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The lugwormArenicola marina: A model of physiological adaptation to life in intertidal sediments

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HELGOL,~NDER MEERESUNTERSUCHUNGEN Helgol~nder Meersunters. 50, 37-68 (1996)

T h e l u g w o r m

A r e n i c o l a m a r i n a :

a m o d e l o f

p h y s i o l o g i c a l a d a p t a t i o n to l i f e i n i n t e r t i d a l s e d i m e n t s

E. Zebe 1 & D. Schiedek 2

llnstitut fur Zoophysiologie, Westf. Wilhelms-Universit~t; Hindenburgplatz 55,

D--48143 Mfinster, Germany

2Institut fiir Ostseeforschung Warnemfinde an der Universit~t Rostock; Seestr. 15,

D-18119 Rostock, Germany

Manuscript received on 30th January 1994; accepted for pubhcation on 1st August 1994

ABSTRACT: The results of more than two decades of intensive research on the physiological and biochemical features of the lugworm are reviewed with the aim of drawing a general and com- prehensive picture of the adaptation of this species to the special conditions of living in the tidal zone, which may also hold true for the majority of invertebrates found in this habitat.

I N T R O D U C T I O N

In his m o n o g r a p h on the l u g w o r m

Arenicola marina,

F. Krfiger (1971) o u t l i n e d the c u r r e n t state of k n o w l e d g e c o n c e r n i n g morphological o r g a n i z a t i o n a n d physiology of this polychaete. Since t h e n the l u g w o r m has, for several reasons, b e c o m e a p o p u l a r object of physiological a n d b i o c h e m i c a l studies:

(1) It is one of the largest a n n e h d s a n d has a rather simple a n a t o m i c a l o r g a n i z a t i o n with a large coelomic cavity e x t e n d i n g t h r o u g h most of the body without s e g m e n t a l subdivisions. This m a k e s e x p e r i m e n t a l m a n i p u l a t i o n s , such as injections, particularly easy.

(2) It is f o u n d i n d e n s e p o p u l a t i o n s in the tidal zone t h r o u g h o u t the North Sea coast. H e n c e the supply, e v e n of large n u m b e r s of specimens, is easy. Moreover, l u g w o r m s c a n be m a i n t a i n e d in c i r u c u l a t i n g sea water for several weeks, which r e n d e r s it feasable to use t h e m in places distant from the coast.

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A. marina

is well a d a p t e d to the special conditions of the tidal zone and, therefore,

exhibits particular physiological properties w h i c h h a v e attracted the interest of a g r o w i n g n u m b e r of scientists.

D u r i n g the last two decades, m a n y e x p e r i m e n t a l i n v e s t i g a t i o n s h a v e b e e n c o n d u c t e d w h i c h h a v e y i e l d e d a w e a l t h of n e w results. As a c o n s e q u e n c e ,

Arenicola

has b e c o m e o n e of the most e x t e n s i v e l y s t u d i e d m e m b e r s of the httoral f a u n a and, therefore, m a y be c o n s i d e r e d a m o d e l w h i c h shows particularly clearly the special physiological properties n e c e s s a r y for h ~ n g i n e x t r e m e habitats such as the tidal zone.

M a n y of the n e w findings h a v e a l r e a d y b e e n treated in reviews stressing special points of view (Sch6ttler, 1980, 1986; Sch6ttler & Bennett, 1991; Kamp, 1992). After two

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38 E. Z e b e & D. Schiedek

d e c a d e s of active research it n o w seems appropriate to give a more g e n e r a l s u r v e y on the physiology of Arenicola b a s e d on the m a n y a n d diverse data g a i n e d since Krfiger's report.

NUTRITION AND DIGESTION

The l u g w o r m is a w e l l - k n o w n e x a m p l e of a direct deposit feeder. It i n g e s t s material from the b a s e of a c o l u m n which e x t e n d s vertically from the horizontal p a r t of its b u r r o w to a f u n n e l - l i k e cavity at the s e d i m e n t surface. This "feeding column" ( m e a s u r i n g 5 m m in d i a m e t e r a n d a b o u t 20 cm in length) is particulary rich in organic m a t e r i a l (Rijken, 1979), p r o b a b l y b e c a u s e the f u n n e l functions as a trap collecting detritus as this settles on the surface of the sediment. T h e f e e d i n g activity of the l u g w o r m causes the material to move from the f u n n e l t h r o u g h the whole l e n g t h of the c o l u m n in a few hours. At the base, the worm p r e f e r e n t i a l l y takes up small particles which easily stick to the m u c u s l i n i n g the papillae of its proboscis, w h e r e a s larger particles are rejected and, as a c o n s e q u e n c e , a c c u m u l a t e in the vicinity of the b u r r o w (Baumfalk, 1979b). Such selective r e m o v a l of certain c o m p o n e n t s from the c o l u m n m a y result in a h e t e r o g e n o u s d i s t r i b u t i o n of particles of different size in the sediment.

In the past, the q u e s t i o n as to w h a t actually is the food of the l u g w o r m has b e e n extensively a n d controversely discussed (Rijken, 1979). Direct utilization of detritus, in particular that of p l a n t origin, a p p e a r s unlikely, b e c a u s e its c o m p o n e n t s are highly resistant to d e g r a d a t i o n . However, b r e a k d o w n of detritus is a c c o m p l i s h e d by bacteria which thrive on the rich deposits p r e s e n t in the sediment. Therefore, A r e n i c o l a p r o b a b l y uses m i c r o o r g a n i s m s as the most i m p o r t a n t source of food. Benthic diatoms a n d m e i o f a u n a f o u n d in the f e e d i n g c o l u m n p r o b a b l y are a n additional food c o m p o n e n t , a l t h o u g h their q u a n t i t a t i v e significance i n this respect is difficult to e v a l u a t e .

The l u g w o r m m a y also filtrate the respiratory w a t e r to obtain p l a n k t o n a n d sus- p e n d e d particles (Krfiger, 1958b; Hobson, 1967). It seems doubtful, however, that this can significantly c o n t r i b u t e to the food b a l a n c e , b e c a u s e the v o l u m e of r e s p i r a t o r y water is small in a b s o l u t e terms: 10-20 m l . g - i . h-1. According to H y l l e b e r g - K r i s t e n s e n (1975), m i c r o o r g a n i s m s should particularly thrive in the "feeding column" a n d h e n c e rapidly increase in their mass due to the f a v o u r a b l e conditions for growth a n d reproduction. However, c o n s i d e r i n g the comparatively fast t u r n o v e r of material c o m p o s i n g the column, it seems difficult to i m a g i n e that this " g a r d e n i n g " could s u p p l y s i g n i f i c a n t q u a n t i t i e s of a d d i t i o n a l food.

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Arenicola marina: a m o d e l of p h y s i o l o g i c a l a d a p t a t i o n 39 the functional differentiation of the gut and, in addition, h a v e q u a l i t a t i v e l y a n a l y s e d the h y d r o l y s i s of v a r i o u s c o m p o u n d s a s s u m e d to b e c o m p o n e n t s of the n a t u r a l food (Ker- m a c k , 1955; L o n g b o t t o m , 1970; H y l l e b e r g - K r i s t e n s e n , 1972). T h e y h a v e d e m o n s t r a t e d that c a r b o h y d r a t e a n d p r o t e i n d e g r a d a t i o n are a c c o m p l i s h e d solely b y p r e p a r a t i o n s of the o e s o p h a g u s d i v e r t i c u l a a n d the s o - c a l l e d stomach. Obviously, t h e s e sections of the gut m u s t b e the c e n t r e s of digestion.

A n a l y s i n g the d i g e s t i v e p r o c e s s e s q u a n t i t a t i v e l y , E b e r h a r d t (1988) o b s e r v e d that g l y c o g e n , s t a r c h a n d m a l t o s e w e r e v e r y r a p i d l y h y d r o l y s e d , w h e r e a s d e g r a d a t i o n of lipids was slow. F u r t h e r m o r e , he d e m o n s t r a t e d an e x t r e m e l y h i g h proteolytic c a p a c i t y a n d a v e r y r a p i d splitting of ~-glucosidic a n d [~-glucosaminic b o n d s as w e l l as of p h o s p h a t e b o n d s w h i c h s e e m typical f e a t u r e s of the a l i m e n t a r y s y s t e m of Arenicola. E b e r h a r d t (1988) also c o n f i r m e d that the d i g e s t i v e e n z y m e s r e s p o n s i b l e for the b r e a k - d o w n of the c o m p o u n d s m e n t i o n e d a b o v e m a i n l y occur in the diverticula of t h e o e s o p h a - gus a n d in the anterior p a r t of the " s t o m a c h " . The e n z y m a t i c p a t t e r n w a s c o n s t a n t a n d did not c h a n g e significantly e v e n d u r i n g e x t e n d e d starvation periods. This i n d i c a t e s that d i g e s t i v e e n z y m e s are virtually c o n t i n u o u s l y s y n t h e s i z e d a n d s e c r e t e d w h i c h c o i n c i d e s w i t h the s u s t a i n e d d i g e s t i o n typical of the n a t u r a l habitat. T h e strikingly h i g h activities of the d i g e s t i v e e n z y m e s are p r o b a b l y n e c e s s a r y r e g a r d i n g the fast p a s s a g e of t h e food t h r o u g h the gut, i.e. the short p e r i o d of digestion. The time m e a s u r e d b e t w e e n the i n g e s t i o n of l a b e l l e d m a t e r i a l a n d its a p p e a r a n c e in the faeces was 30 to 60 m i n u t e s (Jacobsen, 1967; Rijken, 1979). A c c o r d i n g to E b e r h a r d t (1988), the e x t r e m e l y high p r o t e o l y t i c activity m a y b e s e e n in r e l a t i o n to the i n d i r e c t utilization of d e t r i t u s b y Arenicola. Utilizing b a c t e r i a (which are t h e most i m p o r t a n t c o m p o n e n t of food) will d e p e n d on r a p i d p e r m e a t i o n of the p r o t e i n a s e s t h r o u g h the cell wall and, s u b s e q u e n t l y , on effective d e g r a d a t i o n of cell m e m b r a n e proteins. A l t h o u g h t h e r e is no d i r e c t e v i d e n c e s u p p o r t i n g this a s s u m p t i o n , it s e e m s most l i k e l y that the p r o t e i n a s e s of the s t o m a c h are h i g h l y effective in this respect.

