Fine-structural observations on the gill filaments of the freshwater crayfish, Astacus Pallipes Lereboullet

TISSUE & CELL [972 4 (2) 287 299 Published b.i, Longman Group Ltd. Printed in Great Britain

JEAN M. FISHER*

F I N E - S T R U C T U R A L O B S E R V A T I O N S ON THE GILL FILAMENTS OF THE FRESHWATER CRAYFISH, A S T A C U S PALLIPES LEREBOULLET ABSTRACT. The gills of the crayfish are described with special reference to the tissues of the branchial filaments. An account is given of a possible pathway of blood flow through the filaments to ensure maximum oxygenation at the epithelial surface. The branchial epithelium consists o1' a thin, subcuticular layer with an indented apical plasma membrane, and projecting cell bodies. The basal epithelial surface adjacent to the haemolymph possesses the deep membrane infoldings characteristic of resorptive epithelia. Ion and water movement across the epithelium is discussed.

(Ewer and Hattingh, 1952; Allanson and Krijgsman, 1957) and Artemia gill (Croghan, EAr~LY studies of osmotic regulation by 1958). Silver deposit in living and dead freshwater and estuarine crustaceans revealed crayfish was confined to the cuticle of the that the blood was maintained at a higher stem branchial filaments, and was intersalt concentration than that of the external preted as passive inward diffusion of silver medium, although the body surface was in regions where the cuticle was most permeable to both salts and water (Bethe, permeable (Bryan, 1960b). Cuticle thickness 1930; Margaria, 1931 on Carcinus; Krogh, is not related to the animal's ability to 1939 on Eriocheir), Salt loss in dilute media regulate its salt content, a phenomenon was compensated by an inward secretion of which W e b b (1940) decided was a function of salts (Nagel, 1934), which Krogh postulated the branchial epithelium. t o o k place in Eriocheir by the active uptake M o r e direct evidence of the role of of sodium a n d chloride ions against a crustacean gills in ionic and osmotic control concentration gradient. Webb (1940) sugis presented by Croghan's (1958) observagested that the small concentration differ- tions on the effect of K M n Q on the gills of ences between seawater and the body fluids Artemia, and Bryan's (1960b) comparison of of marine crustaceans could also be ex- ion absorption by animals in which the gill plained by such a mechanism. chamber only was perfused, and animals That the gills are the most permeable which were totally immersed in medium. regions of the body surface and therefore Finally, studies on isolated gills confirmed likely to be the site of ionic and osmotic that active uptake of sodium (Koch et al., regulation was deduced by Margaria (1931). 1954 in Eriocheir) and active uptake of Similar conclusions were made f r o m studies chloride ions (Bielawski, 1964; C r o g h a n e t a l . , on iodide absorption (Berger and Bethe, 1965 in crayfish) could occur in these organs. 1931), and silver precipitation in Potamon gill The present w o r k describes some fine* Department of Zoology, The University of structural observations on the b l o o d circulaNewcastle upon Tyne. Present address: Develop- tion and tissues of the branchial filaments of mental Biology Laboratory, University of Newcastle crayfish podobranch gills, and considers the upon Tyne, England. possible roles of these elements in the Manuscript received 8 November 197l. physiological functions of the gill. 287 Introduction

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Fig. 1. Diagram of the gill filament to show the transverse arrangement of tissues, and the blood pathway. Deoxygenated afferent blood (AFF) enters the filament and is forced laterally round the lacunae (L) where it is oxygenated, and thence into the efferent pathway (EFF). CT, connective tissue: E, epithelium: Cu, cuticle. Materials and Methods

P o d o b r a n c h gills were excised from adult specimens of the freshwater crayfish, Astacus pallipes Lereboullet, which had been maintained in tapwater in laboratory aquaria. Fine-structural studies were made of the gill shaft filaments only. Electron microscopy. Material was fixed in Millonig's phosphate-buffered 1% osmium tetroxide (in Pease, 1964) modified by the addition of 5°,~, sucrose at p H 7.3 for 45 mins. Subsequent embedding in Araldite was based oll L u f r s technique (1961). Thin sections were double stained in ethanolic uranyl acetate and lead citrate (Reynolds, 1963), and examined in the AE1 EM6B. Light microscopy. 1-2po thick Araldite preparations were stained in 1% Nile blue sulphate according to McGee-Russell and Smale (1963). Whole gills were processed in Worcester's fluid with subsequent embedding in Paraplast. 5/z sections were stained with either haemalum and eosin or iron haematoxylin. Results

General organisation. Morphological and histological

observations

of the

gills of

Astacus pallipes confirm those previously :recorded by Huxley (1896) for A./tuviatilis, and Bock (1925) for Potamobius astacus.

