Stimulation of hydrocarbon biosynthesis by ecdysterone in the flesh fly Sarcophaga bullata

J. Insect Physiol., 1975, Vol. 21, pp. 1581 to 1586.’ Pergumon Press. Printed in Great Britain.

STIMULATION OF HYDROCARBON BIOSYNTHESIS BY ECDYSTERONE IN THE FLESH FLY SARCOPHAGA BULLAX M. T. ARMOLD* and F. E. REGNIER?. Department

of Biochemistry,

Purdue University,

Lafayette,

IN 47907,

U.S.A.

(Received 7 February 1975) Abstract-Ecdysterone has been shown to stimulate hydrocarbon biosynthesis in Sarcophaga bullata at pupariation. When post-feeding larva were treated with 3H-acetate 10.5 hr aftrr hormone administration, a l-3 times greater quantity of 3H-acetate was incorporated into hydrocarbon in the ecdysterone injected insects than controls. A similar experiment with a 24 hr delay of 3H-acetate administration following hormone treatment resulted in 3.3 times greater incorporation into hydrocarbon of treated animals. Isolated integuments synthesize hydrocarbon from acetate better than internal tissues, and the integuments of ecdysterone-treated insects incorporate acetate into hydrocarbon 9.8 times better than integuments of control insects. This indicates that cuticular hydrocarbon biosynthesis not only occurs in the integument, but that a locus of regulation is present in the integument.

INTRODUCTION

Injection of insects

ARMOLD ANI) REGNIER (1975) have shown that in Sarcophaga bullata Parker the increased rate of

hydrocarbon biosynthesis accompanying pupariation can be delayed or prevented if pupariation is delayed or prevented. This establishes a correlation between these two physiological processes. Pupariation is known to be hormonally regulated (FRAENKEL, 1935), and the stimulation of pupariation of an isolated Calliphora abdomen was used

as the biological assay during the isolation and identification of the moulting hormone ecdysone (KARLSON, .1956). Since early treatment with moulting hormone causes precocious pupariation, it may also stimulate hydrocarbon biosynthesis. This paper examines the effects of ecdysterone on pupariation and hydrocarbon biosynthesis.

MATERIALS

AND METHODS

Sodium acetate (1J”C) (20-45 mCi/m-mole) and sodium acetate (3H) (500-1000 mCi/m-mole) were obtained from Schwartz/Mann. Sodium acetate (3H) (100 mCi/m-mole) was obtained from New England Nuclear. Eastman silica gel thinlayer plates without a fluorescent indicator were obtained from Sargent-Welch Scientific Company. Ecdysterone was obtained from Schwartz/Mann. * Present address : Water Science Subdivision, Environment Canada, 562 Booth Street, Ottawa, Ontario KlA OE7, Canada. t To whom correspondence should be addressed.

Insects were injected with a 250,ul syringe adapted to a micrometer for accurate reproducible injections. Doses were delivered with 30 gauge needles inserted a few mm through abdominal intersegmented folds, posteriorly for single, posteriorly and anteriorly for double treatments. In some cases, injection through the spiracles was used. Test solutions were made up in the buffered saline solution of ZEBE and MCSHAN (1959). Incubation conditions The insects used were day 7, 8, or 9 larvae which had been maintained on wet filter paper since they had been washed from the medium on days 6 or 7. After injection the insects were returned to wet filter paper to prevent release of endogenous moulting hormone which normally ensues in dry environmental conditions. Water was added periodically to maintain the paper in a moistened state. The length of the incubation periods varied with the particular experiment and is described in each experiment. Extraction, purification hydrocarbons

and quantitation

of

Each insect was cut into pieces and added to 1 ml 20% KOH in 50% methanolic water. After overnight digestion at 7O”C, 1 ml hexane was added and the phases mixed on a vortex mixer for 15 set at high speed. After the phases had separated, 200 ~1 of the hexane phase was spotted onto the origin of a 2 cm wide tract of an Eastman silica gel TLC

1581

1582

M. ‘I’. AFWOLD

and F. E.