Thus far, no l y s o z y m e activity has b e e n d e t e c t e d in the l u g w o r m (Eberhardt, 1988). This could b e d u e to t e c h n i c a l difficulties r e s u l t i n g from tests d e v e l o p e d for the s p e c i a l conditions of v e r t e b r a t e s w h i c h are ineffective at a salinity t h r e e times h i g h e r t h a n t h a t in m a m m a l i a n systems. F u r t h e r m o r e , m a r i n e b a c t e r i a m a y h a v e p r o p e r t i e s w h i c h s u b s t a n - tially differ from those of the b a c t e r i a s p e c i a l i z e d in living as m a m m a l i a n p a r a s i t e s .

In a n y case, a n effective d i g e s t i o n of m a s s e s of b a c t e r i a in the gut of Arenicola has b e e n c o n v i n c i n g l y d e m o n s t r a t e d b y the f e e d i n g e x p e r i m e n t s d e s c r i b e d a b o v e (Rijken 1979; Liefken at al., 1979).

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40 E. Z e b e & D. S c h i e d e k

The l u g w o r m (like several other m a r i n e invertebrates) is c a p a b l e of a b s o r b i n g "dissolved o r g a n i c matter" (DOM) from the a m b i e n t water. Thus, it m a y e m p l o y a s e c o n d m o d e of n u t r i t i o n in addition to i n g e s t i n g food, E x p e r i m e n t a l e v i d e n c e shows the u p t a k e of short-chain fatty acids t h r o u g h the b o d y - w a l l (Hoist & Zebe, 1984). Obviously, this absorption r e q u i r e s a sufficient s u p p l y of oxygen, for it was found to cease u n d e r hypoxic conditions.

The rate of p r o p i o n a t e a b s o r p t i o n was h i g h e r t h a n that of b u t y r a t e a n d acetate. U p t a k e of all these fatty acids exhibited s a t u r a t i o n kinetics. Absorption of p r o p i o n a t e was i n h i b i t e d b y the p r e s e n c e of b u t y r a t e a n d acetate (maximal i n h i b i t i o n 78 %). This result suggests that a carrier is involved. After their u p t a k e the fatty acids w e r e rapidly converted, m a i n l y to aspartate, g l u t a m a t e a n d a l a n i n e .

The ability of the l u g w o r m to absorb fatty acids from the a m b i e n t w a t e r m a y b e r e l a t e d to the o c c u r r e n c e of p r o p i o n a t e a n d acetate in its habitat. T h e s e fatty acids originate from the a n a e r o b i c m e t a b o l i s m of m i c r o o r g a n i s m s p r e s e n t i n the sediment. Their c o n c e n t r a t i o n in interstitial w a t e r s o m e t i m e s exceeds 10 ~ m o l . 1-1 (Zebe, u n p u b - h s h e d results). At the present, it is not k n o w n w h e t h e r Arenicola actually m a k e s use of its particular ability; therefore, its significance with respect to nutrition c a n n o t be appraised.

RESPIRATION

The l u g w o r m lives in a virtually anoxic e n v i r o n m e n t a n d must o b t a i n its o x y g e n from the overlying water. This requires c o n t i n u o u s p u m p i n g to irrigate the b u r r o w a n d to m a i n t a i n the respiratory gas e x c h a n g e . T h e m e c h a n i s m of p u m p i n g a n d the b e h a v i o u r i n v o l v e d in irrigation has b e e n e x t e n s i v e l y s t u d i e d b y Wells (1949) a n d Kr~iger (1958b, 1964). P u m p i n g activity proceeds in cycles w h i c h last approximately 40 m i n u t e s in large specimens, while they are c o n s i d e r a b l y shorter in smaller ones. The cycles are p r o b a b l y u n d e r the control of a p a c e - m a k e r situated in the n e r v o u s system. They c a n be o b s e r v e d e v e n in i n d i v i d u a l s t a k e n out of their b u r r o w s a n d p l a c e d in free water.

The p u m p i n g effect results from contractions of the l o n g i t u d i n a l m u s c l e s which cause the d i a m e t e r of the b o d y to increase, until the l u m e n of the b u r r o w is completely closed. The contractions start at the posterior e n d of the m a i n body (i.e. at the b a s e of the "tail") a n d are slowly p r o p a g a t e d towards the "head". The contracted s e c t i o n acts like a piston in a c y h n d e r p u s h i n g the w a t e r i n front of it a h e a d a n d s u c k i n g i n o x y g e n - r i c h w a t e r from b e h i n d , i.e. from a b o v e the s e d i m e n t surface.

The p u m p i n g rate r a n g e s b e t w e e n 8 a n d 26 ml 9 g-1 . h-1 (K_riiger, 1964; M a n g u m , 1976; Baumfalk, 1979a). In Arenicola cristata (100 g w e i g h t as c o m p a r e d to 5 - 1 0 g in A. marina), the rate is a b o u t a fourth of that m e a s u r e d in the latter species. O n the other h a n d , c o n s i d e r a b l y h i g h e r rates h a v e b e e n m e a s u r e d in Abarenicola affinis u n d e r laboratory conditions (Barrow & Wells, 1982). A l t h o u g h the u s u a l irrigation rate seems low, it is sufficient to m a i n t a i n a m e a n pO2 of at least 70 Tort i n the b u r r o w w a t e r ( M a n g u m , 1973). As soon as the p u m p i n g stops, the o x y g e n level d e c l i n e s rapidly.

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Arenicola marina: a m o d e l of physiological a d a p t a t i o n 41 supports this hypothesis. Moreover, it is not k n o w n w h e t h e r lugworms irrigating the b u r r o w s in their n a t u r a l habitat actually p u m p air b u b b l e s together with water. (Aerial respiration has also b e e n reported in Abarenicola pacifica without m e n t i o n i n g concrete e x p e r i m e n t a l results: H o b s o n as cited by May, 1972). More recent studies have s h o w n that in the n a t u r a l habitat Arenicola a c c u m u l a t e s a n a e r o b i c e n d p r o d u c t s d u r i n g low tide (Toulmond, 1973; Pionetti & T o u l m o n d , 1980; Sch6ttler et al., 1983). This seems to p r e c l u d e a significant role of aerial respiration.

Arenicola has 13 pairs of highly b r a n c h e d gills which, in certain p h a s e s of the p u m p i n g cycle, are completely retracted into the coelomic cavity. The respiratory gas e x c h a n g e , however, is not limited to the gill epithelium, b u t takes place also at the u n d i f f e r e n t i a t e d b o d y surface. This has b e e n d e m o n s t r a t e d b y M a n g u m (1976) who a n a l y s e d the o x y g e n c o n t e n t of the w a t e r in different sections of the b u r r o w (Fig. 1). According to her data, more t h a n 75 % of the o x y g e n is i n d e e d t a k e n u p via the gill- b e a r i n g s e g m e n t s (although some of that p r o b a b l y enters the b o d y through the epidermis i n s t e a d of the gill epithelium). M a n g u m estimates that almost e q u a l a m o u n t s of o x y g e n are t a k e n up by the gills a n d b y the u n d i f f e r e n t i a t e d surface. This would m e a n that at least half of the o x y g e n is supplied directly to the cells (mainly of the body-wall), w h e r e a s 50 % or less are carried by the blood to s u p p l y the i n t e r n a l organs such as n e r v o u s system, gut e p i t h e l i u m a n d c h l o r a g o g e n tissue.

By its respiration the l u g w o r m extracts b e t w e e n 32 a n d 40 % (58 % in A. cristat~) of the o x y g e n p r e s e n t in the b u r r o w w a t e r ( M a n g u m , 1976). This seems to be low in c o m p a r i s o n with some fish species that c a n extract more t h a n 90 %. As M a n g u m (1976) emphasizes, the o r g a n i s a t i o n of the gills a n d the circulatory system of Arenicola is rather primitive a n d h e n c e c a n n o t be expected to f u n c t i o n as efficiently as in the more a d v a n c e d

1 4 6

6 2

6

I

6 6

1 0 4

1 3 2

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42 E. Z e b e & D. Schiedek

systems of h i g h e r a n i m a l s such as fish. T h e gills consist of b u n d l e s of r a m i f y i n g a n d highly contractile t u b e s filled with blood. T h e y are emptied, w h e n they a r e w i t h d r a w n into the coelomic cavity as the respective s e g m e n t s increase in diameter d u r i n g p u m p i n g or locomotory activity. It seems d o u b t f u l w h e t h e r in the e x p a n d e d s t a t e there is a n efficient a n d c o n t i n u o u s blood transport. In a n y case, a c o u n t e r - c u r r e n t e x c h a n g e b e t w e e n w a t e r a n d blood h k e that in the fish gill seems impossible in Arenicola ( M a n g u m , 1976).

