Blood sugar, sugar metabolism and related enzymes in the earthworm, Lumbricus terrestris L.

Comp. Biochem. PhysioL Vol. 86B, No. 2, pp. 333-341, 1987

0305-0491/87 $3.00+0.00 © 1987 Pergamon Journals Ltd

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BLOOD SUGAR, SUGAR METABOLISM A N D RELATED ENZYMES IN THE EARTHWORM, L U M B R I C U S TERRESTRIS L. POUL PRENTO Institute of Cell Biology and Anatomy, University of Copenhagen, Universitetsparken 15, Copenhagen, 2100 Denmark (Received 1 May 1986)

Abstract--1. L. terrestris has no trehalose in the blood or coelomic fluid and is without trehalose activity. 2. Glucose-6-phosphatase is also lacking and the concentration of glucose in the blood or coelomic fluid is very low (0.014).05/zg/#l normally). 3. The body wall contains 80% of the lactate dehydrogenase activity of the worm and both the lactate formation and the reoxidation to pyruvate takes place in the body wall muscle itself. 4. In accordance with this the glycogen-storing chloragocytes contain very little or no LDH. 5. Thus the earthworm does not have the lactate-glucose exchange mechanism known from vertebrate liver and muscle and hormonal regulation of extracellular glucose is probably absent. 6. Chloragocytes exhibit significant amylase- and maltase-activities, which may be responsible for glycogen break-down in the absence of the phosphorylase-glucose-6-phosphatase system. 7. There are histological indications that chloragocytes, or their distal parts are released to the coelom to form trephocytes or eleocytes. 8. They may then break up or be transported by the coelomic fluid to other parts of the body and their contents of glycogen and other materials released. 9. Comparative and functional aspects of the above are discussed.

INTRODUCTION

Lumbricus terrestris is a soil organism of great inter-

est, both physiologically and ecologically, and considerable attention has been given to the problem of carbohydrate storage, transport and intermediary metabolism in earthworms. Liebmann (1928, 1931, 1946) held the view that glycogen was mainly stored in the chloragog tissue and that sugar was transported to other tissues by the transformation of chloragocytes to eleocytes (or trephocytes), which migrated into the coelomic space and delivered their storage materials mainly by "fusion" with the target cells. Later authors (e.g. Van Gansen, 1958; Roots, 1960) also regarded the chloragog tissue as the main glycogen-storing tissue, but considered its function more analogous to the vertebrate liver. This implies that the chloragog tissue release mono- or disaccharide into the body fluids and that this is under homeostatic control. This is in accordance with Davis and Slater (1928), who showed that the body wall of L. terrestris follows the vertebrate pattern in that it converts glycogen to lactate anaerobically. Pronounced LDH activity in L. terrestris was also found by Augenfield (1966). In contrast Gruner and Zebe (1978) found that the production and accumulation of lactate by L. terrestris was generally low and that succinate accumulation was the major end result of anaerobic glycogen break-down in L. terrestris. As regards extracellular or "blood" sugar Fairbairn (1958) reported the presence of both glucose and trehalose in earthworms, while Tillinghast et al. (1970) were unable to detect glucose in the blood or

coelomic fluid and furthermore found that glucose-6phosphatase was absent from the whole midgut, as also reported by Van Gansen (1958). More detailed accounts of the state of oligochaete carbohydrate metabolism are given by O'Brien (1957) and by Scheer especially (1969). Laverack (1963) lucidly discusses the chioragocytes in relation to the possible metabolic strategies of the earthworm, including the proposals by Liebmann. While there can be no doubt that earthworms follow the general biochemical pattern for intermediate carbohydrate metabolism, the EmbdenMeyerhof scheme (Dastoli, 1964), the current view of the carbohydrate physiology is obviously confused. I therefore decided to reexamine the carbohydrate physiology of L. terrestris and the distribution of glycogen and relevant enzymes (notably LDH). The primary aim was to obtain some more information as to the probable physiological roles of the chloragog tissue. The following study confirms the low glucose level in the body fluids and the absence of glucose-6phosphatase and it rejects the presence of trehalose. Thus the results rebut the proposal that the chloragog tissue is physiologically analogues to the vertebrate liver as regards carbohydrate metabolism. In doing this it strengthens the views of Liebmann (1931, 1946). MATERIALS AND METHODS

Mature earthworms were collected on grass lawn by use of electric current and kept in sand-free pot mould at 15°C for no longer than two weeks. Before use the worms were

333

334

POUL PRENTO

anaesthetized with 10% ethanol at 15°C, quickly rinsed with detain, water and rolled once on paper toweling.

system was found by chromatography of a dilution series o1 glucose:trehalose (start concentration 2 pg/#l of each).