REGNIER

release of endogenous hormone. After several preliminary experiments it became apparent that when left on filter paper none of the buffer-injected larvae would pupariate and most of the ecdysterone injected larvae did. Two other important observations were made from these experiments: (1) injection beneath the intersegmental folds of the abdomen resulted in much lower mortality than injection into one of the spiracles and (2) among ecdysterone-injected insects, the per cent that pupariated, the degree of synchrony of pupariation, and the time when the majority began pupariation, were all functions of the duration of confinement of post-feeding larvae to wet filter paper before injection. Generally it was observed that maintenance on wet paper for less than 24 hr resulted in experimental populations that were less synchronous, less Experiments with isolated tissues sensitive, and responded later to ecdysone than All dissections were performed under ZEBE and insects maintained on wet filter paper for 24 to 48 hr. Therefore all insects used in subsequent MCSHAN (1959) buffer. The anterior and posterior tips of the insects were removed with iris scissors. experiments were maintained on wet paper for a One or two longitudinal cuts were then made and minimum of 24 hr before injections were made. In the previous studies (JACKSONet al., 1973; the integument layed out to expose the internal tissues. Under 10 power magnification, all tissues ARMOLDand REGNIER,1975), cuticular hydrocarbons were quantitated gas chromatographically. This which were unattached or loosely attached to the integument were removed. The isolated tissues technique is excellent when a sufficient number of insects are available, but may not be sufficiently were then blotted with paper, weighed, and incubated in 0.5 ml buffer containing additions approsensitive to see small differences in amounts of priate to the experiment. At the end of the hydrocarbon from smaller numbers of insects. Therefore, an alternate, more sensitive assay was incubation period 0.5 ml 40% KOH in methanol was added, and the tissues were digested overnight needed, since the administration of ecdysone to large numbers of insects during a short time period at 70°C. The extraction, purification, and quantitation of the hydrocarbons are as described above. was not feasible. Numerous workers have reported the incorporation of acetate into hydrocarbons by insects (CLARK and BLOCH, 1959 ; ROBBINS et al., Statistical analysis of results 1960; LOLJLOUDES et al., 1961; GORDONet al., 1963; PIEK, 1964; VROMANet al., 1965; LAMBREMONTet The standard deviations and tests for significance al., 1966; LAMB and MO~XOE, 1968; NELSON, 1969; were calculated by the procedures described in JACKSONand BAKER, 1970 ; CONRADand JACKSON, SNEDECOR(1956). 1971). Preliminary studies showed that S. bullata could also incorporate acetate into hydrocarbon. RESULTS Therefore the measurement of acetate incorporation was adopted as the assay for the following experiContact with water inhibits pupariation in postfeeding larvae of Sarcophaga peregrina (OHTAKI, ments. Some of the effects of hormones are rapid, and 1966), and post-feeding larvae of Sarcophaga others are slower. Therefore, it was necessary to argyrostoma (ZDAREK and F’RAENKEL, 1970). In establish, by trial and error, not only if, but when, both studies the inhibition of pupariation caused by ecdysterone exerts an effect on hydrocarbon biowater contact could be overcome in isolated abdosynthesis., mens by injections of ecdysone. It has been shown A small population of post-feeding larvae which (ARNOLDand REGNIER,1975) that contact with water had been maintained on wet filter paper was split inhibits pupariation in post-feeding larvae of S. into two groups. One group was injected with bullata. It seems likely that injections of ecdysone 2 ~1 ZEBE and MCSHAN (1959) buffer containing should also overcome the inhibition of pupariation in S. bullata. 2 $i 3H-acetate. The second group was injected with the same materials plus 2 pg ecdysterone. Post-feeding larvae of S. bullata which had been After 22.5 hr the insects were sacrificed and analysed maintained on wet filter paper for varying lengths for the incorporation of 3H-acetate into hydroof time were injected through the spiracles or carbons. The ecdysterone-injected insects incorpobeneath the intersegmental folds of the abdomen rated 1.2 times more acetate than the control insects, with buffer with or without 2 pg ecdysterone. The which differences was insignificant at the 95 percent insects were returned to wet filter paper to prevent

plate without a fluorescent indicator. Authentic hydrocarbon standards were spotted onto other tracts to determine the Rf of hydrocarbons on these plates in the developing solvent hexane. The plates were developed, the Rt of the authentic hydrocarbons determined by visualization in an iodine tank, and the regions of the experimental tracts with the corresponding RT were marked. The tracts were sprayed lightly with water to facilitate scraping (SHAPIRO, 1972), then scraped onto weighing paper. The scrapings were transferred into scintillation vials containing 15 ml of a solution of 5 g 2,5-diphenyloxazole (PPO) per litre toluene. Insects sacrificed immediately after injection served as controls.