U n d e r normoxic conditions (i.e. d u r i n g high tide), the rate of o x y g e n u p t a k e b y the l u g w o r m a m o u n t s to 5-14 ~tl 9 g-1 . h-1 at 6~ 22 ~tl at 14~ a n d 1 1 - 2 7 ~tl at 23~ ( M a n g u m , 1976; Sch6ttler, 1989). W h e n the h a b i t a t e m e r g e s at low tide a n d the s u p p l y of o x y g e n - r i c h w a t e r is interrupted, the l u g w o r m r e d u c e s its m o v e m e n t s to a m i n i m u m . Also, perfusion of the gills virtually ceases a n d the whole circulation d e c l i n e s . As a c o n s e q u e n c e , gas e x c h a n g e stops a n d the pO2 in the blood drops from 6 to 1 Torr, w h e r e a s the pCO2 rises from 0.4 to 2.0 Torr a n d h i g h e r a n d the pH d e c l i n e s from 7.48 to 7.35 (Toulmond, 1973: Wells, 1974).

From studies of Abarenicola pacifica, it a p p e a r s that this species is a b l e to m a i n t a i n a m e a n o x y g e n c o n t e n t of a b o u t 1 ml . 1-1 in the b u r r o w water e v e n at l o w tide, which results in a low respiratory rate (2 ~1 9 g-1 . h - l , t e m p e r a t u r e ?; May, 1972).

Respiratory m e a s u r e m e n t s in the laboratory r e v e a l e d constant rates of o x y g e n c o n s u m p t i o n at pO2 r a n g i n g from 120 to 500 Torr (Toulmond & Tschernigovtzeff, 1984; Conti & T o u l m o n d , 1986; Dejours & T o u l m o n d , 1988). This is a c c o m p l i s h e d b y variation of the p u m p i n g activity. At pO2 h i g h e r t h a n 160 Torr the irrigation rate is reduced, w h e r e a s it is i n c r e a s e d w h e n the pO2 declines b e l o w 160 Torr. At pO2 b e l o w 120 Torr, p u m p i n g activity decreases rapidly, until, at a b o u t 50 Torr, it stops completely. The course of o x y g e n c o n s u m p t i o n is similar, b e i n g c o n s t a n t at high o x y g e n levels a n d d e c r e a s i n g parallel to the pO2 (Shumway, 1979).

Other p a r a m e t e r s such as t e m p e r a t u r e , body mass, season a n d r e p r o d u c t i v e state m a y also i n f l u e n c e the l u g w o r m ' s rate of o x y g e n c o n s u m p t i o n .

In the u p t a k e of o x y g e n via the gills, the h e m o g l o b i n p r e s e n t in the b l o o d plays a k e y role. This has b e e n d e m o n s t r a t e d b y a p p l y i n g c a r b o n monoxide w h i c h inhibits the b i n d i n g of o x y g e n b y h e m o g l o b i n . T h e respiratory rate was t h e n m a r k e d l y r e d u c e d (maximal i n h i b i t i o n : 40 % ; Krfiger, 1959; M a n g u m 1976). At 6 ~ the i n h i b i t o r y effect was m u c h smaller t h a n at 20 ~ M a x i m a l i n h i b i t i o n occurred at pO2 b e t w e e n 120 a n d 30 Torr (150 a n d 40 Torr in A. cristata), w h e r e a s it was comparatively low at h i g h pO2. These results indicate a function of h e m o g l o b i n in o x y g e n u p t a k e especially at i n t e r m e d i a t e pO2 (as it p r e s u m a b l y prevails in the b u r r o w u n d e r n o r m a l conditions), w h e r e a s the significance of the p i g m e n t declines at h i g h e r as well as at very low o x y g e n levels and, in addition, at low t e m p e r a t u r e s . With respect to the role of h e m o g l o b i n , a n o t h e r result is especially i n t e r e s t i n g ; w h e n gill f u n c t i o n was p r e v e n t e d by ligations, this n o t only resulted in a decrease of the respiratory rate, b u t it w a s virtually identical i n e x t e n t with the i n h i b i t o r y effect of CO on h e m o g l o b i n ( M a n g u m , 1976).

CIRCULATION AND BODY FLUIDS

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Arenicola manna: a model of physiological a d a p t a t i o n 43 6 to 10 times larger t h a n that of blood (Toulmond, 1971a), has a static function. In addition, in p u m p i n g a n d locomotory activities, it functions as a c o m p o n e n t of a hydraulic system, b e i n g shifted from o n e part of the b o d y to another. This is possible b e c a u s e in the l u g w o r m the coelomic cavity is not s u b d i v i d e d by dissepiments, as in m a n y p o l y c h a e t e species, except in the "tail".

Due to high c o n c e n t r a t i o n s of extracellular h e m o g l o b i n in the blood (10-14 g - 100 m1-1) its o x y g e n capacity is high (7 ml - 100 m1-1 in A. marina a n d 13 m l . 100 m1-1 in A. cristata: T o u l m o n d , 1973; M a n g u m , 1976). O n the other hand, no h e m o g l o b i n a n d very little protein are p r e s e n t in the coelomic fluid; the o x y g e n capacity is accordingly low.

The m o l e c u l a r o r g a n i z a t i o n of h e m o g l o b i n has b e e n extensively a n a l y s e d in the A m e r i c a n species A. cristata (Waxman, 1971). It is a n extremly large molecule (2850 kDa) c o n t a i n i n g 96 h e m e groups which is m a d e up of two identical disc-shaped halves, each of these c o m p o s e d of six s u b u n i t s of 230 kDa. The s u b u n i t s consist of four p o l y p e p t i d e chains (2 of 13 kDa a n d 2 of 14 kDa) a n d two h e i n e groups. In Abarenicola pacifica, the h e m o g l o b i n m o l e c u l e is slightly smaller t h a n in A. cristata: 2600 kDa (Garlick & Terwil- liger, 1977).

Characteristic properties of l u g w o r m h e m o g l o b i n are a high o x y g e n affinity (p50 -- 2.0-2.5) a n d a p r o n o u n c e d cooperativity. T h e cooperativity is h e t e r o g e n o u s ; it i n c r e a s e s with rising o x y g e n a t i o n and, in addition, with rising pH in the physiological r a n g e b e t w e e n 7.1 a n d 7.4 (Weber, 1978). Furthermore, a n o r g a n i c a n i o n s can i n f l u e n c e o x y g e n affinity as well as cooperativity of Arenicola h e m o g l o b i n , b u t the significance of this effect with r e g a r d to its role u n d e r the conditions of the n a t u r a l habitat a p p e a r s doubtful (Everaarts & Weber, 1974; K r o g h - R a s m u s s e n & Weber, 1979). It seems n o t e w o r t h y that the analytical data of h e m o g l o b i n s o b t a i n e d from various l u g w o r m species differ some- what (Garlick & Terwilliger, 1977).

Because of the strongly sigmoid dissociation curve, comparatively large q u a n t i t i e s of o x y g e n c a n be b o u n d or r e l e a s e d in r e s p o n s e to small c h a n g e s of pO2.

The h e m o g l o b i n s of some species of Arenicola differ m a r k e d l y with respect to the Bohr effect. It is particulary large in A. marina, b u t rather small in A. cristata, w h e r e a s the values m e a s u r e d in Abarenicola claparedi a n d Arenicoloides branchiaHs are i n t e r m e d i - ate. W e b e r (1972) p r e s u m e s that these differences are d u e to the a d a p t a t i o n of each species to the particular conditions of its habitat. O n the other hand, M a n g u m (1976) concludes from her results that in Arenicola the functional significance of the Bohr effect with regard to o x y g e n transport is p r o b a b l y low.

The pO2 in the blood of the l u g w o r m is low: i n p o s t - b r a n c h i a l blood, v a l u e s r a n g i n g b e t w e e n 3 a n d 6 Torr h a v e b e e n m e a s u r e d at high tide a n d 1 Torr at low tide (Toulmond, 1973, 1975). T h e s e low o x y g e n levels result from the high o x y g e n affinity of h e m o g l o b i n and, in addition, especially from the m i x i n g of o x y g e n a t e d a n d n o n - o x y g e n a t e d blood in the primitive circulatory system, as has b e e n e m p h a s i z e d b y M a n g u m (1976).