The gills are a filamentous, 'trichobranchiate', type and lie in the branchial chamber protected by the branchiostegites. There are a maximum of three gills arising from each thoracic appendage except the first maxilliped and the fourth walking leg: one podobranch arises from the base of the appendage, and two arthrobranchs arise from the arthroidal membrane between the base of the appendage and the body. The podobranch alone carries a convoluted lamina on the posterior surface; its function is probably to direct the flow of water through the branchial chamber over the gill filaments. Each gill consists of a central shaft, the axis, bearing numerous (200--300) blindending tubules, arranged roughly in alternate rows. As outgrowths of the body wall, the gills are protected by a cuticle; in this region however it lacks the heavy calcareous deposit, and in the filaments is usually less than 2/~ thick (Fig. 10). Epithelium lies immediately beneath the cuticle; in most preparations these two elements tended to separate. In the axis the epithelial cells are cubical in shape, but in the filaments the epithelium has two distinct components: a thin, continuous layer which lines the cuticle, and mushroom-shaped cell bodies arising from the thin layer. The 'crowns' of each cell body are embedded in connective tissue (Figs. I, 6), resulting in the formation of a

GILL F ILAM E N T S OF CRAYF1SH

289

system of blood filled lacunae (Bock, 1925) (Fig. 4). Connective tissue divides the axis longitudinally into two canals, the narrower afferent, which receives deoxygenated blood from the sternal sinus, and the efferent, which discharges oxygenated blood into the intersegmental channels. According to Bock, the septum separating these two canals extends the length of the axis into the apical filament, and prevents direct communication between the afferent and efferent axial canals. In addition, a third canal, Bock's "peripheral" canal, is formed outside the main channels; this canal receives blood directly from the filaments and discharges into the efferent axial canal. The filament afferent channel arises from the connective tissue of the axial afferent canal. At the filament base the channel is

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Fig. 2. Diagram of a longitudinal section of a filament to show blood flow from the afferent axial canal (Aac) into the filament afferent channel (Fac); fi'om here it is forced laterally round the filaments into the filament efferent channel (Fee) and down into the axial peripheral canal (Apc). Connective tissue (CT) prevents back flow from the filament afferent chmmel into the peripheral canal, and the septum (SI ensures separation of oxygenated and deoxygenated blood. Cu, cuticle; E, epithelium.

closely aligned to the cubical epithelium of the filament-axis junction (Fig. 3), but along the length of the filament the connective tissue flattens out to form the septum, separating afferent and efferent blood flow; it spreads out laterally from the septum becoming thinner as it overlies the epithelial

cell bodies, but except at the filament afferent channel base, it stops short at a point opposite the septum so that the lateral communication exists between blood flow and the epithelium (Fig. 4). At the filament apex, the connective tissue becomes rather diffuse, and shows a few intercellular spaces (Fig. 2). The whole system is well irrigated with blood which contains numerous flee blood cells (Tait, 1918; Tait and Gunn, 1918) (Fig. 6l. Circu[ation lhrottgh the .filaments. The supply of oxygenated water between the gills is maintained by the paddle-like action of the scaphognathite of the maxilla. This causes water to be drawn in under the ventral edges of the branchiostegite into the chamber, forward, over and between the gills and expelled from the anterior end beneath the head near the base of the antennae (Huxley), The heart, lying in the pericardium, pumps blood to the tissues via the arteries, from where it is drained into large sinuses. From the sternal sinus blood flows to the gills where it is oxygenated and returned via intersegmental channels to the pericardium. An account of flow through the gills as a whole has already been given by Bock (1925); h o w e v e r h is description of circulation through the filaments does not altogether explain the important relationship between the epithelium and blood flow. The following account, based on some histological and electron microscope observations, suggests a possible pathway for flow through the filaments themselves. Blood from the afferent axial canal enters the filament through the narrow filament afferent channel, which prevents a back leakage of blood into the peripheral canal by its close association with the cubical epithelium of the filament-axis junction (Fig. 2). The position of the connective tissue along the length of the filament suggests that blood must be forced laterally round the epithelium and through the lacunae, where it can be oxygenated (Figs. 1, 2, 4). F r o m the lacunae the blood can flow into the filament efferent channel, from where it can go only into the peripheral canal of the axis, and follow the pathway outlined by Bock. Fine structure Connective tissue. Estimations from low magnification electron micrographs show the

