feeding and digestion in corophium volutator (crustacea: amphipoda)

Marine Biology 89, 183-195 (1985) Marine . . . . . . . . . . . . Biology

| Springer-Vedag 1985

Feeding and digestion in Corophium volutator (Crustacea: Amphipoda) *

J. D. Icely 1 and J. A. Nott 2

1 Natural Environment Research Council, Unit of Marine Invertebrate Biology, Marine Science Laboratories; Menai Bridge, Auglesey LL59 5EH, North Wales, UK

2 Marine Biological Association of the United Kingdom; Citadel Hill, Plymouth PL1 2PB, Devon, England

Abstract Introduction

New observations on the feeding behaviour of Corophium volutator, collected from the Menai Strait, Anglesey, UK in 1981, show that detritus drawn into one entrance of the U-shaped burrow is pushed out at the opposing entrance either uningested or as faeces. Periodically, the amphipod turns around in the burrow and recommences feeding, so both the uningested material and the faeces may be reworked by the mouthparts. A feeding individual produces, on average, 16 faecal pellets every l0 min, at a seawater temperature of 18 ~ When fed on detritus dyed with azo-carmine, approximately 12 pellets are produced before the foregut, intestine and hindgut are completely clear of previously ingested material and the dye appears in the faeces. Dye which enters the ventral caeca takes between 12 and 48 h to clear this tissue and, furthermore, it is only cleared when the amphipod is feeding. It is concluded that ingested coarse material is subjected only to a primary phase of digestion in the stomach, and that it takes 4 to 24 min to clear the gut. Fine material which enters the ventral caeca is subjected to a secondary phase of digestion and absorption which takes 24 to 48 h. Particles of thorium dioxide and ferritin incorporated in the diet are absorbed by the B- and.ageing RF-cells in the ventral caeca. Non-specific esterases occur in the apical regions of the same cells, the mature cells of the anterior dorsal caeca and the cells at the anterior of the intestine. Protease activity is greatest in the lumen of the ventral caeca and the large vacuole of the B-cells. Apart from the lumen of the posterior caeca, there is some protease activity throughout the lumen of the gut. Carbo- hydrates were localised primarily in the R/F-cells towards the proximal region of the ventral caeca. Lipid was confined to the R/F-cells in the same region.

* Please address all requests for reprints to Dr. J. A. Nott in Ply- mouth

The morphology and fine structure of the alimentary canal in Corophium volutator (Pallas) have been described in some detail by Icely and Nott (1984), and from this some aspects of the function have been identified. There are two main factors which dominate the operation of the digestive processes. First, the diet consists of particulate material, much of which is indigestible, and second, the secretion of enzymes and the absorption of nutrients are confined to the mid-gut because the fore- and hindgut are lined with cuticle.

In the stomach, the dietary particles of mud are processed by a complex array of muscles, filters and channels to remove organic nutrients which are transferred to the caeca for further digestion and absorption. The residual particles in the stomach are passed along to the midgut and hindgut as faeces.

The surface area of the midgut is increased by paired anterior dorsal, ventral and posterior caeca. The ventral caeca are the largest and they are lined with the R/F- and B-cells which are comparable to the R-, F- and B-cells in the digestive glands of other Crustacea (Gibson and Barker, 1979).

In the present study, the function of the digestive system was further investigated. The turnover of food material has been assessed from observations on feeding behaviour and the rate of faecal production. The digestive and absorptive processes have been investigated by the incorporation of inert tracer particles in the diet and by histochemical tests for enzymes and stored carbohydrates.

Materials and methods

Source and maintenance of Corophium volutator

Adult individuals (body length greater than 5 mm) of both sexes were taken from the foreshore of the Menai Strait,

184 J.D. Icely and J. A. Nott: Feeding and digestion in Corophium volutator

Anglesey, UK, and maintained in mud and running seawater at 15 ~ 2 C ~ After acclimation to laboratory conditions for a week or more, specimens at intermoult were used for experimentation.

Observations on feeding behaviour and faecal production

Amphipods were placed in petri dishes containing a thin layer of mud, seawater and artificial burrows or in dishes containing mud and seawater only. The artificial burrows were horizontal, straight, clear tubes of hard plastic, length 2 cm, diameter 3 ram. They were readily occupied by Corophium volutator, although the natural burrows are vertical and U-shaped. The behaviour of amphipods occupying artificial burrows was similar to those in natural burrows.

