The midgut epithelium of the American cockroach: Acid phosphomonoesterase activity during the formation of autophagic vacuoles

J. Insect Physiol., 1968, Vol. 14, pp. 55 to 62. Pergamon Press. Printed in Great Britain

THE M I D G U T E P I T H E L I U M OF T H E AMERICAN COCKROACH: ACID P H O S P H O M O N O E S T E R A S E A C T I V I T Y D U R I N G THE FORMATION OF A U T O P H A G I C VACUOLES E R N E S T F. C O U C H * and R I C H A R D R. M I L L S Department of Biology, Tulane University, New Orleans, Louisiana 70118 (Received 8 August 1967)

Abstraet--Ultrastructural examination of the midgut epithelium of Per/planeta americana (L.) has shown that starvation causes a cyclic change in the

number of autophagic vacuoles. A peak in number of vacuoles occurs 4 days after starvation is initiated. Correlated with these changes is a rise and fall of acid phosphatase activity with a peak identical to that of the vacuoles on day 4. Histochernical localization of the enzymatic activity within the vacuoles suggest that they are autophagic in nature. The midgut acid phosphatase has been partially purified and characterized.

INTRODUCTION THE CYTOLOGY of the insect midgut epithelium has been studied by a number of authors and the process of enzyme secretion has been correlated with changes in cellular structure. On the basis of these histological investigations it has been postulated that both merocrineand holocrinesecretion of digestiveenzymesoccur (PRADHAN, 1940; OWSLEY, 1946, GOODCHILD, 1952). The holocrine secretions arise in vacuoles which supposedlyrelease their contents at the cell surface. On the other hand, TSCHANG(1929) and HENSON(1930) presented cytologicalevidence that these vacuolesdo not containdigestiveenzymesbut instead containbreakdown or hydrolyticproducts of the cell. In complementaryfashion, RAFIQKHANand FORD(1962) have shown that digestiveenzyme production is not correlated with the formation of vacuoles. The above lines of evidencesuggest that the vacuolar system is composedof cytolysome-like bodies as described by NOVIKOFF and ESSNER(1962). In blattids, two investigationshave been conducted. SHINODA(1927) stated that merocrine secretion occurs in Blatta sp. normally, but if the animals were fed after a period of starvation, holocrine secretion occurs. DAY and POWNING (1949) present evidence that is in disagreement with this deduction. T h e y found that in Blattella germanica the formation and secretion of these bodies did not conform with enzymatic activity. T h e present study is an attempt to follow the cytological changes in the midgut of Periplaneta americana (L.) with the electron microscope and to compare the rise * Present address: Department of Biology, Texas Christian University, Fort Worth, Texas.

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ERNEST F. COUCHANDRICHARD R. MILLS

and fall of these secretory granules with levels of acid phosphatase activity. A high level of acid phosphatase activity in association with the secretory granules would be interpreted as evidence of cytolysome activity. MATERIALS AND METHODS Adult female cockroaches maintained as previously described (MILLS,1966) were used in all experiments. Midguts were removed by clipping the posterior tip of the insect, cutting the right side of the abdominal cuticle, and pulling the alimentary canal out with a toothpick. Extraneous fat body, tracheae, and Malpighian tubules were dissected away and the midgut extirpated from the remainder of the gut with a razor blade. When the tissue was to be used for electron microscopy studies it was fixed in situ at 4°C in 1% osmium tetraoxide buffered by phosphate (MmLONIa, 1961). Fixation was continued after the dissection of the midgut for 90 rain. The tissues were embedded in Araldite 502, using DDSA as a plasticizer and DMP-30 as a catalyst (Lurr, 1961). Sections were stained with uranyl acetate and lead citrate (REYNOLDS, 1963). The grids were viewed with either a RCAEMU-2 or RCA-EMU-3f electron microscope. For orientation purposes sections 0"5 or 1/~ thick were stained with Azure B, methylene blue, or toluidine blue 0 and observed with the light microscope. The acid phosphatase enzyme for the biochemical study was prepared as follows: whole midguts were homogenized in a ground-glass homogenizer containing deionized water. The extract was centrifuged at 12,000g for 10 min in a Servall RC-2 refrigerated centrifuge and the pellet discarded. Prehminary experiments revealed that the supernatant contained endogenous inorganic phosphate which prohibited the completion of the reaction at zero-order kinetics. Subsequently, the supernatant containing the enzyme was chromatographed on a 1.3 x 26 em P-60 polyacrylamide gel column. Elution was with 1-5% KCI and 1 ml fractions were collected. The procedure and column preparation were the same as employed previously (MILLS et al., 1966). The reaction mixture consisted of: 0.2 ml 0.05 M beta-glycero-phosphate, 0.2 ml 0-1 M tr/~-acetate buffer, pH 5.2, 0"1 ml 0.01 M MgCI,, and 0.2 ml of the enzyme preparation. Incubation was at 37°C for 30 min and the reaction was stopped by the addition of 0.2 ml 33% tri-chloroacetie acid. Protein was removed by centrifugation and the inorganic phosphate (P~) hydrolysed was determined by the method of FIsIca~ and SUBBAROW(1925). Controls containing only enzyme and only substrate were always incubated simultaneously and values from these tubes were subtracted from the experimentals. Protein was quantitated as shown by LowRY et al. (1951) and bovine serum albumin was used as the standard protein. Localization of acid phosphatase was accomplished in the following manner: Gut tissue was fixed in situ at 4°C in 3.2% glutaraldehyde buffered by 0.2 M cacodylate buffer, pH 5.0 (660 miUiosmoles as determined by a Fiske osmometer). Fixation was continued for 20 min after dissection, and the glutaraldehyde was removed by washing in tr/~-acetate buffer. The tissue was sliced into thin sections

