Insect Biochem., x974, Vol. 4, PP. 423 to 428. Pergamon Press. Printed in Great Britain 423

P R O T E I N S Y N T H E S I S I N W I N G D I S K S OF G A L L E R I A

M E L L O N E L L A : E F F E C T S OF E C D Y S O N E A N D F A T B O D Y

I N V I T R O

JAMES BENSON* AND HERBERT OBERLANDERt

Department of Biology, Brandeis University, Waltham, Massachusetts o2x54, U.S.A.

(Received 5 February x974)

ABSTRACT

The effects of alpha-ecdysone, beta-ecdysone and fat body on protein synthes- is in Galleria mellonella wing disks cultured in vitro were investigated. The eedysones stimulated protein synthesis, but did not maintain it at the higher levels. Fat body enhanced the response of disks to alpha-ecdysone, but this effect was not related to stimulation of morphogenesis by fat body.

ALTHOUGH morphogenesis of imaginal disks has been examined in vitro, we know little about the biochemical changes which occur in such tissues. There have been reports on DNA synthesis in lepidopteran disks in vitro (Oberlander, i969a, I969b, x972), and on DNA, RNA and protein synthesis in dipteran disks in vitro (Fristrom, x972; Ohmori and Ohtaki, i973). We have extended our studies with Galleria mellonella, and report here on the effects of alpha-ecdysone, beta-ecdysone, and fat body on protein synthesis in wing disks cultured in vitro in a chemically-defined medium.

MATERIALS AND M E T H O D S EXPERIMENTAL ANIMALS

The greater wax moth, Galleria mellonella, was reared at 3 °0 C and 55--75 per cent RH. Eggs were placed on medium (2 lb. Gerber Mixed Cereal, 20 g. Difco yeast extract, 2 g. methylhydroxy- benzoate, 2oo ml. honey, I2o ml. glycerine, 80 ml. distilled water) in perforated plastic petri dishes (size zoo x 20 cm.). Soon after hatching the young larvae were transferred to larger plastic boxes (x8o x I3o x xoo ram.) containing the same medium. Newly ecdysed larvae were removed every 24 hr from this box of several hundred larvae as they entered the next to final instar, and were transferred back to the original size petri dish containers at a density of about 30 animals/ dish. Final instar larvae used for experiments were selected from these dishes and maintained at a density of x4 animals/dish. Animals in the third day of the final instar were chosen in the weight range of o'16 to o'x8 g. and fifth-day animals in the range of o'x9 to o'2i g.

In vitro Paoc~Dum~s Left and right wing disks of each animal were cultured separately in control and experimental

dishes. In this way two identical sets of cultured disks were obtained and differences due to individual variation between animals were eliminated. Normally there were 6 disks in each culture

* Present Address: Bangor Hall, University of Maine at Bangor, Bangor, Maine 04401, U.S.A. t To whom requests for reprints should be sent. Present address: Insect Attractants, Behavior

and Basic Biology Research Laboratory, Agricultural Research Service, USDA, P.O. Box I4565, Gainesville, Florida 326o4, U.S.A.

424 BENSON AND OBERLANDER Insect Biochem.

dish. When larval fat body was required for culture it was obtained by the same procedure as used for disks. Only fat body originating in the thorax was utilized, but that included numerous leaves of tissue that ran toward the posterior from points of attachment in the thorax. Larvae used as donors of fat body were of the same age as those used for disks. All culture media contained 5 ° units/ml, of penicillin-streptomycin (Grand Island Biological Co.). Crystalline alpha-ecdysone (Zoecon Co.) and beta-ecdysone (Rohto Pharmaceutical Co.) were dissolved in io per cent ethanol in sterile glass distilled water at a concentration of i mg./ml. All cultures were incubated at 25 ° C and IOO per cent RH. Tissue culture work was carried out in a room sterilized by ultra-violet irradiation and electrostatic precipitation of filtered air.