Histochemistry

Glycogen

Mid-regions of worms were quick-frozen at -70~C, freeze-sectioned at 8 pm and used the same day for enzyme histochemistry. Midregions for glycogen histochemistry were quick-frozen at - 7 0 ° C and fixed at - 4 0 ° C in Lison's Gendre-fluid (Pearse, 1968), chemically dehydrated and cleared with 2,2-dimethoxypropane (Prento, 1978) and paraffin embedded. Glycogen was detected by use of the PAS reaction with or without prior digestion by amyloglucosidase (Sigma A 9268) or human saliva. The reaction for glucose-6phosphatase was performed according to Lazarus and Barden as described by Pearse (1968). Lactate dehydrogenase (LDH), malate dehydrogenase (MDH), NADH-cytochrome c reductase (NADH-Dp) and succinate dehydrogenase (SDH) reactions were performed according to Kiernan (1981) on sections pretreated with ice-cold acetone for 120min and 0.03 mg Meldolas Blue (C.I. 51175; Basic blue 6) was added per ml reaction medium (Kugler and Wrobal, 1978). Distribution of blood vessels in the gut was visualized by the hemoglobin peroxidase reactin according to Lepehne and Pickworth (Lillis and Fullmer, 1976). Material for electron microscopy was fixed in cold, buffered 4% glutaraldehyde:3% formaldehyde for 4 hr and in cold 2% osmium tetroxide for 3 hr. The sections were stained with uranyl acetate and lead citrate.

Tests for body fluid sugars Thirty unfed and nine starch fed worms were used. The worm was opened in the region of the anterior gut and fluid withdrawn by means of a 20/~1 pipette (coelomic fluid and digestive fluid) or a 5 pl pipette (blood). Before opening the blood vessel the area was cleaned of coelomic fluid by means of filter paper. Digestive fluid was always removed last. 2 p l samples of coelomic fluid from blowfly were used throughout as positive controls for trehalose and glucose (Normann and Duve, 1969; Duve, 1972). The samples were used directly or pretreated.

Anthrone or enzymatic detection In some cases protein was removed by pipetting the sample into two volumes of absolute ethanol followed by 70°C for 15 rain and centrifugation. In other cases the samples were led through a Folch methanol:chloroform:water phase separation procedure (Kates, 1972) for complete removal of protein and lipid. Samples for enzymtic sugar detection were always used directly. Total sugar and trehalose were assayed by the anthrone method as modified by Wyatt and Kalf (1957). Glucose was assayed by the glucose oxidas~peroxidase~O-dianisidin method (GOD) according to Bergmeyer (1974) and by the Haemo-Glukotest (Boehringe~Mannheim, 20-800). Trehalose was further assayed by incubation of 100 #1 coelomic fluid samples with a trehalase preparation (see later) followed by the GOD procedure.

Chromatography Sugars were identified and roughly quantitated by chromatography of 2 #1 samples on silica gel (Merck, Darmstadt, Art. No. 5735) with 1-butanol: acetone:water (4: 5:1 ) as a solvent. In some cases samples were applied repetitively to the same starting point. Migration distance was 12cm (about 2 hr). The spots were developed by spraying with anthrone:conc.sulfuric acid:ethanol and heating at 100°C for 15 rain. The detection limit of the chromatographic

*The spectrophotometer is a grant from the Danish Natural Science council (Grant No. 11-0362).

Glycogen was isolated from whole worms by the method of Wiens and Gilbert (1967) and weighed. The relative glycogen contents of rinsed midgut, body wall an coelomic material was determined according to Bergmeyer and Bernt (Bergmeyer, 1974) by the perchloric acid-amyloglucosidase-GOD procedure. All steps prior to protein denaturation were performed at 10°C, as temperatures near zero seemed to activate glycogen break-down.