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44 E. Z e b e & D. S c h i e d e k

point (6.3 a n d 8.0; W e b e r & Pauptit, 1972). S D S - e l e c t r o p h o r e s i s of A. pacifica m y o g l o b i n , h o w e v e r , r e v e a l e d a s i n g l e b a n d i n d i c a t i n g an a p p a r e n t m o l e c u l a r m a s s of 15.3 kDa (Garlick & Terwilliger, 1977). Since the o x y g e n affinity of the m y o g l o b i n s is c l e a r l y h i g h e r t h a n that of h e m o g l o b i n , t h e y could e n h a n c e the transfer of o x y g e n from t h e b l o o d to the muscles. V e r y h k e l y , t h e s e p i g m e n t s a r e involved in the i n t r a c e l l u l a r d i s t r i b u t i o n of o x y g e n b y m e a n s of facilitated diffusion. Moreover. an e q u a l l y i m p o r t a n t function of l u g w o r m m y o g l o b i n p r o b a b l y consists in t a k i n g up o x y g e n directly from the a m b i e n t water, a n d this o b v i o u s l y p r o c e e d s at a h i g h rate.

O n l y few d a t a a r e a v a i l a b l e on the t r a n s p o r t of CO2 in the lugworm. It a p p e a r s that it occurs m a i n l y as h y d r o g e n c a r b o n a t e , w h e r e a s a few p e r c e n t of the total a r e t r a n s p o r t e d in p h y s i c a l solution a n d b o u n d to h e m o g l o b i n by c a r b a m i n o formation. A t a pCO2 of 3 T o r t (the m a x i m u m v a l u e m e a s u r e d in p r e b r a c h i a l blood; T o u l m o n d , 1973), the COz c a p a c i t y of Arenicola b l o o d a m o u n t s to 7 vol% (Weber, 1978).

A c c o r d i n g to T o u l m o n d (197 lb), t h e r e is a p r o n o u n c e d H a l d a n e effect, i.e. o x y g e n a - tion of h e m o g l o b i n results in a d e c r e a s e of C O 2 - b i n d i n g capacity, w h i c h favours its r e l e a s e in the gills. R a p i d h y d r a t i o n of CO2 is a c c o m p h s h e d b y a specific c a r b o a n h y d r a s e in l u g w o r m b l o o d (Wells, 1973). Unlike that of v e r t e b r a t e blood, it is not b o u n d to cells or o t h e r structural e l e m e n t s , b u t occurs in solution in the " p l a s m a " . On t h e o t h e r hand, no c a r b o a n h y d r a s e activity has b e e n d e t e c t e d in gill tissue a n d coelomic fluid.

Arenicola c a r b o a n h y d r a s e (like that in v e r t e b r a t e blood) is i n h i b i t e d b y a c e t a z o l a m i d e (Wells, 1973). W h e n the i n h i b i t o r was a p p l i e d to a e r o b i c l u g w o r m s , the p H of b l o o d a n d c o e l o m i c fluid d e c r e a s e d m a r k e d l y w i t h i n one hour, w h e r e a s n o effect was o b s e r v e d in a n a e r o b i c s p e c i m e n s . This illustrates the role of c a r b o a n h y d r a s e in the e l i m i n a t i o n of COz.

A N A E R O B I C M E T A B O L I S M

In the n a t u r a l habitat, the o x y g e n s u p p l y of the l u g w o r m is cut off t w i c e d a i l y w h e n , at low tide, the w a t e r r e c e d e s for s e v e r a l hours. (Some p l a c e s m a y e v e n o c c a s i o n a l l y e m e r g e for s e v e r a l t i d a l cycles.) This m e a n s that the a n i m a l m u s t r e g u l a r l y switch from the n o r m a l a e r o b i c m e t a b o l i s m to a n a e r o b i o s i s w h i c h is c h a r a c t e r i z e d b y a v e r y low yield of ATP. T h e transition r e q u i r e s a p e r i o d of s e v e r a l hours a n d p r o c e e d s in a s e q u e n c e of p h a s e s w i t h t y p i c a l m e t a b o l i c c h a n g e s . This has b e e n a n a l y s e d in d e t a i l b y Sch6ttler et al. (1984b).

At the o n s e t of h y p o x i a (in the s o - c a l l e d transition p h a s e w h i c h l a s t s a b o u t t h r e e hours), ATP is r e s y n t h e s i z e d m a i n l y b y t r a n s p h o s p h o r y l a t i o n from p h o s p h o t a u r o c y - a m i n e , the s p e c i a l p h o s p h a g e n of the l u g w o r m w h i c h is p r e s e n t i n t h e b o d y - w a l l m u s c u l a t u r e in c o m p a r a t i v e l y h i g h c o n c e n t r a t i o n (Surholt, 1977b, 1979). Its b r e a k d o w n results in a n i n c r e a s e of i n o r g a n i c p h o s p h a t e w h i c h c a n b e o b s e r v e d in i n t a c t a n i m a l s b y m e a n s of 13p-NMR s p e c t r o s c o p y (Kamp & J u r e t s c h k e , 1989a). C o n c o m i t a n t l y , d e g r a d a - tion of g l y c o g e n is a c t i v a t e d as s h o w n b y 13C-NMR t e c h n i q u e ( J u r e t s c h k e & Kamp, 1990a). As a c o n s e q u e n c e , the r a t e of p y r u v a t e f o r m a t i o n i n c r e a s e s , b u t t h e r e is no a c c u m u l a t i o n of lactate, b e c a u s e the activity of LDH is v e r y low in Arenicola. Instead, p y r u v a t e s e r v e s as a s u b s t r a t e in two o t h e r r e a c t i o n s (Fig. 2).

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Arenicola marina:

a m o d e l of physiological a d a p t a t i o n 45

A. E a r l y P h a s e

g l y c o g e n

L"2 ATP - - ~ l F

P'taur~

I/2 F-I,6-P raurocyamine~'-" ~ ATP

NADIt j

, ~ ATP

PEP NADH ; ---~ ATP

D - a l a n i n e

Cyto6ol malaZe

\

M i lochond don ~I~

B. P r o l o n g e d A n a 9

glycogen

I/2 A T P ~

1/2 F. 1,6.P

NADH ~ '

' ~ A T P

i

oxaloa,~late

~,~ NADII

malale

t" t, Cytosol

J mitochondrial

malate ~ pyruvale - - - ~ ~x:~Ze

A ' r P

S u c c i n a t e

ATP at--- ~

propionate

Fig. 2. Pathways of metabolism in

Arenicola marina

in different phases of anaerobiosis (from Sch6ttler & Bennett, 1991). A: transition phase, B: prolonged anaerobiosis

A major q u a n t i t y of p y r u v a t e functions as a n acceptor of the a m i n o group i n the reductive t r a n s a m i n a t i o n of aspartate w h o s e utilization is also i n d u c e d by hypoxia. In this process, three different e n z y m e s cooperate: aspartate aminotransferase, a l a n i n e a m i n o - transferase a n d m a l a t e d e h y d r o g e n a s e (Felbeck, 1980; F e l b e c k & Grieshaber, 1980). Since NADH is reoxidized, the redox b a l a n c e is m a i n t a i n e d :

(1) aspartate + oxoglutarate > g l u t a m a t e + oxaloacetate (2) g l u t a m a t e + p y r u v a t e > a l a n i n e + oxoglutarate (3) oxaloacetate + N A D H > m a l a t e + NAD +

aspartate + p y r u v a t e + N A D H > a l a n i n e + m a l a t e + NAD +

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46 E. Z e b e & D. Schiedek

participate in the t r a n s a m i n a t i o n reactions. It is n o t e w o r t h y that, in addition to L-alanine, also D - a l a n i n e is a c c u m u l a t e d in a p p r o x i m a t e l y e q u i m o l a r a m o u n t s (Felbeck, 1980), which is d u e to the p r e s e n c e of a r a c e m a s e (Sticher, 1985).

Unlike a l a n i n e , which a c c u m u l a t e s in the cytosol, the s e c o n d product of the reaction, malate, is t a k e n up by the mitochondria. T h e r e it u n d e r g o e s further transformations, m a i n l y to succinate, which is the characteristic product of aspartate utilization. The details of this process will b e d e s c r i b e d later.

It is n o t e w o r t h y that in the transition p h a s e the rate of ATP resynthesis b y transphos- phorylation a n d glycolysis is clearly lower t h a n that of ATP c o n s u m p t i o n . This is i n d i c a t e d by a d e c l i n i n g e n e r g y charge (Sch6ttler et al., 1984b). (The e n e r g y charge reflects the e n e r g e t i c state as r e p r e s e n t e d by the e n e r g y available in the a d e n i n e n u c l e o t i d e pool. It is d e f i n e d as

[ATP] + 0.5 [ADP l E C =

[AMP] + [ADP l + [ATP]

In the body-walI of Arenicola it is 0.86 to 0.89 u n d e r normoxic conditions. T h e EC declines w h e n utilization of ATP exceeds resynthesis.)

A b o u t three hours after the o n s e t of hypoxia, the so-called switching p h a s e begins. Characteristically, a n i n c r e a s i n g proportion of p h o s p h o e n o l p y r u v a t e (PEP), resulting from the d e g r a d a t i o n of glycogen, is t h e n c o n v e r t e d to oxaloacetate, a n d the p h o s p h a t e group transferred to GDP (Fig. 2). Conversely, p y r u v a t e formation d e c l i n e s to a corre- s p o n d i n g extent, which results i n d e c r e a s e d a l a n i n e accumulation. C o n c o m i t a n t l y , utili- zation of p h o s p h a g e n a n d a s p a r t a t e g r a d u a l l y ceases as the c o n c e n t r a t i o n s of these substrates d e c l i n e to low levels (Sch6ttler et a l , 1984b).