MEASUREMENT OF PROTEIN SYNTHESIS The specific activity of proteins synthesized by imaginal wing disks in culture was determined by

calculating the incorporation of radioactive amino acid per microgram total protein. Disks were labelled by addition to the culture medium of 5/~Ci DL-valine-3, 4-all, 3"78 mg./mCi (New England Nuclear Corp., lot No. 334-237) for 5 hr. Total protein was determined by the Folin-Lowry procedure. The OD was read at 750 nm. on a Beckman DB-G spectrophotometer using a quartz micro-cuvette with I cm. light path. These values were compared to a standard curve produced with known amounts of bovine serum albumin (Armour Pharmaceutical Co., lot No. D 5406). The protein solution set aside for isotope counting was mixed with an equal volume of 15 per cent trichloroacetic acid (TCA) and stored on ice for I hr. The TCA precipitate was retained on a Millipore filter type MF-GS (MiUipore Corp.) following vacuum filtration of the sample and 3 washes of the sample tube and filter with cold 7 per cent TCA. The filter was dried under a heat lamp and added to a scintillation counting vial with IO ml. of counting fluid consisting of 4 g. of 2,5-diphenyloxazole (PPO) per liter of toluene. Samples were counted in a Beckman LS-255 scintillation counter at room temperature with a 48 per cent efficiency as determined by the external standard method. Specific activity of disk protein was expressed as counts per minute per microgram total protein.

Protein synthesis was also examined by autoradiography. The tissue was labelled with 5/~Ci of tritiated valine. After incubation of the tissue with the isotope for 3½ hr, the tissue was rinsed in fresh medium and fixed in Bouin's solution for at least I hr. Tissue was double-embedded in agar and paraffin and sectioned at 5/~m. Slides were coated with nuclear track emulsion NTB 3 (Eastman Kodak Co.) and processed according to the manufacturer's instructions. Results were evaluated by examining randomly-selected areas of labelled cells and assigning a value denoting the density of the label: o-none, l-light, 2-moderate, 3-dense.

RESULTS

PROTEIN SYNTHESIS

This was examined in disks which were removed from larvae on the fifth day of the last instar and cultured in Grace's medium with and without ecdysone for various times. After a 5-hr incubation with radioisotope, the specific activity of proteins synthesized by disks during that period was determined by the procedure described in the Methods section.

STABILIZATION

Fig. I shows the declining specific activity of proteins synthesized by freshly-excised wing disks as they are allowed to remain in culture uatreated for an extended period. The initially high rate of protein synthesis falls off rapidly and stabilizes at a level which is maintained for at least 2 weeks. Frequent changes of the medium do not substantially increase the disk's longevity in culture and were therefore not performed routinely. The greatest change in the rate of protein synthesis occurs in the first 2 days of culture, and

1974, 4 PROTEIN SYNTHESIS IN Galleria mellonella 425

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F I G . I . - - S u m m a r y of protein synthesis in cultured wing disks. Alpha-ecdysone (i/~g/ml) was added at the start of culture in one group (O), and on day four in two other groups (i/zg/ml = O, 3/xg/ml. = &). Control = Q. The bars indicate standard error.

by the fourth day the basal level is reached. For convenience this phenomenon is called 'stabilization', and stabilized disks are those which have been in culture long enough for them to reach a new equilibrium presumably uninfluenced by their in vivo state.

STABILIZATION WITH HORMONE PRESENT

To see how long the stimulation by hormone persisted and to learn more about the dynamics of the stabilization process, fresh disks were placed in medium containing I/zg.]ml. of alpha-ecdysone which was then continuously present during the stabilization period. Results are shown in Fig. I. The values for the experimental disks are higher than those of the controls at the beginning, but the difference gradually diminishes, and by the fourth day both have stabilized at the same point.

HORMONE STIMULATION AFTER STABILIZATION

Disks were allowed to stabilize in culture for 4 days before either i or 3/~g. of alpha- ecdysone was added to the experimental media and the same volumes of io per cent ethanol to the controls. The results are shown in Fig. 1. Three #g. was proportionately more effective than i #g. in its ability to elevate the rate of protein synthesis. Both dose levels produce their maximal effect in the first 24 hr after introduction into the culture, and the effects of both diminish thereafter at about the same rate.