The rate of faecal production was assessed in ten amphipods; four male and six female. A petri dish containing one specimen in an artificial burrow was placed under a binocular microscope where the temperature was maintained at 15 ~ to 18~ When the amphipod com- menced feeding, the production of faecal pellets was recorded for a half-hour period. For one individual, this procedure was repeated after an interval of 6 h.

in a solution of 2.4% glutaraldehyde in seawater at 4~ pH 7.2 to 7.4 and 1 020 to 1 030 mosM. They were rinsed in buffer for l h, dehydrated in ethanol, and embedded in a low-viscosity resin. Sections (0.05 to 0.1/~m) for transmission electron microscopy were stained in uranyl acetate and lead citrate. Particles of thorium dioxide or ferritin were identified by energy-dispersive X-ray microanalysis.

Histochemical tests

Non-specific esterases (Gomori, 1952; Pearse, 1972)

Amphipods which had been feeding were starved for 24 h to clear some of the coarser material from the gut. Each individual was embedded in sodium carboxymethyl cellu- lose on a block holder and frozen rapidly in liquid nitrogen. Sections (10ttm) were cut with a steel knife at -20 ~ in a Slee cryostat, picked up on glass coverslips and incubated in a medium of either a-naphthyl acetate or a mixture of this compound with fl-naphthyl propionate. Both media contained 0.5 M. Tris buffer at pH 7.0. When the substrates were hydrolysed, the reaction product was identified by the azo dye, fast-blue RR. Medium containing the dye but not the substrate was used as a control.

Feeding experiments with particles of inert tracers

Azo-carmine

Particulate azo-carmine was used to mark the passage of ingested material through the gut. The smaller particles of the dye passed through the two-part filter system of the foregut (Icely and Nott, 1984) into the lumen of the ventral caeca.

The number of faecal pellets necessary to clear the gut completely of coarse material was counted in three individuals with full guts. These amphipods were fed for 5 rain on mud and azo-carmine and the number of pellets counted before the dye appeared in the faeces.

The time taken to clear the dye from the lumen of the ventral caeca was measured by feeding 60 amphipods on dyed mud until a scarlet colour could be observed in the caeca through the translucent cuticle. Fifty of the amphipods were transferred directly to undyed mud and seawater and ten were placed in seawater only for 3 wk before transfer to undyed mud and seawater. They were examined at 12-hourly intervals and the time was noted when the dye had completely cleared the lumen of the ventral caeca.

Thorium dioxide and ferritin

Amphipods were fed for 2 h on mud containing fine particles of thorium dioxide (0.01 to 0.03 ktm) or ferritin. The alimentary canals were dissected out and fixed for 2 h

Proteases (Fratello, 1968," Pearse, 1972)

Sections of frozen tissue (see schedule for esterases in preceding paragraph) were picked up on segments of Kodak Vericolor II, Professional 5025, Type S, film. The gelatin of the film emulsion was dampened with 0.15 M phosphate buffer at pH 7.5, and this provided a substrate for the protease in the tissue. The sections on the film were incubated in a damp atmosphere at 37 ~ for 1 h or more. The activity of the enzyme was reflected by changes in colour as the gelatin from the three different layers of the colour film was digested; unincubated sections dried in the air were used as controls. The areas of digested gelatin were observed by binocular microscope.

Some of the segments of digested film were mounted on aluminium stubs with double-sided tape, coated with platinum by DC sputtering, and examined by scanning electron microscopy.

Thio-semicarbazide/siIver proteinate method for carbohydrates

Carbohydrates were identified by the aldehyde groups which occur on sugar residues after oxidation with periodic acid. These react with thio-semicarbazide to form a product which stains with silver proteinate. The method and the necessary control procedures have been described by Thiery (1967) and Lewis and Knight (1977).

J. D. Icely and J. A. Nott: Feeding and digestion in Corophium volutator 185

Results

Observations on feeding behaviour from artificial burrows

Corophium volutator used the second antennae to draw detrital material towards the entrance o f the burrow. Within the burrow, the amphipod might scoop up the detritus with the gnathopods, although the more usual behaviour was to retreat towards the middle of the burrow and draw the material in by a current of water set up by the pleopods. The particulate matter was filtered with the gnathopods and passed to the other mouthparts for further sorting and ingestion. This method of feeding could continue for 4 h. These observations were similar to those made by Hart (1930) and Meadows and Reid (1966).