The switching at the so-called PEP b r a n c h p o i n t is p r o b a b l y c a u s e d by a shift in the relative activities of p y r u v a t e k i n a s e a n d PEP c a r b o x y k i n a s e which c o m p e t e for the same substrate, PEP. U n d e r aerobic conditions, PK prevails a n d p y r u v a t e is the sole product. In hypoxia, the ratio of the two activities g r a d u a l l y c h a n g e s at the e x p e n s e of PK a n d e v e n t u a l l y PEP is c o n v e r t e d solely to oxaloacetate.

The role of PEPCK in the a n a e r o b i c d e g r a d a t i o n of g l y c o g e n has b e e n c o n v i n c i n g l y d e m o n s t r a t e d b y Sch6ttler & W i e n h a u s e n (1981), who i n h i b i t e d the e n z y m e b y m e a n s of 3-mercaptopicolinic acid. T h e i n h i b i t o r y effect consisted not only of a striking d e c r e a s e in the formation of m e t a b o l i t e s d e r i v e d from oxaloacetate (succinate, p r o p i o n a t e a n d ace- tate; see below), b u t also of a n a c c u m u l a t i o n of lactate, i. e. a m e t a b o l i t e w a s p r o d u c e d that in Arenicola n o r m a l l y does n o t occur in significant amounts. E v i d e n t l y i n h i b i t i n g PEPCK e v o k e d the catalytic action of PK, t r a n s f o r m i n g PEP to p y r u v a t e as u n d e r aerobic conditions. It also indicates that other carboxyIating e n z y m e s such as p y r u v a t e carboxyl- ase are not i n v o l v e d in the formation of oxaloacetate.

A particularly i m p o r t a n t feature of the switching p h a s e is the r e d u c t i o n of the metabolic rate ( m e a s u r e d b y the rate of ATP formation) to o n e fifth or less of the n o r m a l aerobic level (Sch6ttler et al., 1984b) as is s h o w n in T a b l e 1. As a c o n s e q u e n c e , the e n e r g y b a l a n c e stabilizes as i n d i c a t e d b y a n {almost) i n v a r i a b l e e n e r g y charge. U n q u e s - tionably, the dramatic drop i n t h e m e t a b o l i c rate is essential to the l u g w o r m s ability to survive several days of anoxia.

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A r e n i c o l a marina: a m o d e l of physiological a d a p t a t i o n 47 Table 1. Metabolic rate and energy balance of Arenicola marina during normoxia and consecutive

phases of hypoxia (modified from Sch6ttler, 1986, and * Kamp, 1992)

Turnover % of total

Glycogen ATP PTC Glycolysis Succinate

+ vol. FA

Normoxia < 1.6' 60"

Hypoxia

0-3 h 8.2" 40 23 59 18

3-12 h 2.2* 11 6 21 73

12-24 h 1.6" 9 100

24-48 h 1.4" 8 100

Turnover: ~tmol 9 g 1 d.w. - h t % of total: percentage of contribution to total resynthesis of ATP by the respective pathway (The rates of glycogen degradation and ATP turnover during hypoxia were calculated from the quantities of anaerobic end products accumulated and phosphagen utilized, normoxic ATP turnover from oxygen uptake.); PTC = phosphotaurocyamine; vol. FA = volatile fatty acids (propionate + acetate)

A l t h o u g h this clearly affected s o m e p r o c e s s e s (Kamp & J u r e t s c h k e , 1989a), it a p p e a r s insufficient to a c c o u n t for the o b s e r v e d d e c l i n e of the m e t a b o l i c rate.

N i n e to t w e l v e hours after the onset of hypoxia, the s w i t c h i n g from a e r o b i c to a n a e r o b i c m e t a b o l i s m is complete. Malate is t h e n the sole p r o d u c t of g l y c o g e n d e g r a d a - tion. It does not a c c u m u l a t e in the cytosol; but is t a k e n up by the mitochondria. In t h e s e o r g a n e l l e s all s u b s e q u e n t reactions p r o c e e d w h i c h e v e n t u a l l y result in the formation of the e n d products p r o p i o n a t e and a c e t a t e (Zebe, 1975).

M a l a t e is u s e d as substrate by t h r e e different m i t o c h o n d r i a l e n z y m e s : malic e n z y m e , m a l a t e d e h y d r o g e n a s e and fumarase. T h e reactions w h i c h are c a t a l y s e d by t h e s e e n z y m e s not only occur simultaneously, a l t h o u g h at different rates, but they i n t e r a c t with e a c h other (Fig. 3). S o m e m a l a t e is c o n v e r t e d to p y r u v a t e by malic e n z y m e a n d this m e t a b o l i t e is s u b s e q u e n t l y oxidized to a c e t y l - C o A by p y r u v a t e d e h y d r o g e n a s e . A n o t h e r portion of m a l a t e u n d e r g o e s r e o x i d a t i o n to o x a l o a c e t a t e by m a l a t e d e h y d r o g e n a s e . Citrate s y n t h a s e catalyses the formation of citrate from a c e t y l - C o A and o x a l o a c e t a t e . D e s p i t e the a b s e n c e of oxygen, citrate is d e g r a d e d to succinyl-CoA.

T h e o x i d a t i v e reactions can p r o c e e d , b e c a u s e the N A D H arising is reoxidized. This is a c c o m p l i s h e d by c o n v e r t i n g a m a j o r portion of m a l a t e to f u m a r a t e in the r e v e r s e d f u m a r a s e reaction. F u m a r a t e functions as a h y d r o g e n a c c e p t o r (instead of oxygen) a n d is r e d u c e d to succinate. B e c a u s e s o m e c o m p o n e n t s of the r e s p i r a t o r y chain are i n v o l v e d , s u c c i n a t e f o r m a t i o n is c o u p l e d w i t h ATP synthesis by o x i d a t i v e p h o s p h o r y l a t i o n .

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48 E; Z e b e & D. S c h i e d e k

CO 2

ATP

p y r u v a t e ~ a c e t y l - C o a 2

co2

~ , ~

.-

,

I ~176

I

I . . .

I

!

acetate

[ citrate

I

+'~' '~

[ isocitrate ]

/[NADH* ]'~b"

. . .

/

I ~

_

,

I - - - - "

A D P + Pi " x ~ , [ a l p h a - k e t o g l u t a r a t e [

A T P ~ . N A D + "

...

]p,

"

'

u

...

h

_

CO 2

s u c c i n a t e ,

-,,

I succinyl-CoA ~,/

GTP GDP + Pi

~k,,._~ATP

#

p r o p i o n a t e

Fig. 3. Anaerobic metabolism ot malate in mitochondria of body-wall tissue of A r e n i c o l a marina (from Sch6ttler & Bennett. 1991)

first s e g m e n t of t h e citric a c i d cycle is i n v o l v e d in the formation of s u c c i n a t e . T h e y w e r e also u s e d to formulate the e q u a t i o n s of t h e c o n v e r s i o n of m a l a t e to s u c c i n a t e as follows:

2 m a l a t e - 5 N A D - > s u c c i n a t e + 5 N A D H + 4 CO2 5 m a l a t e a_ 5 N A D H > 5 s u c c i n a t e + 5 N A D -

7 m a l a t e >- 6 s u c c i n a t e + 4 CO2

The e x p e r i m e n t s r e v e a l e d that in t h e p r e s e n c e of N A D H e v e n m i t o c h o n d r i a l frag- m e n t s are a b l e to r e d u c e f u m a r a t e to succinate. T h e r e a c t i o n c a n b e b l o c k e d b y specific inhibitors of s u c c i n a t e - c h i n o n - o x i d o r e d u c t a s e (Fig. 4). This i n d i c a t e s t h a t the s o - c a l l e d c o m p l e x e s I a n d II of t h e r e s p i r a t o r y c h a i n a r e involved.

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A r e n i c o l a m a r i n a :

a model of physiological adaptation

49

rotenone

amytat dicumarol anti mycin CN |

|

|

|

|

0 U

S

U

substrate --4~. I - ~ f l a v o - - ~

c o e n - ~ c v t o -

D, c y t o -

ibcyto -

oxydation ~

~ p r o t e i n ~ z y m e - - ' P c h r o m e

- -

chrome'-- chrome -~

I

NAD O //~/~Q b c a 1/202

p , '

ftavoprotei n S

,It

succinate -~l---P~ fumarate

|

malonate

Fig. 4. Pathway of hydrogen in mitochondria of body-wall tissue of

Arenicola marina

(from Schroff &

Sch6ttler, 1977)

GDP + Pi

propionate acetate

NADH NAD +

succinyI-CoA I

~

~

{

CoA

CO2

[ L-methylmalonyl-CoA [

I

[ D-methylmalonyl,CoA]

co2

_ ~ A D P

§ PI

co2

~

propionyl-CoA ~ )

~

ATP

acetyl-CoA NADH

" ~ ;

N~AD

+ pyruvate

CCh

CoA

alpha-ketoglutarate

Fig. 5. Anaerobic formation of propionate and acetate in mifochondria of body-wall tissue of

Arenicola marina

(from Sch6ttler & Bennett, 1991)

tion of propionyl-CoA and in the concomitanf synthesis of ATP. In the final reaction of the

pathway, propionate is split off, while CoA is transferred to succinate. This provides

succinyl-CoA for another turn of the "succinate-propionate cycle". Propionate

accumulates as an end product typical of anaerobic glycogen degradation.