F1(;. 1A. An autophagic vacuole containing remnants of endoplasmic reticulum (ER) and mitochondria (M). x 9360.

FiG. lB.

An autophagic vacuole that has completed autolysis of the enclosed material. × 1200.

MIDGUT E P I T H E L I U M OF THE AMERICAN COCKROACH

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with a razor blade and incubated on a glass slide with the standard reaction mixture (for the biochemical assay) outlined above at 37°C. After 30 min the tissue was washed with tris-acetate buffer and 0-005 M Pb(NO3) ~ was allowed to stand on the preparation for 5 min. The tissue slices were washed with two or three changes of buffer and post-fixed in unbuffered 1% osmium tetraoxide for 30 min. Controls were treated identically except that no substrate was used. The remaining steps were as shown above except the sections were not stained. RESULTS

Ultrastructure of the autophagic vacuoles Examination of the midgut epithelium of starved animals with the electron microscope revealed that a number of autophagic vacuoles were present. These appear to be engulfing various cytological structures such as the mitochondria and rough endoplasmie reticulum (RER). This is illustrated by Fig. 1A. It is assumed that normally the RER is involved in the synthesis of digestive enzymes and the mitochondria supply the energy (in the form of ATP) for the corresponding amino acid activation. It is further assumed that under conditions of starvation no digestive enzymes are produced. Therefore, in all probability the observed inclusion bodies are degenerative in nature and can be classified as autophagie vacuoles. If the time of starvation is extended, these vacuoles may empty into the lumen of the gut. If food is taken, then the vacuoles appear to disintegrate releasing the enclosed material back into the cytoplasm. Thus, the morphological information indicates that the physiological r61e of the autophagic vacuoles is to conserve the energy of the cell by removing extraneous synthetic machinery.

Cyclic changes in the number of autophagic vacuoles during feeding and starvation Examination of both starved and fed cockroaches showed that a consistently higher number of vacuoles are present in the midgut epithelium of starved animals. There is always a limited number present even in cockroaches allowed to feed at will. This indicates a cyclic nature of vacuole formation which may depend upon the feeding cycle. The number of autophagic vacuoles in the midgut epithelium increase after the first day of starvation. It appears that they reach a peak around the fourth or fifth day. This cannot be stated with great certainty for the entire midgut epithelium, however, since only small sections of tissue were examined and only a limited number of cockroaches were used. Fig. 2 depicts a rough estimate of the number of vacuoles during starvation.

Acid phosphatase activity Acid phosphatase is considered to be intimately concerned with hydrolytic functions in the cell. It is known that lysosomes (i.e. autophagic vacuoles, cytolysomes) are involved in degenerative processes and characteristically are high

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ERNEST

F. C O U C H AND R*C~a~D R. MILLS

in acid phosphatase activity. Since the build-up of the autophagic vacuoles in the midgut appeared to be cyclic in nature, a study on the rise and fall of acid phosphatase activity was instigated. It was surmised that if the peak of enzyme activity corresponded to the period of the greatest number of cytolysomes then this would offer additional proof that these bodies were degenerate in function. I

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This was indeed the case. Acid phosphatase activity increased until the fourth or fifth day. After this time the enzyme concentration began to decline although there was always a consistently high level of hydrolysis. T h e relative concentration of enzymatic activity over a 10-day period of starvation is shown in Fig. 3.