Enzymes Trehalose. The worm was opened, the gut contents removed and the whole mid-region of the worm homogenized in ice-cold demin, water. Low-molecular constituents and glycogen were removed by precipitating the total protein twice with 3.5 moles/1 ammonia sulfate. Finally most of the ammonia sulfate was removed by Sephadex G-25 filtration (Pharmacia) of the redissolved protein. The final product contained 5.4 mg protein per ml. Trehalase from the blowfly was used as a positive control (Duve, 1972) by submitting the thoracic muscle of 25 flies to the same procedure as above. Worm and blowfly preparations were both free of glycogen, but the former exhibited amylase activity. The trehalase assay was performed as described by Duve (1972) on 10-100/~1 samples. These were incubated for up to 60 min at 30°C and then kept in a boiling water bath for 10 min. This destroyed catalase, which is a major protein constituent of the chloragocytes (Prento, 1984, 1986) and so might interfere with the subsequent GOD assay. Glucose-6-phosphatase. Whole, rinsed midgut or chloragocyte brush-off (Prento, 1986) as homogenized at 0°C in 0.25 moles/l sucrose with 1 mmoles/1 EDTA pH 7, centrifuged at 150,000g-rain and the sediment discarded. The supernatant was then submitted to first 500,000g-rain and then 5,000,000g-rain. The two sediments were used for glucose-6-phosphatase assay according to Baginski et al. (Bergmeyer, 1974). In order to check unspecific phosphatase activity samples were also incubated with pnitrophenylphosphate instead of glucose-6-phosphate in the pH 6.5 medium. Mouse liver treated as above was used as a positive control. Amylase-, maltase- and sucrase-activities. Chloragocyte brush off, chloragocyte-free intestinal epithelium (Prento, 1986), typhlosole coelomic contents and body wall were homogenized in 0.9% NaC1 and assayed for amylase activity by use of the Phadebas amylase test (Pharmacia Diagnostics). Maltase and sucrase activities were determined as described for trehalase. Incubation was at pH 7, 30°C for 30 min and after boiling glucose was determined by the GOD assay. Maltase activity was also assayed at pH 3. Lactate dehydrogenase. Homogenates as above, but in sucrose medium were centrifuged at 600,000g-min. The supernatants were assayed according to Bergmeyer and Bernt (Bergmeyer, 1974). Furthermore whole midgut and bodywall of earthworm, mouse liver and mouse leg muscle were submitted to agarose gel electrophoresis at pH 8.6 and the enzyme bands developed by essentially the histochemical procedure, but with four times the substrate concentration. Protein determination Substances in the chloragocyte cytosol and chloragosomes interfere with the biuret and Lowry methods. Protein was therefore determined by use of Bradford's (1976) Coomassie Brilliant Blue G-250 method, which is free of this interference. Bovine serum albumin (Sigma A4503) was used as a standard. All photometric measurements were made by use of a Beckman acta CIII recording spectrophotometer.*)

Earthworm sugar metabolism RESULTS

Histochemistry Predigestion with amylases removed all the PASpositivity except for gland cell secretions in gut epithelium and epidermis, the weak reaction of collagen fibres and a very weak reaction of the chloragosomes. When areas were heavily stained by the direct PAS-reaction, unstained following amylase treatment the staining was judged due to glycogen (Figs 1 and 2). Glycogen was prominent in most cells, even nerve cells. Only the gut epithelium and a proportion of the coelomocytes appeared devoid of glycogen. In the body wall of starch-fed worms glycogen was most prominent in the circular muscle layer and in the peripheral parts of the caissons of the longitudinal fibres. In starch-fed worms the peritoneal cell-lining against the body wall and the septa was intensely positive for glycogen as were the nephridia. Both in unfed and in starch-fed worms the chloragog tissue was glycogen-rich, but after starch feeding the glycogen content was distinctly increased and also the basal parts of the cells were filled with glycogen (Fig. 3). The distribution of glycogen and of dehydrogenases are given in Table 1. There is clearly no

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correspondence between glycogen contents and LDH activity. Most of the SDH and N A D H - D p activities, and probably all of the L D H activity in the branching zone of the chloragocytes are due to muscle fibres. There was no indication of glucose-6-phosphatase activity. The chloragocyte granules (the chloragosomes) were slightly stained in both assays and controls, due to unspecific lead binding.

