successful molting as was the approximately equivalent crude acetate dose (Table t. Fig. 1).

lobsters with emulsions

Number Molts

of Deaths

ecdysterone

or ecdysterone

acetate

in 011

Mean time (days)+ from last treatment to Death Molt

358 + 46.9 359 * 42.6 360 + 44.3

0 0 4

0 5 0

355 + 47.0

4

0

34 + x

349 f 57.3 360 + 43.6

4 3

1 0

51 f 5 43+ 17

‘4 *

1

3x + 23 41

ecdysterone triacetate. * E. ecdysterone: EAc, crude ecdysterone acetate: EAc,, crystalline of single treatment, t A second treatment the same size as the first was given 34 days after the first only animals which did not show significant premolt development.

Amounr to those

Molting Table 2. Dose-response

Treatment (pg EAc,/g) Control 0.60 1.3 2.5 4.0 5.0 * The second

129

lobster

relationship for groups of 10 male lobsters which received tions of ecdysterone triacetate in oil emulsion

Average weight (g k S.D.) 350 351 350 350 351 350

in the American

f + + f + +

treatment

Number Molts

55.5 55.9 55.0 54.1 51.3 49.4

0 0 0 0 3 4

44 41 38 30 32

* f f +

Experiment 3

DISCUSSION

Table 3 shows the EAc,, injected intramuscularly, was at least as effective in ethanol as it was in oil emulsion. The results of this experiment are comparable with those of Experiment 2 in that the lobsters were obtained at the same time, held in the same facility and the experiments run concurrently. In fact the treatment in this experiment may have been more effective than that with oil emulsion, probably because the doses were on the average more precise. While the premolt development induced was more erratic (Fig. 3) than before (Fig. 2) it was not systematically different.

We conclude that ecdysterone-2,3,22-triacetate was the major component of our original crude acetate mixtures with which we first successfully induced premolt development followed by molting with high survival frequency in the lobster (Gilgan & Burns, 1976). The minor components of the crude mixture were probably also active but were not markedly more active than EAc, since EAc, showed about the same specific activity as the mixture. EAc, treatment exhibits a dose-response threshold (Fig. 2) like that of free E (Gilgan & Zinck, 1975), presumably for the same reason, but the transition from effective to ineffective dose was less abrupt. There seems to be a distinction in the treatment of adult lobsters between slightly advancing premolt development, which could give a statistically significant

Experiment 4 The results of this experiment for “winter” lobsters are also directly comparable to those of Experiment 2 and for the same reasons as Experiment 3. The

* The second

of groups

of five male lobsters

Average weight (g zk S.D.)

Control 0.6 1.3 2.5

356 355 353 353 treatment

20+ 11 29 f 6.5

results in Table 4 show that the treatment was about as effective with female, “winter” lobsters as with male, “winter” lobsters (Table 4). However the response threshold of the females was apparently slightly higher than for males since 2.5 pg EAc,/g was less effective at both premolt development and molt induction (Fig. 4). On the other hand, none of the females died after the high dose treatments, all or nearly all molted. The female, “summer” lobsters gave a response similar to that obtained previously (Experiment 1) for males except for the peculiarly large number of deaths. The deaths do not seem to relate to the treatments (Table 4). As observed with the male lobsters, the treatment was more effective with the “summer” lobsters. In this case, a second treatment was not used but generally accelerated premolt development and molting was nevertheless achieved. No definite enhancement of egg-laying was observed with “winter” or “summer” lobsters.

It is apparent both from Table 2 and Fig. 2 that doses less than 2.5 pg EAcs/g were essentially without effect. Two treatments with doses of 2.5pg/g and above were highly effective at inducing premolt development in these “winter” lobsters (Fig. 2) but single treatments with 4.0 and 5.0 pg/g were quite toxic. It should be noted from Fig. 2 that the two highest doses resulted in rapid and relatively complete premolt development after a single dose. The absence of molts among the controls shows that in all cases the molts induced were much earlier than would have occurred naturally, not just slightly more rapid than normal as would have resulted after non-toxic, and hence very small, doses of free ecdysterone. Those lobsters treated with a single 2.5 pg EAc,/g dose might not have molted earlier than the controls. Therefore the 2.5pg/Eac,/g dose would seem to be about at the threshold for an effective two-dose scheme.

Treatment (pg EAc,/g)

12 8.1 2.6 6.4

was 70 days after the first.