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50 E. Z e b e & D. S c h i e d e k

a c e t y l - C o A . A l t h o u g h in this c a s e ATP l i k e w i s e arises ( W i e n h a u s e n , 1981), this is a c c o m p l i s h e d in a different m a n n e r (see below).

T h e r e is e x p e r i m e n t a l e v i d e n c e that i s o l a t e d m i t o c h o n d r i a r e t a i n t h e c a p a b i l i t y to c o n v e r t s u c c i n a t e to p r o p i o n a t e (Schroff & Zebe, 1980). M a x i m a l rates w e r e o b s e r v e d , w h e n m a l a t e a n d ADP w e r e p r e s e n t in a d d i t i o n to succinate. T h e i n d i v i d u a l r e a c t i o n s of the p a t h w a y w e r e d e m o n s t r a t e d b y e m p l o y i n g l a b e l l e d precursors. S c h S t t l e r (1983) i d e n t i f i e d all e n z y m e s i n v o l v e d a n d a n a l y s e d i m p o r t a n t details such as t h e role of the s p e c i a l t r a n s f e r a s e in the splitting of p r o p i o n y l - C o A w h i c h m a k e s p o s s i b l e the r e c y c l i n g of the e n e r g y - r i c h t h i o e s t e r b o n d a n d thus an efficient utilization of s u c c i n a t e in a c o n t i n u o u s " s u c c i n a t e - p r o p i o n a t e cycle".

F u r t h e r m o r e , i n v e s t i g a t i n g the f o r m a t i o n of a c e t a t e in detail, SchSttler (1980, 1986) o b s e r v e d t h a t it r e q u i r e s the p r e s e n c e of s u c c i n a t e in a d d i t i o n to a c e t y l - C o A . This s e e m s to i n d i c a t e t h a t the splitting of a c e t y l - C o A is c a t a l y s e d b y the s a m e C o A t r a n s f e r a s e w h i c h has a low specifity c o n c e r n i n g the a c y l m o i e t y of its substrate. H o w e v e r , this w o u l d p r e c l u d e a s y n t h e s i s of ATP as found b y W i e n h a u s e n (1981). But t h e r e s e e m s to b e a n o t h e r e x p l a n a t i o n of the a p p a r e n t l y i n c o n s i s t e n t results: s u c c i n y l - C o A p r o d u c e d b y C o A t r a n s f e r a s e could b e split b y s u c c i n y l - C o A s y n t h e t a s e (succinyl-CoA --> s u c c i n a t e § CoA + GTP) w h i c h w o u l d result in the s y n t h e s i s of GTP. Since the rate of a c e t y l - C o A splitting h a s b e e n f o u n d to increase, w h e n s u c c i n y l - C o A s y n t h e t a s e w a s a d d e d to a s u i t a b l e r e a c t i o n mixture, and, on the o t h e r h a n d , inhibition of s u c c i n y l - C o A s y n t h e t a s e has the o p p o s i t e effect, the p r e s u m p t i o n a p p e a r s p l a u s i b l e .

In conclusion, it has to b e p o i n t e d out that a n a e r o b i c g l y c o g e n d e g r a d a t i o n , r e s u l t i n g in the f o r m a t i o n of succinate, p r o p i o n a t e a n d a c e t a t e , yields 6 to 7 ATP ( d e p e n d i n g on the v a r y i n g ratio p r o p i o n a t e / a c e t a t e ) as c o m p a r e d to 3 in the p r o d u c t i o n of lactate. T h e fatty a c i d s p r o p i o n a t e a n d acetate, u n l i k e lactate, e a s i l y p e r m e a t e b i o l o g i c a l m e m b r a n e s a n d can thus b e e x c r e t e d into the a m b i e n t water. In this way, n e g a t i v e effects r e s u l t i n g from l a r g e - s c a l e a c c u m u l a t i o n of m e t a b o l i c e n d p r o d u c t s such as acidosis a n d o s m o t i c i m b a l - a n c e are a v o i d e d . This m e a n s , h o w e v e r , a loss of p o t e n t i a l l y e n e r g y - r i c h c o m p o u n d s .

B e c a u s e of the l o n g d u r a t i o n of the s w i t c h i n g period, the f o r m a t i o n of p r o p i o n a t e a n d a c e t a t e starts r a t h e r slowly, so t h a t the a c c u m u l a t i o n of t h e s e m e t a b o l i t e s and, conse- quently, their e x c r e t i o n are d e l a y e d . Therefore, the a p p e a r a n c e of volatile fatty acids in the w a t e r w a s e v i d e n t only after 6 hours of e x p e r i m e n t a l a n o x i a (Surholt, 1977a, b; SchSttler et al., 1984b; Holst & Zebe, 1986). Since l o w tide in the n a t u r a l h a b i t a t u s u a l l y lasts a b o u t 6 hours, the r e l e a s e of p r o p i o n a t e a n d a c e t a t e t h e n w o u l d b e insignificant. This h a s b e e n c o n f i r m e d b y a n a l y s i n g l u g w o r m s i m m e d i a t e l y after their r e m o v a l from the burrows. I n d i v i d u a l s o b t a i n e d after m a x i m a l p e r i o d s of e m e r s i o n (6 to 8 hours) h a d a c c u m u l a t e d c o m p a r a t i v e l y l a r g e q u a n t i t i e s of s u c c i n a t e , w h e r e a s the c o n c e n t r a t i o n s of p r o p i o n a t e a n d a c e t a t e w e r e only slightly i n c r e a s e d (Pionetti & T o u l m o n d , 1980~ SchStt- ler et al., 1984a). Obviously, l a r g e - s c a l e r e l e a s e of v o l a t i l e fatty acids, as o b s e r v e d d u r i n g l o n g p e r i o d s of e x p e r i m e n t a l a n o x i a (Surholt, 1977a, b; SchStfler et aL, 1984b; Hoist & Z e b e , 1986), occurs v e r y rarely, e.g. d u r i n g e x t r e m e p e r i o d s of e m e r s i o n .

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Arenicola marina: a m o d e l of physiological a d a p t a t i o n 51 wall of the lugworm. The p e r c e n t a g e of p r o p i o n a t e a p p e a r i n g in the w a t e r regularly e x c e e d e d that of acetate which also a c c u m u l a t e d in comparatively small quantities.

The rate of fatty acid release was s h o w n to d e p e n d on the c o n c e n t r a t i o n g r a d i e n t b e t w e e n l u g w o r m a n d water. This suggests that it proceeds b y diffusion. However, w h e n a n i m a l s w e r e i n j e c t e d with butyrate, this c a u s e d a s u b s t a n t i a l r e d u c t i o n of p r o p i o n a t e excretion. From the inhibitory effect, it a p p e a r s that carrier molecules could m e d i a t e the release.

In view of its ecophysiology, the ability of the l u g w o r m to m a i n t a i n a n aerobic m e t a b o l i s m at r e d u c e d pO2 was tested to d e t e r m i n e the precise limit at which the first symptoms of h y p o x i a appear. In e x p e r i m e n t s c o n d u c t e d b y i n c u b a t i n g a n i m a l s 24 hours at various controlled l o w - o x y g e n levels, the c o n c e n t r a t i o n s of p h o s p h a g e n , aspartate, a l a n i n e a n d s u c c i n a t e started c h a n g i n g at a pO2 of 45 Torr (SchSttler et al., 1983). At first, the c h a n g e s were detected in the g u t / c h l o r a g o g e n tissue complex, w h e r e a s in the body- wall they occurred b e t w e e n 37 a n d 20 Torr. Very likely, this difference is d u e to differences in the o x y g e n supply to these o r g a n s as well as in the c o n c e n t r a t i o n s of some metabolites, i n c l u d i n g p h o s p h a g e n , whose c o n c e n t r a t i o n in the gut is minimal. At 15 Torr, the m e t a b o l i s m was completely a n a e r o b i c as i n d i c a t e d by the f i n d i n g that the q u a n t i t y of a c c u m u l a t e d e n d products was the same as in full anoxia.

In l u g w o r m s i n c u b a t e d at 22 Torr, symptoms of a n a n a e r o b i c m e t a b o l i s m were e v i d e n t after o n e hour; b u t after 12 hours the e x t e n t to which m e t a b o l i t e levels h a d c h a n g e d was still clearly less t h a n after 12 hours of anoxia, i n d i c a t i n g a partially aerobic metabolism.

Periods of hypoxia lasting several hours m e a n severe stress to a n o r g a n i s m b e c a u s e of the r e s u l t i n g high d e m a n d for stored reserves. In addition, r e d u c i n g the m e t a b o l i c rate to less t h a n 20 % of the n o r m a l rate requires that all functions are d e c r e a s e d to the m i n i m u m n e c e s s a r y to secure survival. In view of these dramatic curtailments, it is particulary i m p o r t a n t to the l u g w o r m to r e - e s t a b l i s h aerobic m e t a b o l i s m as soon as the tide rises a n d o x y g e n - r i c h water b e c o m e s a v a i l a b l e again. Likewise, n o r m a l functions h a v e to be q u i c k l y restored to their optimal levels. S u b s e q u e n t l y , a c c u m u l a t e d m e t a b o l i t e s m u s t b e r e m o v e d a n d utilized reserves r e p l e n i s h e d . All these processes are typical of the so-called recovery phase.