FISHER

290 c o n n e c t i v e t i s s u e to c o m p r i s e 30-50~'~; t o t a l t i s s u e e l e m e n t s in t h e f i l a m e n t . It is c o m p o s e d o f finely a t t e n u a t e d a n d i n t e r l o c k e d cells, surrounded by a thin basement membrane, I n t h e s e p t u m t h e cells f o r m a c o n t i n u o u s sheet the length of the filament; elsewhere the connective tissue spreads out from the sides of the septum and caps the epithelial cell b o d i e s (Figs. 6, 7). The cytoplasm varies in complexity: w h e r e a s s o m e cells a p p e a r to c o n t a i n little more than a small nucleus, small spherical mitochondria and irregularly orientated microtubules, others show abundant smooth r e t i c u l a r profiles, G o l g i c o m p l e x e s , o r single l a r g e v a c u o l e s (Fig. 5),

£~pithelium. T h e e p i t h e l i u m is in c l o s e contact with two extracellular compartments, the external medium and the haemolymph, a n d t h e cells a r e specifically o r g a n i s e d at both surfaces. Basal sm:fiwe. T h e b a s a l s u r f a c e is a l w a y s completely enveloped by a basement memb r a n e a p p r o x i m a t e l y 0'If* t h i c k (Figs. 7, 9, 11). T h e p l a s m a m e m b r a n e d i s p l a y s d e e p a n d complex invaginations, which partition the g r e a t e r p a r t o f t h e cell i n t e r i o r i n t o n u m e r o u s serial or c o n c e n t r i c c o m p a r t m e n t s , p a r t i c u l a r l y w h e r e t h e e p i t h e l i u m is directly in c o n t a c t w i t h h a e m o l y m p h (Fig. 7). T h e m a j o r i t y o f m i t o c h o n d r i a lie c l o s e l y a l i g n e d to t h e b a s a l f o l d s in a m a n n e r s i m i l a r

Fig. 3. Light micrograph of the junction between a filament and the gill shaft. Blood is directed from the axial afferent canal (Aacl into the filament afferent channel (Fae) by connective tissue (CT). The latter initially closely follows the filament epithelium (at El, and forms the septum (SL x 400. Fig. 4. Light micrograph of a filament in transverse section. At points (x) opposite the septum (S) tim epithelium (El has no connective tissue cover. Fac, filament afferent channel: Fee, filament efferent channel: L, lacuna; CT, connective tissue. :, 400, Fig. 5. Connective tissue cells (CT) of high and low organelle density. The cells are separated from the haemolymph of the lacunae (L) and filament channel (FC), and the epithelial cells (El by a thin basement membrane (bin); va, connective tissue vacuole. :, 10,000. Fig. 6. Low magnification survey micrograph near the filament apex to show the arrangement of the tissues. Connective tissue (CT), showing intracellular vacuoles (ra/, surrounds the epithelial cell bodies (El. Tissues arc bathed iu haemolymph containing hyaline blood cells (Hbcl. Lacunae (L) are formed between the connective tissue and epithelium. :. 3000. Fig. 7. The epithelial nucleus (nu) is contained in the cell body region. The highest concentration of basal folds (/~f) and mitochondria (m) occurs in the regions of epithelium facing the haemolymph of the lacunae (L). Hbe, hyaline blood cell; G, Golgi figure; A1, apical leaflets: bin, basement membrane. ~: 7650. Fig. 8. The apical surface of the epithelium. The septate desmosomc (sd) gives way to a convoluted basal fold (bj). Mt, microtubules; r, ribosomes: er, endoplasmic reticulum; AI, apical leaflets: mvb, multivesicular body. x 48,000. Fig. 9. Section of thin epithelium. The apical leaflets (All show slight thickening of their tips and a particulate external surface. L, lacuna; bin, basement membrane. ,: 59,000. Fig. 10. The cuticle. Normally 2~ or less in width. Inner bands show fine striations, whereas the outer layer is strongly osmiophilic, x 22,300. Fig. 11. Distribution of organelles in the epithelial cytoplasm. Rough endoplasmic reticulum (er) may follow the direction of basal folds (/?/); ribosomal particles (r) also appear to be grouped freely. Microtubules (rot) occur both as short scattered profiles, and associated with basal folds (arrowed). L, lacuna: bin, basement membrane; G, Golgi figure; mvb, multivesicular body; ly, lysosome-like body; m, mitochondria. :.: 25,000.