Histological observations The bases of the chloragocytes form, together with ramifications from the gut muscle and cells associated with the blood sinus, an extensive branching zone, (Figs 3, 4 and 5), as did to some degree the base of the gut epithelial cells. The two branching zones were separated from each other by a gap consisting of the epithelial basal lamina, the thin muscle layer and a more or less extensive extracellular space, generally containing a blood sinus (Fig. 6). Free chloragocytes were often observed in starchfed worms, both in the coelomic cavity (Fig. 7) and in the typhlosole and cells containing numerous chloragosome-like granules, but devoid of glycogen, were common. In several instances groups of coelomocytes were found intimately apposed to the chloragog epithelium, suggesting some kind of interaction between the two cell types. Finally the chlo-

c.ch

Fig. I. Cross section of midbody region of earthworm, × 24. Chloragog tissue in typhlosole ch; nephridium n; peritoneal lining p. PAS reaction. Fig. 2. As for Fig, 1. Amylase-PAS reaction. Fig. 3. Basal ramifications ofchloragocyte in starch-fed worm, x 800; PAS reaction. Muscle fibre m; Basal lamina b. Fig. 4. Chloragog branching zone, x 320; reaction for SDH. Corpora of chloragocytes c.ch; Gut epithelium g.

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PouLPRENTO Table 1. Histochemical distribution of glycogen and dehydrogenases in fresh-frozen sections from the the midbody region of starch-fed L. terrestris. ? = positive reaction doubtful; n.i. = not identified; b.d. = below detection Glycogen LDH SDH MDH NADH-Dp Epidermis + + + + + Body wall Circ. muscle +/+ + + + + + + + + Longit. muscle Outer zone + + + + + + + + + + Inner zone + + + + + Coelom Epitb. lining + + + ? (+) n.J. n.J. Most coelomocytes + + + n.J. n.i. Eleocytes + + +/b.d. b.d. n.i. n.i. n.J. Nephridia + + + + + + + n.i. n.i. Neurons, nerve cord + + + + + + + n.i. n.i. Chloragocytes Basal branchings + + + ? + + + Corpora + + + b.d. b.d. b.d. (+) Gut epithelium b.d. + + + + + + ++ +

r a g o c y t e s in s t a r c h - f e d w o r m s s o m e t i m e s e x h i b i t e d a morphology reminescent of an apocrine secretion mechanism or "budding off".

" B l o o d sugars" The method of Wyatt and Kalf for trehalose and t o t a l s u g a r g a v e a n i n t e n s e to m o d e r a t e a n t h r o n e r e a c t i o n o n e t h a n o l p r e c i p i t a t e d b o d y fluids a n d a distinct reaction for both total sugar and trehalose

a f t e r F o l c h p h a s e s e p a r a t i o n . T h e s e r e s p o n s e s were u n s p e c i f i c a n d p r o b a b l y m o s t l y d u e to c h a r r i n g by t h e c o n c . s u l f u r i c acid. B o d y fluid s a m p l e s s u b m i t t e d to t h e blowfly t r e h a l o s e - G O D a s s a y were in all c a s e s n e g a t i v e for t r e h a l o s e . T h e H a e m o - G l u c o t e s t for g l u c o s e w a s n e g a t i v e or, in o n e case, slightly p o s i t i v e ( p r o b a b l y < 0 . 1 0 # g / # l ) o n c o e l o m i c fluids f r o m u n f e d w o r m s . C o e l o m i c fluids f r o m s t a r c h - f e d w o r m s e x h i b i t e d a

Fig. 5. Chloragocyte ramifications in branching zone, ×2400. Chloragosome c; arrows indicate mitochondria. Fig. 6. Blood plexus between gut epithelium and chloragog tissue, x 130. Gut epithelium g. Fig. 7. Chloragocytes released into the coelomic space in starch-fed worm, × 200. Fig. 8. Longitudinal muscle of body wall, × 200. S D H reaction. Arrow points to outer zone with high SDH activity.

Earthworm sugar metabolism

337

Table 2. Chromatography of L . terrestris blood and coelomic fluid (silica gel, I-butanol:acetone:water 4:5:1; migration 12cm; anthrone development). The spots were quantitized by comparison with a chromatographed dilution series of glucose:trehalose Sugar Rr (SD) Blood Starch fed Coelomic fluid Starch fed

Glucose, fructose

Maltose, cellobiose

Sucrose

0.32 0.22 0.16 (0.02) (0.01) (0.01) Sugar concentration in tissue fluid (/zg//zl)

Trehalose, lactose 0.11 (0.01)

0.2~).4

0.1

b.d.*

b.d.