Stimulation of hydrocarbon biosynthesis by ecdysterone in the flesh fly

1583

Table 1. The effects of ecdysterone on hydrocarbon biosynthesis in S. bulluta when substrate and hormone are coinjectedr

Trial 1

Sodium Acetate (%3

(2 wm

22 I,2 mus

II

I.115

+

2%

+ 2 pg Ecaystei-one ____________________________________________________________~___________

confidence level. When the experiment was repeated, the ecdysterone-injected insects and the control insects incorporated essentially the same quantity of aH-acetate into hydrocarbon. Table 1 summarizes these results. The similar rates of incorporation for the test and control insects contrast with the observed differences in rates of hydrocarbon biosynthesis seen previously (ARMOLD and REGNIER, 1975). While the ecdysterone-injected insects had pupariated between the time of injection and the time they were killed, the controi insects were all apparently normal post-feeding larvae at the end of the experiment. One possible explanation for the similar rates of incorporation could be that the substrate was metabolized by the organism before the hormone exerted its effects upon the rate of hydrocarbon biosynthesis. To test this possibility another small population of post-feeding larvae was split into two groups which were injected with 2 ~1 buffer with or without 2 pg ecdysterone. These insects were killed 9 hr after injection before any morphological evidence of pupariation was visible in the ecdysterone-injected insects. After corrections had been made for the amount of radioactivity used in the experiments, it was clear that the insects sacrificed after 9 hr had incorporated approximately the same amount of radioactivity into hydrocarbon as those insects sacrifked 1 day after injection. In addition, the ecdysterone-injected insects and the control insects incorporated virtually the same amount of 3H-acetate into hydrocarbon. Two conclusions are possible from this experiment: (1) the hormone does not appear to exert an immediate effect upon

hydrocarbon biosynthesis and (2) the labelled substrate may be available for hydrocarbon synthesis only for a relatively short period of time before it has been utilized by and lost to other metabolic pools. In an attempt to overcome the problem of other pathways removing the substrate prior to the hormone exerting its effect, a double injection technique was developed. In a feasibility study post-feeding larvae were injected with buffer, then injected with buffer a second time a variable number of hours later. Two injections beneath the abdominal intersegmental folds caused little mortality in the larvae and, furthermore, what appeared to be normal adults developed from the majority of the doubly injected larvae. Once it had been established that injecting material into post-feeding larvae twice within a short period of time would not seriously injure the insects, experiments using two injections were designed to overcome the above problem. Postfeeding larvae were injected with either 2 ~1 buffer or 2 ~1 buffer containing 2 pg ecdysterone. When the ecdysterone-injected larvae began to pupariate, all of the insects were injected with 2 ,ul buffer containing 2 &i “H-acetate. The results of this experiment are shown in Table 2. The incorporation of 3H-acetate into hydrocarbon by the ecdysterone-injected insects was 1.3 times greater than for the control insects, this difference being insignificant at the 95 per cent confidence level. The above experiment was repeated with the Four pCi sH-acetate were fallowing alteration. injected into ecdysterone-injected insects and

M.T.

1584

AFCMOLDAND

F.E.

FCEGNIER

Table 2. The effects of ecdysterone on hydrocarbon biosynthesis in S. bullata when substrate is injected at onset of pupariation

Table 3, The effects of ecdysterone on hydrocarbon biosynthesis in S. bullata when substrate is injected 6 to 12 hr after onset of pupariation in isolated tissues Material in 1st Injection

Trial

Time

2nd

Injection made

after

Quantity mjected

Time

after

Sacrificed 2nd Injection

Number

Of

Average

cd

std.

Tndivicluals Incorporated

1st

(per

sndiv.

Dev.

1

1

Buffer

24 Hours

Sodium Acetat* (%)

Buffer +

24 I1ours

a.3 Ekaysterone

Sodium Acetate ?x)

9

7

1875

!: 379

9

?