DOSE RESPONSE TO ECDYSONE

The rate of protein synthesis after treatment with different doses of alpha- or beta- ecdysone was determined by placing freshly-excised wing disks directly into 1 ml. of Grace's medium containing t, 3, 5, or io #g. of the hormone and measuring the in-

426 BENSON AND OBERLANDER Insect Biochem.

o

7C

60

50

40

50

20

I 0 ~ -

I , I I I [ I I I I I I 2 3 4 5 6 7 8 9 IO

p.g/ml hormone

FIG. 2.--Dose-response of protein synthesis in cultured wing disks to different concentra- tions of alpha- (0) and beta-ecdysone (0). The control cultures received the same volumes of xo per cent ethanol without hormone. The bars indicate standard error.

corporation of [ZH]-valine into proteins after 22 hr. Matched sets of control disks received the same volume of io per cent ethanol as the corresponding experimental disks. The results are summarized in Fig. 2. One tzg. of alpha-ecdysone caused almost a doubling of the rate compared to that in the untreated control. Three/~g. was the most effective dose of those tested here and caused an even greater elevation of the specific activity. Five and Io Fg. exceed what are generally considered physiological doses, and both depress the rate below the control level. When beta-ecdysone (ecdysterone) was tested, the results were similar to those with alpha-ecdysone, but at every dose level its in- fluence was less pronounced. Low doses of beta-ecdysone did not elevate the rate of protein synthesis as much, nor did high doses depress the rate as much as the same doses of alpha-ecdysone.

EFFECTS OF CONDITIONED MEDIUM Matched pairs of disks were put in plain Grace's medium to stabilize while 2 other

media were being prepared. An experimental medium was made by culturing fat body in Grace's medium containing I/zg./ml. of alpha-ecdysone. The control medium contained the hormone alone without fat body. After 2 days, the experimental medium conditioned by fat body was removed to another petri dish with a sterile pipette, leaving the fat body behind. The control medium of the same age was also pipetted to another petri dish. The stabilized disks were now transferred individually from their plain Grace's into the control and experimental media, and their rate of protein synthesis determined at 24-hr intervals. The results appear in Fig. 3. The conditions of this experiment differ from the preceeding ones in that the larvae used were from the third rather than the fifth day of the last instar, and the disks were stabilized for 2 days instead of 4. The values obtained here with disks in the fat body conditioned medium are substantially higher at every point than those in the hormone alone. The curve generated by the control disks is a familiar one, resembling those in Fig. i, but stabilizing at a higher specific activity. The situation with the experimental disks is one that has not been seen before: the initially high value of the first 24 hr is sustained for the entire

1974, 4 PROTEIN SYNTHESIS IN Galleria mellonella 427

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6 0 -

5 0 -

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FIG. 3.--Stimulation of protein synthesis by fat body conditioned medium cultured in conditioned medium containing x pg./ml. alpha-ecdysone (0) compared with x/~g./ml. alpha-ecdysone alone (O). The bars indicate standard error.

o

. c o

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FIG. 4.--Autoradiographic determination of protein specific activity in cultured disks. Disks were placed in fat body-conditioned medium containing t #g./ml. of alpha- ecdysone (&), plain medium with 3 pg./ml. of aipha-ecdysone (O), or plain medium with neither hormone nor fat body (O). o = no label, x = light label, 2 = moderate label, 3 = dense label.

5-day period of the experiment. The presence of fat body in the culture modified the conditioned medium in such a way that the stimulation of protein synthesis in the disks by t pg. of alpha-ecdysone was synergistically increased and prolonged. When the fat body conditioned medium was heated to 5o ° C for 3 ° min its ability to stimulate protein synthesis was reduced by 26 per cent when measured after the first 24 hr of culture. Similar treatment of a control medium containing only ecdysone had no effect. A component of conditioned medium which was isolated by chromatography (unpub- lished observations) stimulated morphogenesis but did not stimulate protein synthesis.