0.2~).4 0.3t

b.d.~).l

b.d.

b.d.

0.4-0.5 OAt 0.01-4).05

b.d.

b.d.

b.d.

b.d.

b.d.

b.d.

1~, b.d.

b.d.~). 1 b.d.

2-3 b.d.

b.d. b.d.

ca

Starch fed - HCI:~

ca

Unfed Gut contents Starch fed Starch-soil

*b.d.: below detection limit (less than 0.01/zg//zl body fluid). tResult from Boehringer Mannbeim Haemo-Glukotest. SHCI, 1/15 moles/l, 70°C for 15 min.

slight reaction (about 0.3/~g/#l) and digestive fluids a moderate to intense reaction (1-6 #g//~l). The results from chromatography of body fluids from six starch fed and 10 unfed worms are given in Table 2. The results from blood and coelomic fluid were identical. Glucose was below the direct detection limit (0.05/zg/pl) in unfed worms. Application of several 2 ttl droplets from eight unfed worms to the same starting point gave glucose spots corresponding to 0.01--0.05/~g//~l coelomic fluid. Trehalose was always absent from both blood and coelomic fluid. In several cases a weak but distinct spot was present at the sucrose location. This spot was absent and the glucose spot intensified if the sample was submitted to mild acid hydrolysis before chromatography. This sucrose spot cannot be an artefact as the worms or the body fluids were never exposed to sucrose.

Glycogen The results for glycogen are listed in Table 3. The value for gut epithelium is inferred from the histochemical results. Both the chloragog and the body wall results are too large as these fractions contain most of the glycogen from the septa. The glycogen results for coelomic material are correspondingly too small.

Enzymes In earthworm material glucose-6-phosphatase and trehalose were not detected and there was no sucrase activity. As regards glucose-6-phosphatase activity in the earthworm the ratios glucose-6-phosphate hydrolysis:p-nitrophenyl-phosphate hydrolysis were virtu-

ally the same for both the lysosomal and the microsomal fractions and the activity of the microsome fraction was only about 1/10 that of the "lysosome" fraction. While the total absence of the enzyme is difficult to prove it is safe to conclude that it can be present only in insignificant amounts. The results for amylase and maltase activities and for lactate dehydrogenase (LDH) are presented in Table 4. If the enzyme distribution between chloragog tissue and gut epithelium is about the same in the typhlosole as between the chloragog and epithelial fractions (which from the data is plausible), then the midgut epithelium contains 22% of the total amylase activity, 21% of the total maltase activity and about 7%0 of the total LDH activity. The chloragog tissue then contains 43% of the total amylase activity, 26% of the total maltase activity and about 0.5% of the total LDH activity. The chloragocyte LDH activity may however be due to contamination from gut wall muscle cells. Agar gel electrophoresis revealed "identical LDH isozyme patterns for gut and body wall. Only the bands corresponding to M4 and M3H from mouse liver and muscle were present. For the chloragocyte fractions the maltase activity was about five times higher at pH 3 than at pH 7. For gut epithelium fractions the corresponding ratio was about 2, indicating the possible presence of two different disacccharidases. DISCUSSION

The results prove that trehalose is not "blood" sugar in Lumbricus terrestris. Both the sugar and the

Table 3. Glycogen distribution and amount in mature mould for two weeks at 15°C

Glycogen (n = 3) (%) Total glycogen (n = 3) (mg/g w.w.)

L . terrestris

kept on pot

Gut epithelium

Chloragog tissue

Coelomic material

Body wall

0

42 (9)

10.5 (8)

47.5 (10)

37 (9)

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POUL PRENTO Table 4. Distribution of enzymes in fractions from the mid-body region of L. terrestris

Amylase (n = 4) % RSA Maltase (n = 4) % RSA LDH (n = 4) % RSA

Total activity (U/g w.w.)*

Typhlosole

Peripheral gut

Chloragog fraction

Peritoneal material

Body wall

15 (5) 0.7 (0.1)

17 (8) 1.5(0.1)

33 (1) 5(1)

12 (4) 0.7(0.8)

