The induction of autophagy in isolated insect fat body by β-ecdysone

J. Insecf Physiol.. 1978. Vol. 74. Pp. 439 to 447. c Per,gumon Press Ltd. Printed in Great Britain.

THE INDUCTION OF AUTOPHAGY IN ISOLATED INSECT FAT BODY BY /9-ECDYSONE R. L. DEAN The Cell Science Laboratories, Department of Zoology, University of Western Ontario, London, Ontario. N6A 5B7. Canada

(Received I5 August 1977) Abstract-The fat body in Caipodes undergoes sequential organelle specific autophagy as a first step in the cell remodeling process necessary for metamorphosis to the pupa. This autophagy begins at about 36 hr before pupation and coincides with a critical period after which an isolated abdomen will pupate without further influence from the prothoracic glands. This suggested that autophagy might be induced by ecdysone. Fat body taken before the critical period and cultured in a medium containing I-ecdysone undergoes autophagy. Fat body from the same animal maintained in hormone-free medium retains the pre-critical period morphology with no autophagy. Autophagy is therefore directly induced by ,Secdysone. Fat body taken soon after the critical period continues with the autophagic sequence in hormone-free medium. Therefore the entire autophagic sequence is induced and does not require the continuing presence of hormone. protein storage granule formation and cell dissociation, which occur in fat body at metamorphosis, are also induced by p-ecdysone.

INTRODUCTION THE FAT body of the fifth stage larva of Calpodes ethlius undergoes sequential organelle specitic autophagy as a first step in the cell remodeling necessary for metamorphosis to the pupa (LOCKE and COLLINS, 1965; LOCKE and MCMAHON, 1971). This autophagy begins at about 36 hr before pupation and coincides with the time after which an abdomen isolated from the thorax by ligation will still pupate. After this critical period (LOCKE, 1970) the abdominal tissues continue to develop in the absence of further ecdysone from the prothoracic glands. The coincidence between the critical period for the operation of the prothoracic glands and the start of autophagy, and the fact that fat body autophagy occurs in isolated pre-critical period abdomens when injected with ecdysone (COLLINS, 1969) suggested that autophagy is induced by ecdysone. However, the effects of ecdysone on intact insects may be due to the hormone acting indirectly by stimulating other glands or tissues to secrete substances which cause the observed effects (GILBERT and KING, 1973). Therefore to answer the questions of whether ecdysone directly induces autophagy and whether the autophagic sequence requires the continuing presence of the hormone for its completion, experiments were performed on fat body in vitro. MATERIALS

the experiment could be determined. The posterior part was retained to ensure that the larva was in the pre-critical period as such isolated abdomens should not pupate (COLLINS, 1969). Fat body from the middle part was divided into four and explanted into culture medium, both with and without hormone. for 6, 10, 16, or 24 hr and 48 hr at 25°C before fixation. Fat body was maintained in Grace’s TC medium with 10% foetal-calf serum. The experimental medium contained ,8-ecdysone at a concentration of 1 pg/ml. The continuation induction in vivo

in vitro after ifs

The experiment was repeated with just-postcritical period fat body which was cultured in hormone-free medium only.

AND METHODS

x

houn \n

Grace's TC

Fifth instar Calpodes larvae were reared as described elsewhere (LOCKE and COLLINS, 1965). The induction of autophagy in vitro Pre-critical period larvae (approximately 144 hr after ecdysis to the 5th instar) were ligated into 3 parts as shown in Fig. 1. The anterior part was fixed immediately by injection with ice-cold glutaraldehyde so that the fat body morphology at the start of

of autophagy

medwm

x

hours I”

Grace's TC medium

+

hormone

4s

hours

I”

Grace's TC me&m

4s

burr

I”

GrocerTC dwm

+

hormone

-.