Experiment 2

Table 3. Responsiveness

injec-

Average time (days) from last treatment* to Molt Death

of Deaths

0 1 2 7 I 6

intramuscular

Molts

+ 41.1 &- 41.4 + 41.4 + 48.7

was 70 days after the first

0 1 3 4

injected

with ecdysterone

Number

of Deaths 0 0 0 0

triacetate

in ethanol

Average time from last treatment* to molt

63 482 15 31 + 3.4

hut marginal shortening of the time to molt, and the induction of the presumably irreversible premolt development in a single treatment. Also the sub-threshold treatments seem accumulative (Figs 1-4) since the induced premolt development does not normally reverse. However. after a certain threshold is achieved. either of the amount of ecdysterone or its elfect. then the lobsters stem obliged to proceed with rapid premolt t~e~elopmcnt which, after E treatment at ail effective dose Ievcls, leads normally to their death at molt. In contrast EAc, treatment. at low levels. does not. Free E was much more elective on ;I wcigbt basiq at inducing premolt development than uas EAc, hut for unknown reasons effective doses were ncarlq always lethal (Gilgan & Zinck, 1975). Approxinl~~tely four times as much E must be administered as EAc, (in two doses) as would be required of free E to achieve the same premolt development induction. We have not been able to consistently induce premoit development and molting without using two triacetate treatments. Single doses of EAc, large enough to always induce molting with a single tr~tmcnt were generally toxic. Iike E treatments (Table+ 1 and 3). Usually only “summer” lobsters were sufficiently sensitibe or responsive to molt and surbi\c after a single EAc, treatment (Table 1). EAc, was about as effective with female, adult lobsters in winter and in summer as it had been with males. While there was isolated instances of egg-laying and perhaps egg maturation (green-pigmented protein in the normally blue or colorless hemolymph) the incidence was too low to relate it to treatment. Since the injections of EAc, in ethanoi (Table 3) were as efl’ective (perhaps more so) as those in oil emulsion (Table 2) either the oil emulsion was not stable

or the solubility

of EAc3

in the oil

was not

affect the speed of release. Recent studies in this laboratory (Gilgan & Burns. unpublished results) have shown that an effective dose of EAcj injected into the muscle in ethanol was as rapidly distributed in the hemolymph of a lobster as was E injected directly into the abdominal sinus (Gilgan & Farquhurson, unpublished results). Apparently. theresufficient

tcl

fore, EAc, &ectiveness as compared to E was not related to the speed of release from the injection site. The function of the acetate groups on the EAc, molecule which moderates the prcmolt accelerating effect of E and allows successful molting is still not clear. As mentioned above. the acetates do not apparently affect the rate of release from the injection site. The EAc, appears very quickly in the hemolymph and all tissues must therefore be exposed to the compound via the hemolymph circulation. However, injected radio-labeled EAc, does not persist in the hemolymph as such. nor as E or its metabolites in contrast to the case with E itself(Gilgnn & Burns, unpublished results) where labeled materials persist in the hemolymph. Therefore EAc, is quickly removed by the tissues. There is no indication as to what form is biillogically active: the partial or unhydrolysed ester or the free E. While we were unable to find a dose of EAc, which would cause successful molting after a single treatment with “winter” lobsters. “summer” lobsters generally molted after a single treatment (Tables 1 and 4). It is not clear why this was the case however since “winter” and “summer” lobsters were about the same size, at the same temperatures and in the same intcrmolt stages at treatment. Apparently the “summer” lobsters were just more sensitive and able to respond. This variation in sensitivcity may relate to that observed previously between lobsters in stages D,, and D, from the same group (Gilgan & Farquharson. 1977) and which was then presumed to indicate a probable change in target tissue sensitivity to E. Freeman & Bartell (1976) habe observed that eyestalk extracts appear to reduce the sensitivity of periferal tissues to injected E. The classical molt inhibiting hormone (MIH) was presumed to exert its inhibitor effect only on the Y-organ (Passano, 1960). We assumed little or no molt hormone secretion was taking place in the D; lobsters since the control group showed little inclination to progress to molt. However, since they were clearly in early premolt the MIH should not have been present. Since the D; lobsters were markedly more sensitive to injected F than were the