23 (4) 0.5(0.1)

21 (10)

18(2) 0,7 (0.3)

13(3) 1.1 (0.3)

16(2) 5.1 (0.7)

14(6) 0.9 (0.5)

39(11) 0.5 (0.1)

0.5(0.4)

4 (1) 0.19(0.1)

3 (2) 0.27(0.1)

0.2 (0.1) 0.02(0.01)

5 (3) 0.36(0.2)

88 (5) 2,1 (0.2)

46 (12)

( )=SD. RSA = % enzyme activity/% protein. *1 U ~ 1.0#tool glucose or pyruvate/min. The large variation in U/g wet weight is due partly to differences in the hydration states of the worms, partly to varying amounts of gut contents. For maltase the determination of total activity is relatively inaccurate,

enzyme trehalase, necessary for metabolizing it, are missing. The absence of trehalose is in contrast to Fairbairn (1958). This author, however, reached his conclusion on the basis of 70% ethanol extraction of whole, minced animals and probably one or more intracellular metabolites have been confused with trehalose. Probably the author did not positively identify trehalose in the earthworm, but calculated trehalose as the difference between total "disaccharide" and glucose. This can be highly misleading, as described for the "trehalose" results using the procedure of Wyatt and Kalf (1957). The sucrose contents in body fluids is meaningless as far as the animal itself is concerned. Possibly it is due to the presence of symbiotic microorganisms. The only candidate for a "blood sugar" in the earthworm is glucose. The low glucose level in body fluid (<0.014).05/~g/#1 in normally and 0.24).4#g//~1 in starch-fed worms) is in agreement with Tillinghast et al. (1970), who found glucose to be absent or below 0.1/~g//~l. There was no appreciable difference in glucose concentration between blood and coelomic fluid, indicating, as also found by Tillinghast, that blood and coelomic fluid is in equilibrium. The observation, that glucose-6-phosphatase is absent from the earthworm, is also made by Van Gansen (1958) and Tillinghast et al. (1970). This, taken together with the very low extracellular glucose concentration and the wide variation in glucose level, leads to the conclusion that the earthworm has no regulation of the level of extracellular glucose and that the tissues are not dependent on the presence of extracellular glucose for short term metabolism. The resting rate of respiration in L. terrestris is about 0.040 ml oxygen/g w.w. per hr at 15°C. (Johnson, 1942; Raffy, 1930) or at most 1/5th the human rate. Under normal conditions the worm probably is never without digestible carbohydrate, so a "blood sugar" level of 0.054).2/~g/#l may be plausible. The free glucose may be derived directly from digestion in the gut and need not be released from a glycogen store. Only during burrowing and possibly during aestivation may the release of glucose from the gut epithelium be insufficient, and intracellular glycogen stores in the various organs are then activated. Histochemistry reveals that most of the tissues contain glycogen. The large glycogen stores (about 37 mg/g wet weight) indicate that most of the energy require-

ments are met by the degradation of carbohydrate. This is in accordance with Van Gansen (1956) but in contrast to Gruner and Zebe (1978), who found only 2-12 mg/g wet weight. These authors, however, kept the animals starved for 4 weeks before glycogen extraction, while in this case the worms were not starved and used inside 2 weeks. Probably all cell types except the gut epithelium take up glucose efficiently and build it into glcogen. This local glycogen is then the principal energy store for the cell during periods of low carbohydrates intake by the earthworm. The apparent absence of true physiological homeostasis is in contrast to vertebrates and insects and has to my knowledge not been explicitly described before in animals with circulating body fluids. It is however a logical intermediate between the acoelomate and the physiologically more complex higher invertebrate, even though the situation in the earthworm may be a secondary adaptation rather than genuinely primitative. The earthworm blood circulatory system has a small volume compared to the bulk of the animal. A logical next step for improving the solvent-transport capacity of the circulatory system would be to increase its volume on the expense of the coelomic space, the solution found in vertebrates, or to abolish the separation between the blood circulatory system and the coelomic space, the solution chosen by arthropods. The branching zones of the chloragog tissue and of the gut epithelial base indicate an efficient transport of metabolites from the intestine to the chloragog tissue. This is in accordance with the function of the chloragog tissue as a storage organ for glycogen and other materials derived from the break-down of food in the intestine, but may also be related to the removal of superoxide/hydrogen peroxide from the blood (Prento, 1986). The histochemical distribution of dehydrogenases is peculiar. LDH is probably wholly absent from the chloragocytes. The slight activity of this cytosol enzyme in chloragocyte fractions, and histochemically in the branching zone, is probably due to the gut muscle fibres. Although less than the interspaced muscle fibres, the basal ramifications of the chloragocytes exhibited appreciable SDH-, MDHand N A D H - D p activities (Table 1 and Fig. 4), which agree with the relative abundance of mitochondria in the ramifications (Fig. 5). In contrast the chlo-