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294 to those in the anal papillae (Copeland, 1964), rectal papillae (Berridge and Gupta, 1967; Hopkins, 1966), and branchial epithelium (All, 1966) (Fig. 7). Most profiles reveal them as considerably larger than those of the other tissue elements, l ~ in length. Apical surface. This surface is characterized by the indentation of the cell membrane into villous-like processes, average 0.5~ high (Fig. 9). The tips of these leaflets rest on the cuticle, and the whole of the extracellular surface normally carries a fine particulate coat (Fig. 9), similar to that of the mucosal surface of the toad urinary bladder, where it was thought to be involved in maintaining a permeability barrier (Peachey and Rasmussen, 1961). The apical surface is the only observed site of septate desmosomes (Locke, 1965); these may run a tortuous course parallel to the cuticle before giving way to membrane pairs identical to basal folds (Fig. 8). Cell membrane specializations are a significant feature of the branchial epithelium, in common with other absorptive and secretory epithelia, and are of central interest in the discussion of localisation of active ion uptake sites. The highest concentration of basal folds and associated mitochondria occurs in basal epithelium facing the haemolymph. It was soon recognized that such subcellular components are a characteristic feature of tissues known to be engaged in selective transport of ions and water (Pease, 1956). These membranes are interpreted as close interdigitations between adjacent cells (Doyle, 1960; Tormey, 1963; Hicks, 1965)0 resulting in the formation of long intercellular channels within the cell mass. The channels communicate with one extracellular compartment, that to which fluid and salts are being transported (Diamond and Bossert, 1967); the opposite external compartment is closed off from the intercellular channels by a septate desmosome. It has been suggested that the septate desmosome has a low ion resistance in the cell to cell direction of flow, but acts as a barrier to diffusion between intercellular channels and the external compartment (Loewenstein and Kanno, 1964: Wiener, Spiro and Loewenstein, 1964). Central region. Adjacent cellular compartments occasionally contain different pro-

FISHER portions of elements, but normally the cytoplasm is fairly uniform. The nucleus measures 7-12~ dia., is lobed, and includes large areas of peripheral chromatin and usually a nucleolus. Nuclear pores have been observed, and the outer nuclear envelope may carry ribosomes. Tubular or branched profiles of granular reticulum occur throughout the compartments, often running parallel to basal folds (Fig. 11). Ribosomal particles are usually associated with reticulum, but they may also be flee in the cytoplasm, either singly or in groups. Microtubules are present either as short individiuals, randomly orientated, or in small bundles (Fig, IlL The tendency to form aggregates, which is very marked in the branchial pillar cells of Carc&us (Ali, 1966) suggests that in the crustacean gill these organelles are cytoskeletal in function. This function has already been attributed to similar organelles in certain blood cells (Fawcett and Witebsky, 1964). The Golgi complex usually occupies a position within the central column of the cell body; it may be straight (Fig. 11), crescentic, or S-shaped, and typically consists of five cisternae with expanded ends. These are always associated with vesicles and one or two vacuoles. More than one Golgi may be present in any one compartment. One or more lysosome-like bodies (Novikoff and Essner, 1962), and multivesicular bodies are often observed in the cytoplasm (Fig. 11). Discussion

Genera! aspects The organization of the gill filaments shows that the tissues are designed to serve two major functions: firstly the direction of blood flow through the branchial tissues, and, secondly, the absorption and transport of selected substances from the external medium into the blood. The first function appears to be carried out by the connective tissue: it provides both separation of oxygenated and deoxygenated blood, ensures that the epithelial cells are well irrigated, and supports the cell body epithelium. On existing evidence, the second function is primarily the concern of the epithelium.