A series of experiments using autoradiography to assess protein synthesis confirmed the fundamental difference between stimulation by conditioned medium or by hormone. One set of disks was cultured in conditioned medium, another set was cultured in Grace's medium, and a third set was cultured in Grace's medium containing 3 pg./ml, of alpha- ecdysone. The matched control disks were placed in plain Grace's. Disks were removed for autoradiographic processing every 24-hr. Fig. 4 shows that the conditioned medium originally containing only i pg. of ecdysone caused as much stimulation of protein synthesis as did 3 Pg. of ecdysone alone. Further, the conditioned medium elevated the specific activity more rapidly and again sustained it at that high level for 5 days. The ecdysone curve shows the typical rise and fall seen before with the Folin-Lowry method.

DISCUSSION Our results establish that cultured Galleria wing disks synthesize protein in response

to alpha- or beta-ecdysone. The increase in protein synthesis was greater when disks

428 BENSON AND OBERLANDER Insect Biochem.

were incubated with alpha-ecdysone than when they were cultured with beta-ecdysone. This observation is consistent with previous findings that alpha-eedysone stimulates early phases of metamorphosis as well as or better than beta-eedysone in lepidopterous disks cultured in vitro (Oberlander, i972 ).

Unlike DNA synthesis (Oberlander, x969a ), protein synthesis in the disks is not maintained by the continuous presence of either alpha- or beta-eedysone. Whether DNA synthesis in the cultured disks resulted from continued conversion of alpha- eedysone to low levels of beta-eedysone, whereas protein synthesis was induced by alpha-eedysone itself is not known (but see Marks, i973).

Fat body stimulates traeheole migration and evagination (Richman and Oberlander, I97X ) in Galleria disks incubated with alpha-ecdysone. The results reported here show that fat body increases protein synthesis in disks incubated with alpha-eedysone, and that this increase is not related to fat body-stimulated morphogenesis. However, the ecdysone-indueed protein synthesis may be required for subsequent phases of disk development such as cuticle deposition.

ACKNOWLEDGEMENTS

We thank Dr. John Siddall (Zoecon) for providing the alpha-ecdysone used in these experi- ments, and the National Science Foundation (Grant GB21238) for support.

REFERENCES FRISTROM J. W. (I972) The biochemistry of imaginal disk development. In Biology of Imaginal

Disks (ed. Ursprung and Nothiger) pp. Io9-I54. Berlin: Springer. MARKS E. P. (x973) Deposition of insect cuticle in vitro: differential responses to ~x- and fl-ecdysone.

Gen. comp. Endocr. 2I, 472-477. OBERLANDEa H. (I969a) Effects of ecdysone, ecdysterone, and inokosterone on the in vitro initia-

tion of metamporphosis of wing disks of Galleria mellonella. ~. Insect Physiol. Ij) 297-3o4. OB~a.ANDER H. (x969b) Ecdysone and DNA synthesis in cultured wing disks of the wax moth,

Galleria raellonella. ~. Insect Physiol. I5, I8O3-I8O6. OBVmLANDER H. (I972) ~t-Eedysone-induced DNA-synthesis in cultured wing disks of Galleria

mellonella: inhibition by 2o-hydroxyecdysone and 22-isoecdysone..7. Insect Physiol. x8, 223- 228.

OHMOBI K. and OHTAKI T. (x973) Effects of ecdysone analogues on development and metabolic activity of wing disks of the fleshfly, Sarcophaga peregrina, in vitro, ft. Insect Physiol. I9, 1199- I2IO.

RICHMAN K. and OBERLANDER H. (1970 Effects of fat body on alpha-ecdysone-induced morpho- genesis in cultured wing disks of the wax moth. Galleria melltmella, s ~. Insect Physiol. x7, 269- 276.

Key Word Index: Protein synthesis, wing disks, Galleria meUonella, ecdysone, fat body.