Earthworm sugar metabolism ragocyte corpus appears devoid of dehydrogenase activities. So, while the chloragocyte stores glycogen in relatively huge amounts it is poor in the metabolic machinery for utilizing it efficiently. The low dehydrogenase contents and the concomitant low metabolic activity was also remarked upon by Van Gansen (1958) and Urich (1965). Urich found that about 10% of the chloragog protein was mitochondrial. This value seems too large considering the histochemical results described here and indicates, aside from probable contamination from the mitochondria-rich gut muscle during the isolation procedure, that the dehydrogenase content of the chloragocyte mitochondria is very low. This may be supported by Lindner (1965), who found the chloragocyte mitochondria very poor in crista. In conclusion, the corpus of the chloragocyte seems to be merely a storage organ, while the basal part of the chloragocyte with its branchings contains most of the metabolic machinery and is concerned with the uptake of substances released from the intestine or otherwise present in the blood, see Prento (1986). The outer zone of the longitudinal body wall muscle is especially rich in mitochondrial dehydrogenases, Fig. 8. This zone is also the best oxygenated during aerobic conditions and probably the LDH in this region is responsible for most of the lactate dehydrogenation and subsequent mitochondrial oxidation, while the LDH in the deeper muscle layers is predominantly pyrovate-reducing. So the pyrovate to lactate to pyruvate interaction we find in vertebrate muscle and liver is in L. terrestris exclusively in the muscle, as also indicated by the fact that about 90% of the mid-body LDH is in the body wall. This agrees well with the exclusive presence of the M4 and M3H isozymes and is in accordance with the findings of Davis and Slater (1928), Dastoli (1964) and possibly Augenfield (1966). In contrast Gruner and Zebe (1978) found little or not lactate accumulation in L. terrestris during extreme hypoxia. However, their work deals with general metabolic adaptation to anoxia, which may be more complex than simply lactate formation. The present paper shows that LDH is present in fairly large amounts in the body wall, indicating that lactate production is part of the normal physiology of L. terrestris and surely important during burrowing activity. As regards carbohydrates metabolism the claloragog tissue has generally been considered analogous to vertebrate liver because of its high glycogen contents (e.g. Van Gansen, 1958; Roots, 1960). However, Tillinghast et al. (1970), on the basis of the low blood sugar concentration and the absence of glucose-6phosphatase, concluded that the chloragog tissue could not be entirely analogous to vertebrate liver. Obviously the tissue relationships as regards "blood" sugar, glycogen, glucose-6-phosphatase, LDH and the other dehydrogenases preclude any real analogy between the chloragog tissue and liver. By far the most plausible physiological relation between the chloragog tissue and the other tissues of the earthworm is the one originally suggested by K/ikenthal (1885) and further developed by Liebmann (1928, 1931, 1948). Liebmann (1931) described the release of chloragocytes into the coelomic space, where they are