6263

* 16.96

(4 pci)

(4 Vi)

______________________~__~~~~~~~~__~~~~___~_~__~~__~~~~~~~~~~~~~~~~~~~~~~~~_~___~~~___~~_______________

Buffer

24 IIDUrs

Sodium Acetate &I)

1

. Difference in incorwxation

9

7

931

c

547

(2 p.Ci)

in Trial 1 simificant at 99.9% confidencelevel, Trial 2 at 97.5%

control insects 24 hr after the initial injection of buffer with or without ecdysterone. At this time the ecdysterone-injected insects were 6 to 12 hr past the onset of pupariation, while none of the control insects had pupariated. Nine hr after the injection of 3H-acetate the insects were killed and treated as above. The hormonally treated insects incorporated 3H-acetate into hydrocarbon 3.3 times better than the control insects. Statistical analysis of these data show that the observed difference is significant at the 99.9 per cent confidence level. The above experiment was repeated, and the results of both trials are shown in Table 3. The above experiments show that insects injected with ecdysterone incorporate acetate into hydrocarbon better than insects which do not receive the hormone. Attempts were next made to determine where cuticular hydrocarbon biosynthesis occurs in S. bullata. Post-feeding larvae were injected with 2 ,ul buffer with or without 2 pg ecdysterone. The next day the insects were dissected and tissues separated into two groups. At the time of dissection the ecdysterone-injected insects were 12 to 22 hr past the onset of pupariation. The

cuticle and epidermis with attached muscle and some tracheae made up one group of tissues and were designated the integument. The fat body, gut, Malpighian tubules, and all other internal tissues which were not attached or were loosely attached to the integument were pooled and designated the viscera. Since the dissections occurred in buffer, the haemolymph was diluted greatly and would not contribute to either group of tissues. The isolated tissues from three insects were then incubated in 05 ml buffer containing 30 $Zi 3H-acetate. The results of these experiments are shown in Table 4. The preparations of isolated integuments incorporate 3H-acetate. into hydrocarbon better than the preparations of viscera. Furthermore, the isolated integuments of the ecdysterone-injected insects incorporate “H-acetate 9.8 times better than the isolated integuments of the control insects. Even though there was considerable variation among the individual determinations, the difference was significant at the 95 per cent confidence level. Since the variance in the above experiment was high, the experiment was repeated using 20 ,nCi l*C-acetate rather than the 3H-acetate. As shown

Stimulation of hydrocarbon biosynthesis by ecdysterone in the flesh fly

1585

Table 4. The effects of ecdysterone on hydrocarbon biosynthesis in S. bullata in isolated tissues

Table 5. The effects of ecdysterone on hydrocarbon biosynthesis in S. bullatain isolated integuments Time between Injection and Dissection

Tissue

IntepmentS

Incubation Pericd

NumberOf Determinations

Std. De".

*

25 aOUrS

12 Hours

5

43,508

25 Hfmr5

12 Iialrs

5

475,503

+

15,580

from Buffer Injected msects

Integuments from

+ 341,500

Emiysterone Injected Insects

in Table 5, the ecdysterone-injected insects incorporate acetate 10.9 times better than the control insects. The difference is significant at the 95 per cent confidence level. DISCUSSION Ecdysterone stimulates hydrocarbon biosynthesis in S. bullata. This stimulation does not occur until1 pupariation begins, and is more pronounced if the substrate is provided 6 to 12 hr past the onset of pupariatian. Since acetate may not he a physiological substrate of hydrocarbon biosynthesis, this may have a direct bearing on the rates of incorporation into hydrocarbon. It is also possible that massive injections of ecdysterone cause nonphysiological changes, or alter the proper sequence of events in the moulting process. It is known that insects administered with ecdysone usually die after they apolyse (SCHNEIDERMAN, 1972), and in these experiments many of the ecdysterone-injected insects formed abnormal puparia which appeared to undergo normal tanning, but failed to contract properly. The ecdysterone-injected insects died before the pupal-adult ecdysis. Isolated integuments synthesize hydrocarbon from acetate better than the internal tissues, and the ingeguments of ecdysterone-treated _ insects _ incorporate acetate into hydrocarbon much better than the integuments of the control insects. This

indicates that cuticular hydrocarbon biosynthesis not only occurs in the integument, but that a locus of regulation is present in the integument. Of course other mechanisms of regulation, such a increases in substrate availability by mobilization of lipids or sugars from the fat body, could also be involved. Since ecdysone has been shown to stimulate transcription and translation (KARLSON and SEKERIS, 1966), it is possible that hydrocarbon biosynthesis is stimulated by protein synthesis. The observation that a number of hours pass after injection of the hormone before an increase in hydrocarbon biosynthesis occurs is comparable to delays seen for other ecdysone-stimulated enzyme activities (KARLSON and SEKERIS, 1962). Acknowledgement-This work was supported by National Science Foundation Grant No. BMS 74-14103.

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ARMOLD

1586

M. T. ARMOLD

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m

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