Fig. I. Diagrammatic representation of the treatment of fat body which was fixed immediately or maintained in vitro for two time periods with and without hormone. The abdominal tip was retained. 439

440

Tissuepreparation

R. L. DEAN forlight and electron microscopy

Fat body was fixed for l-4 hr in ice-cold 5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) containing 2% sucrose, washed in 0.05 M cacodylate buffer containing 10% sucrose and postfixed for I hr in 1% osmium tetroxide in 0.05 M cacodylate buffer containing 4% sucrose. The tissue was stained en bloc overnight in 2% uranyl acetate, dehydrated through a graded ethanol series and embedded in araldite. Sections for light microscopy were stained with toluidine blue. EM sections were stained with uranyl acetate and lead citrate.

RESULTS (1) The induction of autophagy in vitro

Profiles of pre-critical period fat body, fixed at the start of the experiment, contain large pale areas of glycogen and much lipid stored in large droplets. Thin branches connect the cytoplasm around the nucleus to that at the edge of the cell. At this time the cytoplasm appears dense and homogeneous under the light microscope (Fig. 2, inset). The dense RER synthesizes the protein which is packed into large vesicles by the Golgi complex and transported to the haemolymph. Microbodies containing a characteristic dense core (LOCKE and MCMAHON, 1971) are abundant (Fig. 2). Fat body taken from the same larva and maintained for 48 hr in vitro with 1 aglml of B-ecdysone contains numerous small spherical granules around the nucleus and in the glycogen storage area. The peripheral cytoplasm remains homogeneous (Fig. 3 inset). The granules are autophagic vacuoles containing mostly RER. This morphology is like that seen in the final stage of organelle specific autophagy which occurs in the last I2 hr before pupation in vivo. Normally this stage of RER destruction follows a period of blood protein sequestration into multivesicular bodies. The protein is hydrolysed in heterophagic vacuoles the remnants of which appear in Fig. 3. There are no microbodies present in the tissue, these organelles are the first to be isolated and destroyed in intact larvae soon after the critical period. Although histochemical localization of hydrolytic enzymes in autophagic vacuoles has not been demonstrated in this study the degeneration of isolated organelles appears normal. Newly isolated RER retains its characteristic appearance (Fig. 4) but as a number of such isolation bodies coalesce to form vacuoles it is presumed that enzymes added from primary lysosomes begin to degrade the contents (Fig. 5) until the autophagic vacuoles contain no recognizable organelles (Fig. 6). As noted in the light micrograph (Fig. 3, inset) the peripheral cytoplasm remains unaffected by autophagy. Figure 7 shows such an area in which the Golgi complexes are now producing small vesicles. The densely stained vesicles on the secretory face of the CC are presumed to be primary lysosomes. Some mitochondria are not destroyed during the autophagic sequence (Figs. 3 and 7) and give rise to the adult population by division (LARSEN, 1970).

The autophagy does not result from cell injury or pathological culture conditions as none occurs in fat body maintained for 48 hr in hormone-free medium (Fig. 8). This tissue resembles pre-critical fat body except that microboclies are reduced in number and tend to lack the dense core of coiled tubules (LOCKE and MCMAHON, 1971) and the Golgi complex no longer produces the large blood protein vesicles. This suggests that factors present in the haemolymph but missing from the culture medium are necessary for the maintenance of microbody stability and for massive protein synthesis. Maintenance of form may also require exogenous factors as there is a pronounced tendency for organelle types to aggregate and for microfibrils to occur in bundles in vitro.