Some additional aspects of feeding behaviour were noted. Amphipods feeding at one entrance to a burrow would periodically turn round and push out accumulated sediment and faeces through the opposing entrance. Then they would commence feeding in this new position. The same behaviour was observed in individuals living in natural sediment both in the laboratory and on the shore.

Feeding amphipods defecated frequently, each time after the following sequence of movements. The abdomen was brought in line with the rest o f the body, the uropods were spread out laterally and the telson flicked vertically. The faecal pellet was ejected into the current o f water produced by the beating action o f the pleopods. This current transported the pellet towards the opposing entrance to the burrow. It was probable that some of the faeces were reingested when the amphipod turned round to remove material and feed at the opposing entrance.

When water was removed from the petri dish, isolating the water remaining in the burrow, feeding ceased. The pleopods maintained a current o f water within the burrow, presumably, for respiratory purposes. When mudflats on the shore at the Menai Strait were uncovered, the amphipods continued feeding, but they used water on the surface and in the interstices o f the mud for transporting detritus to the mouthparts.

Turnover o f material in the gut

Turnover o f material in the gut o f Corophium volutator involves two separate processes (Icely and Nott, 1984). Primary digestion occurs in the foregut and produces residual material at a rate which can be estimated from the production of faecal pellets. Secondary digestion occurs in the ventral caeca and produces a quantity of residual material which can be followed by watching the move- ment of particulate dye from the lumen of the ventral caeca. The dye enters the caeca from the foregut, but it is not absorbed by the digestive epithelium.

Faecal production

Table 1 shows that ten amphipods produced, on average, 16 (range 5 to 27) faecal pellets every 10 min at a temperature of 18 ~ C.

The rate of ingestion by Corophium volutator was assessed from the rate of faecal production. The length and diameter of the pellets were usually regular, and averaged 0.50 and 0.14 ram, respectively. Thus, substitut- ing in the formula s~ x radius 2 x length x 0.001, the volume for a single faecal pellet was calculated as 7 .7x 10 .6 ml. From this it can be calculated that the rate of ingestion was 7 .47x 10 .4 ml h -1 within a range of 2.46 to 11.09 x 10 -4 (Table 1).

Using azo-carmine as a marker in the food for three individuals, it was established that twelve pellets were produced before the dye appeared in the faeces. On this evidence, it was calculated that the average time for coarse material to pass through the gut was 9 rain from a range of 4 to 24 min (Table 1). These results are relevant only to individuals feeding actively. In non-feeding individuals, faecal production is much more erratic and some retain a full gut for at least 24 h. Even if defecation does occur, material is always retained within the hindgut.

Table 1. Corophium volutator. Production of faecal pellets by feeding amphipods (18 ~ nd: no data

Amphipod Sex Body No. length

(mm)

No. of pellets produced and (time in min) for particles to pass through gut

first l0 min second 10 rain third 10 min total 30 min

Faeces vol. h -1 x 10 -4 (ml)"

1 c? 6 6 (20) 5 (24) 5 (24) 16 2 c~ 7 18 (7) 19 (6) 24 (5) 6t 3 c? 7 20 (6) 25 (5) 27 (4) 72 4 ff 7 24 (5) 22 (5) nd 46 (20 min) 5 2 8 21 (6) 20 (6) 17 (7) 58 6 2 9 16 (7) 26 (5) 21 (6) 63 7 ~ 8 10(12) 18 (7) 1t(11) 39 8 ? 8 17 (7) 12 (10) 6 (20) 35 9 ~ 8 17 (7) 13 (9) nd 30 (20 min)

10 ~ 8 15 (8) 12 (10) 12 (I0) 39 10 (repeat after 6 h) i5 (8) 12 (10) 16 (7) 43

Mean= 16 + 17 + 15= 48

2.46 9.39

11.09 9.70 8.93 9.70 6.01 5.39 6.93 6.01 6.62

7.47

a see "Results - Turnover of material in the gut - Faecal production" for calculation


Table 2. Corophium volutator. Clearance of azo-carmine from ventral caeca during feeding. In starved amphipods, the dye remains in the caeca