GILL FILAMENTS OF CRAYFISH This tissue has two components: a thin, continuous peripheral epithelium, and a projecting cell body epithelium. Considering its large surface area and its proximity to the two extracellular compartments, the thin epithelium must be particularly involved with gaseous exchange. Assuming that the branchial cuticle is freely permeable to gases, the effective width of the thin epithelium, from the external medium to the blood, compares favourably with that of other known respiratory tissues: the water/blood pathway in a cichlid fish is 0-3 20t~, and the air/blood pathway in human lung is 0.3 2-5t~ (Schultz, 1962); in the gills of the pollack, 1.0-3.0~ (Hughes and Grimstone, 1965); and the eel gill tissue width is 5-0~ (Steen and Kruysse, 1964). In A s t a c u s gill the water/ blood pathway measures approx. 1.5-5.65~, made up of the following elements: cuticle 0.75-2-5t~; thin epithelium 0.7-3.0t< basement membrane 0.05-0.15 t~. With dimensions of this order oxygen can be absorbed by simple diffusion, facilitated by continuous blood circulation which creates a permanent oxygen concentration gradient. Ion a n d water m o v e m e n t in the ct'a~.¢3'h

Adaptation from a marine to a freshwater environment has necessitated the acquisition of means to maintain salt balance at low external concentrations. In the Crustacea this has been achieved by a partial reduction in the permeability of the body surface to salts, and the development of ion uptake mechanisms which operate at low external concentrations (Shaw, 1961b). In freshwater species the sodium uptake mechanism reaches saturation at a lower concentration than that of marine crustaceans. Shaw (1964) suggests that this is due to the presence of an uptake mechanism with a high affinity for the transported ion. Activation of this system could result from changes in the concentration of blood sodium, or the concentration difference between blood sodium and chloride ions. Since the balance of charges between the internal and external media must be maintained, it has been suggested that ion exchange mechanisms are in operation (Krogh, 1939; Maluf, 1940; Shaw, 1960a, b; Bryan, 1960b). In support of this idea, it has been shown that ammonium ions in the external medium can inhibit sodium influx;

295 similar results were achieved on the uptake of chloride in the presence of bicarbonate ions (Shaw, 1960a, b). The inference is that a carrier in the cell membrane having a high affinity for sodium ions on the outside would exchange sodium for ammonium ions on the inside where there would be a higher affinity for the latter. Active chloride uptake would operate in a similar manner, except that normal chloride uptake by exchange of chloride for chloride ions, under conditions of chloride deficiency, would switch to an exchange of chloride for bicarbonate ions (Shaw, 1964). In any attempt to correlate the fine structure with what is already known of the physiology of the gill, attention is obviously drawn to the specializations of the cell membrane, the basal folds and the apical leaflets. B a s a l f o l d s as active sites'. Theoretical models have now been devised to account for solute and water transport across membranes against steep electrochemical and osmotic gradients (Curran and McIntosh, 1962; Diamond and Bossert, 1967); so far they have been applied to lateral epithelial membranes of gall bladder (Kaye et al., 1966; Diamond and Tormey, 1966; Tormey and Diamond, 1967); reptilian kidney (Schmidt-Nielsen and Davis, 1968); insect rectal papillae (Berridge and Gupta, 1967); and basal membranes of Malphigian tubules (Berridge and Oschman, 1969). Diamond and Bossert (1967) have suggested that steep osmotic gradients, 'a standing osmotic gradient', could be set up in the closed end of basal infoldings as a result of active ion uptake. This could lead to passive inflow of water. Osmotic equilibration could occur progressively along the channel length, so that the emerging fluid would be isotonic with that extracellular compartment. The results of combined physiological and electron microscopy studies on several resorptive epithelia show that the width of the membranes is significantly affected by changes in ionic and osmotic concentration, (Schmidt-Nielsen and Davis, 1968; Diamond and Tormey, 1966; Kaye et al., 1966; Berridge and Gupta, 1967; Hopkins, 1967). Whether or not crayfish branchial epi~ thelium behaves according to the system postulated by Diamond and Bossert must