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52 E. Z e b e & D. S c h i e d e k

Altogether, these results i n d i c a t e that a fully aerobic m e t a b o l i s m is restored w i t h i n 60 m i n u t e s after r e a d m i s s i o n of oxygen, a n d possibly m u c h earlier. It is likely t h a t w i t h i n this period all functions are also e l e v a t e d to their optimal rates. Removal of t h e m e t a b o l i t e s a c c u m u l a t e d d u r i n g 24 hours of a n o x i a (a rare e v e n t u n d e r n a t u r a l conditions!) takes m u c h longer, which, however, does n o t s e e m to affect vital processes.

It is not k n o w n w h e t h e r in the initial p h a s e s of recovery the total m e t a b o l i c rate rises a b o v e the n o r m a l aerobic level, r e s u l t i n g i n a n i n c r e a s e d o x y g e n u p t a k e . A t t e m p t s to d e t e r m i n e w h e t h e r there was a n o x y g e n d e b t i n

Abarenicola paci[ica

after 3 days of a n o x i a did not show u n e q u i v o c a l results (May, 1972).

Rapid recovery d e p e n d s on the r e s u m p t i o n of the n o r m a l p u m p i n g activity by the l u g w o r m as soon as the tide rises. H o w does the a n i m a l b e c o m e aware of the p r e s e n c e of o x y g e n - r i c h water? T h e r e is e v i d e n c e that the c a u d a l e n d of

Arenicola

(which is u s u a l l y k e p t in a position close to the surface of the sediment) is highly sensitive to c h a n g e s of the pO2. (Toulmond et al., 1984; Dejours & T o u l m o n d , 1988). Obviously, special c h e m o r e c e p - tors p r e s e n t there are s t i m u l a t e d and, via a p a c e m a k e r in the central n e r v o u s system, control the p u m p i n g activity.

Occasionally, the l u g w o r m is also likely to e n c o u n t e r so-called f u n c t i o n - r e l a t e d a n a e r o b i o s i s in a d d i t i o n to the r e g u l a r periods of hypoxia at low tide. This stems from excessive m u s c l e activity, as is r e q u i r e d w h e n the worm after h a v i n g b e e n e x p o s e d at the surface m u s t rapidly re-dig into the sediment. In such a situation, ATP is m a i n l y r e s y n t h e s i z e d b y t r a n s p h o s p h o r y l a t i o n from p h o s p h a g e n . In addition, a n a e r o b i c glycoly- sis is initiated as well as utilization of aspartate ( S i e g m u n d et al., 1985), w h i c h typically results in the a c c u m u l a t i o n of a l a n o p i n e a n d succinate together with small a m o u n t s of malate. This is similar to the c h a n g e s o b s e r v e d in the transition phase after the onset of e n v i r o n m e n t a l anaerobiosis, except that s t r o m b i n e is t h e n formed i n s t e a d of a l a n o p i n e .

Physiological conditions in f u n c t i o n a l a n d e n v i r o n m e n t a l a n a e r o b i o s i s differ s u b s t a n - tially with respect to the glycolytic rate, i.e. the q u a n t i t y of p y r u v a t e p r o d u c e d d u r i n g a certain period of time. Kreutzer et al. {1989) p o s t u l a t e d from theoretical c o n s i d e r a t i o n s that at low glycolytic rates (characteristic of e n v i r o n m e n t a l anaerobiosis) t h e a m i n o acid p r e s e n t in the h i g h e s t c o n c e n t r a t i o n should b e preferentially u s e d as s u b s t r a t e in the c o n d e n s a t i o n reaction. Since this is g l y c i n e in

Arenicola,

it w o u l d favour formation of strombine. O n the other h a n d at high glycolytic rates (as they occur in w o r k i n g muscles), w h e n comparatively large a m o u n t s of p y r u v a t e are produced, the most active e n z y m e p r e s e n t w o u l d d e t e r m i n e its transformation. Since a l a n o p i n e d e h y d r o g e n a s e exceeds s t r o m b i n e d e h y d r o g e n a s e b y a factor of 10, a l a n o p i n e should arise as the p r e d o m i n a n t e n d p r o d u c t i n f u n c t i o n a l anaerobiosis. T h e results of comparative analyses u s i n g 25 different species of m a r i n e i n v e r t e b r a t e s h a v e fully confirmed this hypothesis (Kreutzer et al., 1989). In view of the f r e q u e n t switching of the l u g w o r m b e t w e e n aerobic a n d a n a e r o b i c metabolism, the m e c h a n i s m s of m e t a b o l i c control i n v o l v e d are particularly i n t e r e s t i n g . In this context, the special properties of two i m p o r t a n t r e g u l a t o r y e n z y m e s h a v e b e e n analysed.
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Arenicola marina: a model of physiological a d a p t a t i o n 53 d e g r e e of GP p h o s p h o r y l a t i o n did not c h a n g e significantly after various periods of hypoxia, a l t h o u g h the rate of glycogenolysis i n c r e a s e d substantially (Kamp, 1986; H u n - feld & Kamp, 1990). In vitro p h o s p h o r y l a t i o n of (unphosphorylated) GP b was a c h i e v e d by e m p l o y i n g r a b b i t p h o s p h o r y l a s e k i n a s e (Kamp & WinnemSller, 1992). As it t u r n e d out, p h o s p h o r y l a t i o n was t h e n not complete, b u t e v i d e n t l y h a d resulted in the formation of a h y b r i d form c o m p o s e d of one p h o s p h o r y l a t e d a n d one u n p h o s p h o r y l a t e d s u b u n i t . It was d e s i g n a t e d GP a / b (Kamp & W i n n e m S l l e r , 1992). A completely p h o s p h o r y l a t e d e n z y m e has not b e e n o b t a i n e d in vitro n o r was it detected in vivo.

In the b o d y - w a l l m u s c u l a t u r e of the lugworm, GP a/b is the p r e d o m i n a n t form. Its catalytic activity was s h o w n in vitro to d e p e n d on the c o n c e n t r a t i o n s of i n o r g a n i c p h o s p h a t e a n d AMP (Kamp, 1986). According to Kamp (1992), it is very likely that in vivo GP a/b activity is also r e g u l a t e d b y p h o s p h a t e a n d AMP. (The affinity of this form for both c o m p o u n d s clearly exceeds that of GP b.). U n d e r normoxic conditions, the levels of p h o s p h a t e a n d AMP in the tissues (as calculated from 31P-NMR data) are very low., i.e. too low to activate GP a/b. After the onset of hypoxia, the c o n c e n t r a t i o n of p h o s p h a t e rises s u b s t a n t i a l l y (due to the utilization of p h o s p h a g e n ) , which results in the activation of GP a/b. The increase of AMP has a s u p p o r t i n g effect (Kamp, 1992). According to the results of Kamp a n d coworkers, other regulatory m e c h a n i s m s a p p e a r insignificant for controlling GP activity in the body-wall of Arenicola. In this respect, a n o t h e r o b s e r v a t i o n is important: the activity of g l y c o g e n s y n t h a s e of the l u g w o r m also d e p e n d s on the c o n c e n t r a t i o n of phosphate, b u t is affected in a different way, as an i n c r e a s e causes i n h i b i t i o n (Hunfeld & Kamp, 1990). Possibly, a n i n t e r r e l a t i o n exists b e t w e e n the control of phosphorylase a n d g l y c o g e n synthase, as has b e e n d e m o n - strated in vertebrates.

Pyruvate k i n a s e has a central role in a n a e r o b i c metabolism, b e c a u s e it is e s s e n t i a l to the switching at the PEP b r a n c h - p o i n t . Its activity m u s t c h a n g e to a great e x t e n t a n d in a precisely controlled m a n n e r . U n d e r normoxic conditions, the glycolytic flux is at a m i n i m u m and, c o n s e q u e n t l y , PK activity very low. After the onset of hypoxia, however, large q u a n t i t i e s of metabolites d e r i v e d from p y r u v a t e a c c u m u l a t e , i n d i c a t i n g a s u b s t a n - tially i n c r e a s e d glycolytic rate a n d a c o r r e s p o n d i n g l y high PK activity. W h e n a n a e r o b i o s i s c o n t i n u e s for several hours, p y r u v a t e formation gradually declines, until it ceases completely a n d the switching b e c o m e s fully effective.

From the few existing studies on l u g w o r m PK, it a p p e a r s that the e n z y m e m a y be r e g u l a t e d in various ways. A n a l y s e s of the kinetics of PK reaction h a v e s h o w n an i n f l u e n c e of c e r t a i n effectors (Englisch, 1989). This m a y s u g g e s t that the rise of PK activity in the early p h a s e of a n a e r o b i o s i s could result from allosteric effects of c h a n g i n g c o n c e n t r a t i o n s of p h o s p h o t a u r o c y a m i n as well as of fructose-1,6-bisphosphate. While the i n h i b i t o r y effect of the former declines as the p h o s p h a g e n is degraded, a rising level of the latter causes a n activation.