(2) The continuation of autophagy in hormone-free medium

Figure 9 shows fat body early in the autophagic sequence fixed soon after the critical period. Small vacuoles in the nuclear area contain easily identifiable mitochondria which represent the start of the second phase in the autophagic sequence. It is extremely difficult to catch the tissue in the first phase during which all the microbodies are isolated and destroyed presumably because it occurs rapidly. After 48 hr in hormone-free medium tissue from the same animal contains numerous autophagic vacuoles in various stages of development (Fig. 10). Themostrecentlyformedcontainpredominantly RER. The residual heterophagic vacuoles indicate that protein sequestration and hydrolysis has already occurred. The authophagic sequence which is presumably induced by exposure to ecdysone in vivo therefore continues in vitro in the absence of hormone. DISCUSSION Massive autophagy has been reported in many tissues of holometabolous insects at metamorphosis. These include fat body (LOCKE~~~SYKES, 1975; S~~sand Kovkcs. ~~~~;ISHIZAKI, 1965). silkglands (MATSUURA et al., 1968; CHINZEI, 1975), salivary glands (SCHIN and CLEVER, 1975), gastric caecae and Malpighian tubules (RASCH~~~GAWLIK, 1964). perirectal tubules (BYERS, 1971). mid-gut (RADFORD and MISCH, 1971), oenocytes (LOCKE, 1969) and epidermis (SEDLAK and GILBERT, 1976). It has often been suggested that autophagy is hormone dependent and autophagy has been induced experimentally by ecdysone injection in Calpodes fat body (COLLINS, 1969), the epidermis of Hyalophora (SEDLAK and GILBERT, 1976) and the mid-gut of Sarcophaga (RADFORD and MISCH, 1971). Autophagy is induced in vitro in the fat body of Mamestra in the presence of n-ecdysone (SASS and KovAcs, 1975). The experiments on Calpodes show unequivocally that autophagyresultsfromdirect stimuby @-ecdysone that once the whole of organelle autophagy proWithout further Observations made this study confirm that other important in fat metamorphosis are by p-ecdysone. protein sequestra-

Fig.

2.

F’re-critical

period fat body fixed at the start of the experiment. Mic = microbody, M = mitochondrion, N = nucleus.

GC = Golgi

complex,

Fig. 3. Autophagy induced in tissue from the same larva as in Fig. 2 after 48 hr in vitrowith ,&ecdysone. AV = autophagic vacuole, HV = heterophagic vacuole, M = mitochondrion.

Figs. 4. 5 and 6. Sequence Fig.

7. Peripheral

cell area

of organelle

not affected complexes

digestion

(RER)

in autophagic

vacuoles

by autophagy after 48 hr in vitro with (GC) producing 1’ lysomes.

in vitro.

,&ecdysone.

Golgi

Fig. 8. Pre-critical

period

in hormone-free

morphology medium.

retained

by fat body from

Mic = microbody.

same larva as in Fig. 2 after 48 hr

M = mitochondrion,

N = nucleus.

Fig. 9. An early

stage in fat body autophagy fixed soon after the critical period. autophagic vacuoles (AV) around the nucleus.

Fig.

autophagy

10. Intense

Mitochondria

within

in fat body from the same larva as in Fig. 9 after 48 hr in hormone-free medium.

The induction of autophagy in isolated insect fat body by /?-ecdysone tion intomultivesicularbodiesisanintrinsicproperty of fat body cells but the protein is only converted to storage granules after ecdysone stimulation (COLLINS. 1969). Second, the dissociation of fat body cells which precedes their redistribution around adult tissues occurs only after ecdysone stimulation (JUDY and MARKS, 1971: OBERLANDER, 1976). However, these events do not occur at the end of each larval stage when the titre of ecdysone rises to cause ecdysis. The autophagy which occurs at metamorphosis is therefore probably induced by ecdysone only after a decline in the juvenile hormone titre has occurred. There is some indirect evidence for this in Mumesfra where topical application of a JH analogue inhibits fat body autophagy (SASS and Kovkcs, 1975). In Plodia, JH directly inhibits fat body in vitro (OBERLANDER, 1976). “Relatively few studies relate to the effects of hormones on non-epidermal tissues during metamorphosis and evidence for direct action on any specific tissue is generally wanting” (DOANE, 1973). Since normal fat body metamorphosis can be duplicated in vitro the system provides an opportunity to study both hormone action and the control of the autophagic process in detail. Acknow/edgements--Supported by NRC grant number A6607 to M. LOCKE. I thank M. LOCKE for guidance and criticism of the manuscript and P. FLOYD for assistance.

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