Treatment Total no. of amphipods

No. ofamphipods showing clearance during:

0-12 h 12-24 h 24-48 h >~ 48 h

Food available immediately after ingestion of dye

After ingestion of dye amphipods starved for 3 wk then fed again to effect clearance of dye

50 0 32 15 3

10 10 0 0 0

Clearance of dye from lumen of ventral caeca:

Table 2 summarises the length of time taken by the amphipods to clear particles of azo-carmine from the lumen of the ventral caeca. Fifty individuals were fed immediately after they had ingested the dye, and it was found that 64% cleared the dye within 12 to 24 h and 30% within 24 to 48 h. The remaining 6% which were unable to clear the caeca within 48 h were not feeding, either because they were about to moult or because they had been injured. The 10 individuals which were starved for 3 wk after they had ingested the dye were unable to void it during this period. Finally, after they were fed, the dye in all ten amphipods was cleared within 12 h.

Distribution of ingested azo-carmine, thorium dioxide and ferritin

Particulate azo-carmine, thorium dioxide and ferritin were ingested readily with the food and passed through the stomach into the lumen of all regions of the midgut, apart from the posterior caeca. Azo-carmine was not absorbed by the tissues of the midgut. Thorium dioxide (Fig. 1A) and ferritin came into contact with the microvillous border of the anterior dorsal caeca and intestine, but they were seldom absorbed.

In the ventral caeca, both thorium dioxide (Fig. 1 B, C) and ferritin (Fig. 1 D) occurred in the lumen, adjacent to the microvillous border. They were seldom absorbed by the R/F-cells (Fig. 1 B, D), except where these were ageing and the microvilli breaking down (for description of cell types, see Icely and Nott, 1984). Particles were more readily absorbed by B-cells, wherein they appeared either individually or in small clumps throughout the apical complex (arrows in Fig. 1 C and in B-cell of Fig. 1 D). They were rarely observed in the large vacuole of the B-cell.

Location of non-specific esterases

The reaction product for non-specific esterases was apparent in the anterior dorsal caeca, particularly in the apical region of the cells (dc in Fig. 2 A). Reactivity continued into the intestine, but it was progressively reduced towards the posterior region (i in Fig. 2 B) and was absent in the posterior caeca (pc in Fig. 2 C). There was some sign of activity in a layer ventral to the cuticle of the hindgut (h in Fig. 2 C).

The esterases were located in the R/F- and B-cells along the entire length of the ventral caeca (vc in Fig. 2 A inset and in Fig. 2 B, C). The reaction product was particularly dense in the apical region of the epithelium, but it was lacking in the vacuole of the B-cells (v in Fig. 2 B).

Location ofproteases

Digestion of gelatin by proteases occurred along the length of the lumen in the funnel of the stomach (fu.st) and in the anterior dorsal caeca (dc in Fig. 3A), and was continued into the intestine (i in Fig. 3B). There was no digestion in the lumen of the posterior caeca (pc in Fig. 3 C), and only a limited amount in the hindgut (h in Fig. 3 C). Activity was greatest along the length of the ventral caeca (vc); compare Fig. 3A, B and C with the control in Fig. 3 D.

Sites of protease activity were examined in greater detail by scanning electron microscopy (Fig. 3 E, F and G). This showed that B-cells could be distinguished by cavities in the epithelium of the ventral caeca (v in Fig. 3 F and G). The solid contents of the vacuoles in the B-cell were not extracted during preparation because frozen tissue was placed directly onto photographic film without immersion in any fluids. The cavities were not formed as a result of water loss from the vacuoles because they were not

Fig. 1. Corophium volutator. Transmission electron micrographs (TEM) showing inert particles in sections of gut. (A)-(C) Thorium dioxide in midgut, tissue fixed in glutaraldehyde; (D) ferritin in a ventral caecum, fixed in glutaraldehyde and stained with uranyl acetate and lead citrate. Particles identified by X-ray microanalyis. (A) Apical region of cells in an anterior dorsal caecum showing particles confined to lumen (arrows) (TEM • 17 000). (B) Apical region of young R/F-cell in ventral caecum, with particles aggregated between microvilli (arrows), but absent from cytoplasm (TEM x 39 000). (C) Apical complex of an ageing B-cell, with particles aggregated at luminal surface and scattered within cytoplasm (arrows) (TEM x 35 000). (D) Apical region of R/F- (R/F) and B-cell (B), with particles aggregated in lumen and in cytoplasm (arrows); there are more particles in B-cell than in R/F-cell; mv: microvilli (TEM • 66 500)


Fig. 1

188

observed in control sections, where protease activity was inhibited.