Partial purification and some properties o/the enzyme Crude homogenates of the midgut contained a high titre of endogenous inorganic phosphate. This prohibited the completion of the reaction at zeroorder kinetics. Thus, the homogenate was centrifuged and the supernatant chromatographed on a 1.3 x 26 cm column containing P-60 polyacrylamide gel. This separated the enzyme from the endogenous inorganic phosphate as shown by Fig. 4 and some purification of the enzyme was achieved. T h e pH optima was determined to be 5.2 by incubation in a graded series of 0"1 M tr/~-acetate buffers (Fig. 5). Magnesium was stimulatory but not essential. Further purification of the enzyme and a more detailed characterization are in progress (MxLLS, unpublished).

59

MIDGUT EPITHELIUM OF THE AMERICAN COCKROACH |

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Cytological localization of acid phosphatase Since the rise in acid phosphatase activity corresponded to the peak in number of autophagic vacuoles, histochemical localization at the electron microscope level was used to test for enzymatic activity in the vacuoles. From Fig. 6 it can be seen that phosphatase activity at pH 5.2 is concentrated in these structures. Acid phosphatase was also localized on the membrane(s) of the epithelial cells adjacent to the haemolymph and some other regions of the cell.

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pH FIG. 5. pH optimum of acid phosphatase from the midgut epithelium. This evidence supports the contention that the autophagic vacuoles are hydrolytic in nature and are involved in degradation rather than synthesis. DISCUSSION Examination of the midgut epithelium of the American cockroach with the electron microscope shows conclusively the formation of autophagic vacuoles. From Fig. 1A it can be seen that such organelles as the mitochondria and rough endoplasmic reticulum are being engulfed. Fig. 1B shows the degeneration of the enclosed material. T h e nature of the material being discarded is unknown, but it is feasible to believe that degraded products useful to the cell diffuse from the vacuole into the cytoplasm. A d d phosphatase is generally believed to be a hydrolytic enzyme and active in the degeneration of tissues. The enzyme has been found to be concentrated in the autophagic vacuoles, which provides further evidence that they are degenerative in nature and not associated with the production of digestive protein.

FIG. 6. Localization of acid phosphatase in the autophagic vacuoles in the midgut epithelium, x 27,000.

MIDGUT E P I T H E L I U M OF THE AMERICAN COCKROACH

61

Furthermore, the bodies observed in the electron micrographs closely resemble previously described autophagic vacuoles both in structure and formation (NovmoFF et al., 1964). The enzyme has been studied by biochemical means and its pH optima found to be 5.2. Some purification has been achieved and added magnesium proves to be stimulatory though not essential. Its physiological function has not been clearly established in this particular investigation, but its hydrolytic ability on both proteins and smaller substrates is now commonly accepted by most investigators. It is probable that the rough endoplasmic reticulum containing both polysomal RNA and protein is degraded by the cytolysome. Thus the function of acid phosphatase in the hydrolysis of phosphoprotein and ribose-phosphates is not hard to visualize. In addition, it is known that a nucleotidase (active at acid pH) is present in the midgut (MILLS, unpublished) which may also be located in the cytolysomes. The evidence presented here supports the contention of T s c ~ G (1929) and HENSO~q (1930) that the vacuolar secretions into the lumen of the midgut are breakdown products of the cell, and not digestive enzymes. More conclusive proof is shown by the investigations of DAY and POWNING (1949) and R~IQ K r I ~ and FORD (1962), who found that the production of digestive enzymes did not correspond to the rise in vacuole formation. Similar studies using Periplaneta are in progress. Acknowledgements--The authors are indebted to Dr. D. E. COPELANDfor reading the manuscript and for making his facilities available to us. The work was supported in part by a USPHS-NIH-GM-669 training grant in cell biology which also provided a fellowship for E. F. C.

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NOVlKOFF A. B., ESSNER E., and QUINTANAiNT. (1964) Golgi apparatus and lysosomes. Fed. Proc. 23, 1010-1022. OWSLEY W. B. (1946) The comparative morphology of internal structures of the Asilidae (Diptera). Ann. ent. Soc. Am. 39, 33-68. P r ~ D ~ S. (1940) The alimentary canal and pro-epithelial regeneration in Coccinella septempunctata with a comparison of carnivorous and herbivorous coccinellids. Quart. J. m/at. Sc/. 81, 451-478. R~ZQ KHAN M. and FORD J. B. (1962) Studies on digestive enzyme production and its relationship to the cytology of the midgut epithelium in Dysdercus fasciatus Sign. (Hemiptera, Pyrrhocridae). J. Insect Physiol. 8, 597-608. RmrNOt-DS E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. SmNODA O. (1927) Contribution to the knowledge of intestinal secretion in insects--II. A comparative histo-cytology of the mid-intestine in various orders of insects. Z. Zellforsch. 5, 278-292. TSCH~a~G Y. T. (1929) L'histogen~se et l'histophysiologie de l'~pith~lium de l'intestin moyen chez un l~pidopt~re (Galleria mellonella). Bull. biol. 12, 1-144.