Molting

in the American lobster

C and De lobsters, it seemed to indicate that the MIH might act on the target tissues. This would require two MIH, one to control E secretion and one to control tissue sensitivity. The sensitivity variations between “winter” and “summer” lobsters in the present studies, and after temperature changes (Gilgan & Farquharson, 1977; Aiken & Waddy, 1975) could also be due to a hormonal control of tissue sensitivity, separate from that which controls E release (or synthesis) and thus premolt development. The same sensitivity differences as we have observed also could be produced by variations in the ability of the lobster to metabolize and/or excrete the ecdysteroids. A major effect of the acetate groups therefore may be to limit the rate of metabolism of the E and the type of metabolite to which it is converted. These experiments show that ecdysterone triacetate can be used to force male and female lobsters to molt precociously and successfully. The treatment with EAc, was markedly more successful than that with the presumed natural hormone, ecdysterone. Generally two low dose EAc, treatments were required to force molting but one large treatment was toxic. It therefore appears that the premolt development induced by the first dose does not reverse over a long time interval. This suggests that the hormonal form of EAc, is bound at an active site for a prolonged period.

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setal development and molt prediction in the American lobster (Homarus americanus). J. Fish. Res. Bd Can. 30, 1337-1344. AIKEN D. E. & WADDY S. L. (1975) Temperature increase can cause hyper-ecdysonism in American lobsters (Homarus americanus) injected with ecdvsterone. J. Fish. Res. Bd Can. 32, 1843%i845. FLINT R. W. (1972) Effects of eyestalk removal and ecdysterone infusion on molting in Homarus americanus. J. Fish. Res. Bd Can. 29, 1229-1233. FREEMAN J. A. & BARTELL C. K. (1976) Some effects of the molt-inhibiting hormone and 20-hydroxyecdysone

131

upon molting in the grass shrimp, Pulaemonetes pugio. Gen. camp. Endocr. 28, 131-142. GALBRAITH M. N. & HORN D. H. S. (1969) Insect moulting hormones: Crustecdysone (20-Hydroxyecdysone) from Podocarpus elatus. Aust. J. Chem. 22, 1045-~1057. GILCAN M. W. & ZINCK M. E. (1975) Response of the adult lobster (Homarus americanus) to graded and multiple doses of ecdysterone. Comp. Biochrm. Phxtiol. S2A, 26 l-264. GILGAN M. W. & BURNS B. G. (1976) The successful induction of molting in the adult male lobster (Homurfrs americanus) with a slow-release form of ecdysterone. Steroids 27, 571&580. GILGAN M. W. & BURNS B. G. (1977a) On the reduced sensitivity of the adult male lobster (Homurus umericanus) to ecdysterone at reduced temperatures. Camp. Biochem. Physiol. 58A, 33-36. GILGAN M. W. 8~ BURNS B. G. (197713) Molt induction in lobsters (Homarus americanus) by intramuscular injection of ecdysterone triacetate. Evperientiu 33, 1114-l 115. GILGAN M. W. & FARQUHARSON T. E. (1977) A change in the sensitivity of adult male lobsters (Homarus americanus) to ecdysterone on changing from mtermolt to active premolt development. Camp. Biochr,m. Physiol. 58A, 29-32. GILGAN M. W., FARQUHARSON T. E. & BURNS B. G. (1977) The effect of cc-ecdysone, ecdysterone and inokosterone treatment, separately or in combinations, on premolt development and molting in adult male lobsters (Homurus americanus). Comp. Biochem. Physiol. 56A, 43-49. IMAI S., FUJIOKA S., NAKANISHI K., KOREEDA M. & KUROKAWA T. (1967) Extraction of ponasterone A and ecdysterone from Podocarpuceue and related plants. Steroids 10, 557-565. IMAI S., TOYOSAT~ T., SAKAI M., SATO Y. FUJI~KA S., MURATA E. & GOTO M. (1969) Isolation of cyasterone and ecdysterone from plant materials. Chum. pharm. Bull., Tokyo 17, 34&342. PASSANO L. M. (1960) Molting and its control. In The Physiology of Crustacea (Edited by WA~RMAN T. H.). Vol. 1, pp. 473-536. Academic Press, New York.

RAO K. R.,FINGERMAN S. W. & FINGERMAN M. (1973) Effects of exogenous ecdysones on the molt cycles of fourth and fifth stage American lobsters, Homurus americanus. Camp. Biochem. Physiol. 44A, 110551120. TAKEMOTOT., OGAWA S. & NISHIMOT~ N. (1967) Isolation of the mouhing hormones of insects from Achyunthi.s Radix. J. pharm. Sot. Japan 87, 3255327.