Location of glycogen

Glycogen was located throughout the tissue of the midgut. In the epithelium of the intestine, the particles, in the form of aggregates (arrows in Fig. 4A) were distributed throughout the cytoplasm. In the anterior dorsal and posterior caeca the particles were finely dispersed (Fig. 4 B).

In the ventral caeca, the distribution of glycogen was irregular. Distally, it occurred throughout the cytoplasm of the R/F-cells, but there were marked differences in the amounts in different cells (R /F in Fig. 4 C). Towards the proximal region, greater aggregations occurred adjacent to the microvilli (Fig. 4D) and the lipid globules (Fig. 4E). Some glycogen was found in the apical complex of the B- cells, but little occurred in the basal cytoplasm (Fig. 4 F).

Deposits of glycogen were identified by digestion with amylase.

Location of lipid

Lipid was observed in only the R/F-cells, particularly those in the proximal region of the ventral caeca.

J. D. Icely and J. A. Nott: Feeding and digestion in Corophium volutator

Discus~on

Source of food and feeding behaviour

Fenchel etal. (1975) investigated the diet o f Corophium volutator and found that it ingests particulate matter, organic detritus and diatoms within the size range 4 to 63/~m. By experiment, they showed that this amphipod can ingest bacteria suspended in seawater, providing they are mixed with clay and silt particles. Within the gut, Fenchel (1972) found that all the protozoans, 98% of the bacteria and over 80% of the diatoms are extracted from ingested material.

The present study confirms the size range of the particles which is similar to that observed in three other species of Corophium (Taghon, 1982; Miller, 1984). Our observations on the feeding behaviour of C, volutator are similar to those described by Har t (1930) and Meadows and Reid (1966) for the same species, al though the ingestion of faecal material has not been suggested previously. Miller (1984) described similar feeding behaviour and showed that particles transferred to the mouthpar ts by the gnathopods can be sorted further by the maxillipeds. Rejected material is deposited anteriorly and selected material is transferred to the mouth via the first and second maxillae and the mandibles. Nielsen and Kofoed (1982) observed a marked increase in the content of organic material in the faeces compared to that in the

Captions to figures on pages 189-191

Fig. 2. Corophium volutator. Light micrographs (LM) showing non-specific esterase activity as areas stained black in fresh, frozen, transverse-sections of gut. Insets also show dense layers of pigment granules around ovary (ov) and under exoskeleton (ex); outline boxes show areas selected for higher magnification. (A) Funnel of stomach (fu.st) and densely stained proximal end of anterior dorsal caeca (dc); lu: lumen of caecum (LM x 500); (Inset A) tissues in funnel region, including densely stained ventral caeca (vc) (LM • 70). (B) Posterior region of intestine (i) and ventral caeca (vc); apical region of caecal epithelium and apical complex of the B-cells (*) are densely stained; the B-cell vacuoles (v) are not stained; lu: lumen of ventral caecum (LM x 500); (Inset B) tissues in region of intestine (LM x 70), (C) Hindgut (h) with some staining (*) and ventral caecum (vc) which is densely stained; posterior caeca (pc) are not stained (LM • 500); (Inset C) tissue in hindgut region (LM x 70)

Fig. 3. Corophium volutator, Protease activity. Fresh, frozen, transverse-sections incubated on photographic film; dark, sunken areas represent sites of protease activity. (A) Anterior to midgut where there is apparently more activity in ventral caeca (vc) than in funnel of the stomach (fu.st); dc: dorsal caecum, ov: ovary (LM x 50). (B) Posterior ofmidgut where activity is greater in ventrat caeca than in intestine (i) (LM X 50). (C) Anterior region ofhindgut (h); tissue is damaged and the protease from ventral caeca is displaced into ventral region of hindgut (*); there is no activity in dorsal regions of hindgut; posterior caeca (pc) shows no activity (LM x 50). (D) Control preparation from mid-region ofmidgut; section has been dried out at room temperature to de-activate the enzyme and it shows only minimal surface pitting (LM x 50). (E) Towards posterior of midgut; after incubation the preparation was viewed in the scanning electron microscope (SEM); sunken areas representing sites of protease activity are similar to those in (B) (SEM x 90). (F) Large vacuoles of B-cells 03) in ventral caecum are shown by sunken areas (v) which could represent protease activity; R/F: R/F-cell (SEM x 635). (G) Detail of a vacuole in a B-cell (SEM • 13 500)