T h e r e is e v i d e n c e , however, that further r e g u l a t o r y m e c h a n i s m s exist w h i c h effec- tively r e g u l a t e PK action b y c h a n g i n g its affinity for PEP. As the results of SchSttler (1980) a n d Englisch (1989) indicate, the affinity clearly declines d u r i n g anaerobiosis. This seems to b e d u e to some e x t e n t to the d e c r e a s i n g pH, b u t in addition, modifications of the m o l e c u l a r structure of PK m a y b e i n v o l v e d which a u g m e n t this effect.

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54 E. Z e b e & D. Schiedek

(1) P h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n : the affinity for PEP of PK e x t r a c t e d from l u g w o r m s s u b j e c t e d to anoxia was s h o w n to increase by t r e a t m e n t with phosphatase. A n a l y s e s of purified Arenicola PK b y isoelectric focussing revealed that t h e e n z y m e is not h o m o g e n o u s . At least five discrete b a n d s c o n t a i n i n g e n z y m a t i c activity h a v e b e e n o b s e r v e d w h i c h m a y r e p r e s e n t different d e g r e e s or stages of phosphorylation. It was impossible, however, to detect q u a n t i t a t i v e differences b e t w e e n the b a n d p a t t e r n s of normoxic s p e c i m e n s a n d those exposed to anoxia.

(2) C h a n g e s b y a g g r e g a t i o n or dissociation of s u b u n i t s of the e n z y m e molecule: According to gel filtration analyses, PK m a y occur (in vitro[) in forms w h i c h differ with respect to m o l e c u l a r mass, i.e. in the n u m b e r of subunits.

(3) C h a n g e s in the association of PK with structural e l e m e n t s of the cell: In normoxlc lugworms, PK a p p e a r s to b e m a i n l y associated with cellular particles, while d u r i n g a n a e r o b i o s i s the e x t e n t of association declines.

T h u s far, it has not b e e n possible to isolate a n d characterize different forms of PK in Arenicola. T h e significance of the various alterations of its m o l e c u l a r s t r u c t u r e r e m a i n s unsolved. It seems, however, that i n the l u g w o r m the r e g u l a t i o n of PK is h i g h l y complex. In conclusion, it should b e stated that, until now, there is no i n d i c a t i o n of a c h a n g e of PEPCK activity with respect to the switching from aerobiosis to anaerobiosis.

INFLUENCE OF HYDROGEN SULFIDE

The a n o x i c s e d i m e n t of the tidal zone is rich in sulfides i n c l u d i n g free h y d r o g e n sulfide. In the interstitial water, the c o n c e n t r a t i o n of the latter m a y be as h i g h as 1 m m o l 9 1-1 ( G r o e n e n d a a l , 1979; VSlkel & Grieshaber, 1992). H2S is extremely toxic, b e c a u s e it inhibits cytochrome c oxidase (although reversibly) a n d thus p r e v e n t s a e r o b i c m e t a b o - hsm. In r e c e n t years, it has b e c o m e evident, however, that m a n y a n i m a l s a r e tolerant of H2S, at least to some degree, a n d h e n c e are able to live in H2S e n v i r o n m e n t s , i n c l u d i n g the tidal z o n e (Vismann, 1991; Giere, 1992). Therefore, the q u e s t i o n arises: H o w does H2S affect the l u g w o r m ? W h a t a d a p t a t i o n s allow it to cope with this i m p o r t a n t ecofactor? Ecological studies i n the Dutch W a d d e n Sea have r e v e a l e d that H2S h a s a n obvious i n f l u e n c e o n Arenicola: T h e p o p u l a t i o n d e n s i t y was f o u n d to b e i n v e r s e l y r e l a t e d to the H2S c o n c e n t r a t i o n at the d e p t h of the b u r r o w s ( G r o e n e n d a a l , 1979).

In l a b o r a t o r y e x p e r i m e n t s , l u g w o r m s survived a b o u t 90 hours of i n c u b a t i o n i n o x y g e n - f r e e s e a w a t e r to which sulfide (10 m m o l 9 1-1) h a d b e e n a d d e d (10~ G r o e n e n - daal, 1980, 1981). It was f o u n d that H2S rapidly p e n e t r a t e d into the tissues, until a n e q u i l i b r i u m b e t w e e n the external a n d i n t e r n a l c o n c e n t r a t i o n s was r e a c h e d . Typically, the latter w a s only 50 p e r c e n t (or e v e n less) of the former, w h i c h is d u e to t h e fact that the i n t e m a l p H is m a i n t a i n e d 0.2 units b e l o w that of t h e m e d i u m . As a c o n s e q u e n c e , the c o n c e n t r a t i o n of u n d i s s o c i a t e d H2S rises w i t h i n the b o d y a n d thus favours its o u t w a r d diffusion. U n d e r anoxic conditions, no f u n d a m e n t a l chemical c h a n g e s of sulfide occurred n o r was it b o u n d b y c o m p o n e n t s w i t h i n t h e l u g w o r m s body.

(19)

Arenicola marina: a m o d e l of physiological a d a p t a t i o n 55 cient as i n d i c a t e d b y rising i n t e r n a l H2S concentration. As a result, cytochrome c oxidase was i n h i b i t e d a n d the l u g w o r m h a d to resort to a n a e r o b i c m e t a b o l i s m i n i t i a t i n g the a c c u m u l a t i o n of succinate.

In a detailed study, V61kel & G r i e s h a b e r (1994) tested the capacity of H2S oxidation. T h e y found that u n d e r normoxic conditions the limit was a n external H2S c o n c e n t r a t i o n of a p p r o x i m a t e l y 0.3 m m o l 9 1-1. W h e n l u g w o r m s were i n c u b a t e d in water c o n t a i n i n g h i g h e r H2S concentrations, the i n t e r n a l H2S level rose to 0.5 mmol or e v e n higher ( d e p e n d i n g on the season, i.e. the physiological state of the animals). This resulted in a n i n h i b i t i o n of the aerobic m e t a b o l i s m as i n d i c a t e d by a rising succinate concentration. W h e n the authors i n c r e a s e d the e x t e r n a l sulfide from 0.76 to 1.27 m m o l . 1-1, H2S in the coelomic fluid did not increase as expected, b u t did n o t c h a n g e significantly. Therefore, they a s s u m e that a n o t h e r mode of sulfide detoxification m i g h t exist in a d d i t i o n to thiosulfate formation.

A rise of the i n t e r n a l H2S c o n c e n t r a t i o n w a s also observed w h e n the pO2 of the a m b i e n t water declined. Obviously, the capacity of H2S oxidation declined s u b s t a n t i a l l y as soon as the high d e m a n d for o x y g e n was not met.

Thiosulfate was s h o w n to be the m a i n p r o d u c t of sulfide oxidation. In l u g w o r m s i n c u b a t e d for 8 hours at 1 mmol 9 1-1 H2S, its level rose almost linearly with time to a b o u t 4 m m o l . 1-1. In addition, sulfite (0.04 m m o l - 1-1) a p p e a r e d as a minor oxidation product, b u t no sulfate w a s detected.

Sulfide oxidation proceeds in the mitochondria. S t u d y i n g the o x y g e n u p t a k e of m i t o c h o n d r i a p r e p a r e d from b o d y - w a l l tissue, V61kel & G r i e s h a b e r (1994) d e m o n s t r a t e d a dramatic i n c r e a s e in the respiratory rate u p o n a d d i n g H2S to the i n c u b a t i o n mixture. O x y g e n c o n s u m p t i o n was t h e n similar to that o b s e r v e d with succinate plus ADP. Sulfide- related o x y g e n uptake, however, p r o v e d to b e i n d e p e n d e n t of ADP. A stoichiometric ratio sulfide to o x y g e n to thiosulfate 1 : 1 : 0 . 5 w a s m e a s u r e d . Sulfide-related o x y g e n u p t a k e was s h o w n to be i n h i b i t e d by c y a n i d e a n d azide (the inhibitory effect of KCN e x c e e d i n g that of azide), b u t the i n h i b i t i o n was not complete and, in addition, it was clearly less t h a n i n the case of succinate oxidation. E v e n w h e n succinate respiration was completely inhibited, oxygen u p t a k e could still be s t i m u l a t e d by sulfide. This result suggests that different e n z y m e s m a y b e r e s p o n s i b l e for succinate a n d sulfide oxidation, respectively. V~lkel & G r i e s h a b e r (1994) propose that a n alternative t e r m i n a l oxidase be p r e s e n t in l u g w o r m mitochondria w h i c h is n o t i n h i b i t e d b y high H~S c o n c e n t r a t i o n s a n d w h i c h has a lower sensitivity towards c y a n i d e a n d azide t

Figure

Fig. 1. pO2 measured in different parts of the burrow of Arenicola cfistata (from Mangum, 1976)
Fig. 2. Pathways of metabolism in Arenicola marina in different phases of anaerobiosis (from Sch6ttler & Bennett, 1991)
Table 1. Metabolic rate and energy balance of Arenicola marina during normoxia and consecutive phases of hypoxia (modified from Sch6ttler, 1986, and * Kamp, 1992)
Fig. 3. Anaerobic metabolism ot malate in mitochondria of body-wall tissue of Arenicola marina (from Sch6ttler & Bennett
+6

References

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