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transformed into eleocytes. These nutrient-transport cells migrate into the varius tissues, to which they somehow deliver their contents. He also described how from time to time the distal end of the chloragocyte is "pinched off" and falls into the coelom, where it is ingested by coelomocytes. The author was concerned with lipid transport in earthworms, but obviously the same phenomena may be relevant as regards sugar (glycogen) transport. The migration or release of chloragocytes into the coelomic space can be inferred from histological observations in this paper. In sections of starch-fed worms, numerous chloragocytes, singly or in groups, can be seen free in the coelomic space (Fig. 7) and in the typhlosole free chloragocytes can be found in the coelomic crevices. As the chloragocytes are not motile, they are probably transported by the pumping action of the coelomic fluid, when the worm moves. The newly released chloragocytes are very rich in glycogen and are very little modified, while later stages are more or less depleted of glycogen. The presence of free chloragocytes in the coelomic space was also noted by Stein and Cooper (1978). In starch-fed worms the cells of the peritoneal lining, especially of the septa, are very rich in glycogen. The fate of this glycogen is uncertain. Liebmann (1928) found that in the body wall the eleocytes formed aggregates especially in the neighbourhood of the septa and in the connective tissue separating the bundles of longitudinal or circular muscle. These are also the localities where the neighbouring muscle tissue seems to store most glycogen. On the other hand, as reviewed by Jamieson (1981), several types of coelomocytes are derived from the somatopleure and it may be that the glycogen is part of a storage-transport system similar to the proposed chloragocyte-eleocyte system, but perhaps more concerned with the energy requirements of the body wall muscle. There still remains the question of how the chloragocyte glycogen, and the glycogen of the coelomic lining, is broken down and released. In this context it is interesting that the chloragocytes and the coelom contain exceptionally high amylase and maltase activities, see Table 4. As all the sessile chloragocytes are equally glycogen-rich, amylase and maltase are probably somehow activated after "budding off", the release of the chloragocyte into the coelomic space or the uptake of chloragocyte material into coelomocytes. The low pH optimum of the maltase suggests that it is related to or identical with a lysosomal ct-glucosidase as found by Joris (1964). Possibly the maltase and amylase activities are due to the same enzyme. Valembois and Cozaux (1970) found in the earthworm Eiseniafoetida, by use of autoradiography that amino acids and glucose, given orally or injected into the coelomic space, were first accumulated in the chloragocytes. This in accordance with the results of Tillinghast et al. (1970) and supports a nutritionstorage function of the chloragog tissue. Valembois and Cozaux further found that after some hours a significant proportion of the radioactive material was located in coelomic cells, which they concluded were eleocytes. This too is in accordance with the chloragocyte-eleocyte/trophocyte scheme by Lieb-

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mann (1942), with the observations by Stein and Cooper (1978) and with the data presented here. Liebmann followed these events mostly by studying the fate of lipid droplets and chloragosomes. It seems a major part of the lipid is transformed into phospholipids and not used catabolically. This may fit well with a relatively inefficient transport mechanism for lipids and merits further investigation. Liebmann (1931) found that chloragocytes or eleocytes were especially abundant in the gonads and that lipid droplets and chloragosomes were taken up apparently unmodified by the developing eggs. The Ca-phosphate content of chloragosomes (Prento, 1979) is significant. Phosphate is one of the limiting factors of plant growth and probably only present in low amounts in the natural food of L. terrestris. In contrast neutral lipids are relatively abundant, so an effective mechanism of phosphate uptake and storage may be necessary to ensure adequate phospholipid synthesis and thereby membrane biosynthesis and growth. The phosphate store may also be necessary for the increased nucleic acid biosynthesis during production of eggs and sperm. It is here of interest that after injection of tritiated thymidin into the mid-green coelomic space of mature earthworms the label was not detected in nuclei of the mid-region, but only in spermatozoa of the seminal vesicles (Prentg, unpublished observation). All these data together point to the chloragog tissue being a storage tissue primarily involved in growth, gamete maturation, wound healing and regeneration, that is anabolic processes, while the catabolic needs of the various tissues, including the body wall muscle, are met by the more or less constant influx from the gut of glucose and other low-molecular substances. This implies that hormonal regulation of "blood sugar" may be absent and that the competitive uptake of glucose and other materials into the chloragog tissue, and the subsequent release of eleocytes is necessary for the adequate local supply of materials for growth and other anabolic processes. While some of the findings of Liebmann, and some observations not cited here (Liebmann, 1942, 1946) may perhaps find other interpretations, the general scheme, midgut epithelium--chloragocyte-transport of bulk nourishment ("eleocyte"), is in accordance with the data propounded in this paper, while their role in the maintenance of "blood sugar" level is not. This points to important differencies in physiological strategies between vertebrates or arthropods on the one hand and oligochaetes on the other hand. The schemes proposed by Liebmann and by the present author should therefore be thoroughly tested, not only on oligochaetes, but also on other annelids and lower invertebrates, where Liebmann (1946) found trephocytes or eleocytes to be common. Acknowledgement--I have to thank Alice Kristiansen for skilled technical assistance. REFERENCES

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