Fig. 4. Corophium volutator. Transmission electron micrographs (TEM) showing glycogen in sections of midgut fixed in glutaraldehyde and stained with silver proteinate. (A) Apical region of epithelium of intestine, showing aggregations of glycogen particles (arrows); my: microvilli (TEM • 40 000). (B) Distal region of a posterior caecum showing scattered particles of glycogen in epithelium (arrows) (TEM x 26 500). (C) Distal region of a ventral caecum, where frequency of particles varies in different R/F-cells (R/F); within individual ceils the particles (arrows) are evenly distributed (TEM x 11 500). (D) Apical region of an R/F-ceil from mid-region of a ventral caecum showing a substantial concentration of glycogen (arrows) (TEMx28 000). (E) Adjacent R/F-cells showing a difference in abundance of glycogen; it aggregates around extracted lipid droplets (li) (TEM x 18 000). (F) Basal region of epithelium; glycogen is more abundant in R/F-cell than in B-cell 03) (TEM x 18 000)


Fig. 2

190 J.D. Icely and L A. Nott: Feeding and digestion in Corophium volutator

Fig. 3


Fig. 4

192

Table 3. Turnover of coarse material in the gut ofamphipods

J. D. Icely and J.A. Nott: Feeding and digestion in Corophium volutator

Species Diet Conditions Clearance time when gut full (hours)

Source

Gammaridea

Gammarus pulex Marinogammarus

obtusatus

Marinogammarus pirloti

Corophium volutator

Hyperiidea

Parathemisto gaudiehaudi

Concentrated artificial diet

Concentrated artificial diet

Cladophora sp.

Ulva sp.

Range of algae on shore

Range of algae on shore

Particles of mud

Ammodytes sp. larvae Ammodytes sp. larvae

Amphipods previously starved? 2.5-5

Amphipods starved for 4 h 2.5 before feeding

Amphipods starved for 4 h 5 before feeding

Amphipods starved for 4 h 6-9 before feeding

? 8-12

? 8-I2

Amphipods fed at 15~ ~ 0.06-0.4

Amphipods fed at 4 ~ 72 ; Amphipods fed at 14 ~ 4 J

Martin (1965)

Martin (1966)

Present study

Sheader and Evans (1975)

food, and they concluded that this is derived from browsing on those particles that are too large or too small to be ingested. They suggested that, under experimental conditions, browsing provides up to 90% of the organic carbon ingested by the amphipod. Organic matter is probably scraped off the surfaces of the particles with the tooth-like setae on the first gnathopods.

Some of the feeding behaviour in Corophium volutator is similar, also, to the amphipods Marinogammarus (Eu- limnogammarus ~ ) obtusata and Marinogammarus ( Chaeto- gammarus 1) pirloti (Martin, 1966) in that they can feed over a period of low tide provided that there is some water left on the surface.

Turnover of material in the gut

Doyle (1979) concluded that the rate of ingestion of a female Coro])hium volutator is correlated positively with the nutritional value of the food, when the diet contains varying proportions of natural sediment and glass spheres. The maximum rate of ingestion is 4 x 10 .4 ml h -1, which comes within the range measured for ten individuals in the present study (Table 1), but is less than the average of 7.47x 10 .4 m l h -1. This may reflect differences in body size, since our smallest individual was 6 mm in length whilst Doyle's specimen was only 4.5 mm. However, the amphipod Hyalella azteca, which also feeds on sediment, shows a significant reduction in the rate of faecal production with an increase in body size (Hargrave, 1972) as measured by the proportion of the body dry weight egested each day.