296 remain a matter for speculation until more electron microscopy has been combined with physiological work. At least it satisfies certain cytological conditions: fluid from the external compartment (freshwater) can enter the epithelium only through the apical folds since the intercellular spaces between lateral cell membranes are rendered impermeable from the exterior by septate desmosomes. These lateral membranes traverse the epithelium forming long intercellular channels which open at the haemolymph surface under the thin basement membrane, and which are closely followed by exceptionally large mitochondria, suggesting sites of high metabolic activity. The role o f apical leaflets. There is much less available data on the role of apical leaflets and related microvilli-like structures, although there is a little evidence to suggest that ion pumps may be present, and that this cell surface may act as a physical barrier to the passage of fluid (Sohal and Copeland, 1966; Hopkins, 1966). The possibility that ion pumps may be present at the apical surface of crustacean gill epithelium cannot be discounted: certain regions of the branchial epithelium of the freshwater crab, Potamonautes, (unpublished data) are structurally very similar to the apical surface of insect anal papilla epithelium described by Sohal and Copeland. In addition, Croghan et al. (1965), measuring the electrical potential difference across the .epithelium in isolated podobranchs of the crayfish, suggested that a sodium pump could exist in the inner cell membrane and a chloride pump in the outer. In conclusion, the following proposal is tentatively advanced to account for active :solute transport in crayfish gill filaments. Under conditions of activation of the ion uptake mechanism, that is, in low external salt concentration, the apical leaflets would expand to form sub-cuticular spaces and increase membrane surface area, thus increasing the availability of ions. Fluid would enter the epithelial cells through the leaflets, where chloride exchange may take place. Sodium ions could be actively absorbed from this intracellular fluid by the basal membranes and transported into the inter.cellular channels where osmotic gradients could be set up as described by Diamond and Bossert. Water could enter until this corn-

FISHER partment became isotonic with the haemolymph. At high external salt concentration, when the uptake mechanism is suppressed, cuticular permeability and surface area could be reduced by closing or flattening the leaflets; regulation of the blood ion concentration would then depend on the activity of the excretory system.

Acknowledgements I should like to thank Professor J. Shaw for continual encouragement and criticism of this manuscript, and Mr G. Howson for photographic assistance. This work was supported by a grant from the Science Research Council awarded to Professor J. Shaw.

Summary I. The tissues of the branchial filaments of the freshwater crayfish, Astacuspallipes, have been investigated by histology and electron microscopy to determine their roles in relation to respiratory and ion uptake mechanisms known to exist in the gills. 2. The general organization of tissues in the gill as a whole is consistent with Bock's earlier report on Potamobius astacus. His account of flow through the gills appears to be valid for Astacus; however the model for flow through the branchial filament has been expanded to explain how blood flows from the connective tissue channels into the epithelial lacunae where it can be oxygenated. It is proposed that blood flowing in the filament afferent channel is forced laterally round the lacunae where gaseous exchange takes place; from here it flows into the filament efferent channel and out into the axial peripheral canal. 3. Three tissues occur along the length of the filament: blood containing corpuscles, connective tissue, and the epithelium which is separated from the external medium by a cuticle of less than 2t4. Connective tissue cells possess finely attenuated and interlocked cytoplasm, often sparsely populated by organelles.

GILL FILAMENTS OF CRAYFISH lts organization into the septum and round the epithelial cell bodies suggests that its functions are to direct blood flow efficiently round the epithelium where it can be oxygenated, and to separate oxygenated from deoxygenated blood. 5. The epithelium has two components: a thin, continuous layer immediately below the cuticle, and probably the site of gaseous exchange: and regularly spaced cell bodies which are embedded by their crowns in connective tissue. The spaces thus formed between the connective tissue and the epithelium are the

297 'lacunae'. The basal epithelial surface is covered by basement membrane; where it faces the haemolymph it possesses deep membrane infoldings and large orientated mitochondria. The apical surface is indented to form short leaflets Lateral cell junctions at this surface possess septate desmosomes. 6. It is proposed that the epithelial basal infoldings are involved in ion exchange mechanisms in the gills. Active uptake of ions together with passive inflow of water could be explained by the 'standing osmotic gradient' hypothesis advanced by Diamond and Bossert.

298

FISHER References

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