1 See Lincoln (1979) for authority

The experiments with azo-carmine show marked differences in the passage times of coarse and fine material through the gut. Coarse material takes only 4 to 24 rain, whilst the fine material that enters the lumen of the ventral caeca takes 24 to 48 h. Indeed, the duration of the complete cycle of digestion is reflected by the passage of fine rather than coarse material. When the amphipods are not feeding, the passage of coarse material is more erratic and takes longer. Nielsen and Kofoed (1982) measured a minimal time of 20 rain.

Corophium volutator takes less time to pass particles of sediment through the gut than Martin (1965, 1966) found in the essentially herbivorous amphipods Gammarus pulex, Marinogammarus obtusatus and M. pirIoti, and as Sheader and Evans (1975) found in the carnivorous amphipod Parathemisto gaudichaudi (Table 3). In most of these studies, the time related to the passage of the coarse material and is, therefore, a measure of the period of primary digestion.

Martin (1965) found in GammaruspuIex that the fraction of a Bynogen diet (proprietory food rich in essential food substances) that passed straight through the gut had a clearance time of 5 h, whilst that fraction which entered the hepatopancreas took up to 24 h to enter this epithelium and other storage tissues in the amphipod. Primary digestion took longer than in Corophium voluta~or, but the time span for complete digestion of this different diet was similar. Indeed, in the carnivorous decapods Carcinus maenas and Penaeus semisulcatus, Hopkin and Nott (1980) and A1-Mohanna (1983), respectively, have found that primary digestion takes under 12 h, but complete digestion takes 24 to 48 h. It can be suggested, therefore, that the times of complete digestion for all the amphipods in Table 3 could be much longer and show much greater similarity than those for primary digestion.


In Corophium volutator, exchange of material between the lumen of the hepatopancreas and the rest of the gut is dependent on the feeding activity of the amphipod, since ingested azo-carmine is not removed from the hepatopancreas in starved individuals. This is not the case in Carcinus rnaenas, where particles introduced into the hepatopancreas with the diet are removed into the midgut and eventually defecated, whether the crab is starved or fed again (Hopkin, 1980; Hopkin and Nott, 1980). Presumably, this difference reflects the mode of feeding; food is always available for Corophium volutator and it can feed continuously, whilst Carcinus maenas hunts for its food and can only feed sporadically. In non-feeding individuals of both species there is residual material in the hindgut which is not defecated until they feed again.

Absorption of particles, location of enzymes, carbohydrates and lipid

Foregut

Primary digestion occurs in the foregut, where particles of food are supplied with enzymes and fluids from the ventral or hepatopancreatic caeca (Icely and Nott, 1984). Fine and soluble materials enter the caeca through a system of filters that block particles greater than 0.06/~m. There is no evidence for the absorption of tracer particles by the epithelium of the foregut. Some non-specific esterase activity occurs in the epithelium below the cuticle of the foregut, but this is probably concerned with the formation of cuticle.

Stores of lipid and glycogen do not occur in the epithelium of the foregut of intermoult Corophium volutator. However, in Gammarus pulex both are deposited in the foregut and hindgut, particularly at the premoult stage (Martin, 1965).

Anterior dorsal caeca, intestine and posterior caeca

The anterior dorsal and posterior caeca show differences in fine structure (Icely and Nott, 1984), and this is reflected in the localisation of digestive enzymes and particulate tracers in the lumen and/or the epithelium of the anterior dorsal caeca and the intestine, but not in the posterior caeca. The contributions of the anterior dorsal caeca and the intestine to the processes of digestion and absorption are limited in comparison with the ventral caeca. The volume is relatively small and the cells un- differentiated. The turnover of material in the lumen is faster and the digestive enzymes show less activity, particularly towards the posterior end of the intestine. Tracer particles are not as readily assimilated. The basal lamina is structurally more complex and thicker than that of the ventral caeca and could, thereby, reduce exchange of material with the haemocoel. Lipid does not occur in the anterior dorsal caeca and the intestine, but glycogen does

occur in all regions of the midgut. Acid phosphatase occurs in the amorphous material underlying the microvilli of the intestine, where it is probably involved with processes of absorption (Icely, 1981). Mabillot (1955), Martin (1964) and Sheader and Evans (1975) found that histochemical studies on the anterior dorsal caeca and intestine produce similar results in Gamrnarus pulex, Ma- rinogammarus obtusatus and Parathemisto gaudichaudi, respectively.

Both ultrastructural (Icely and Nott, 1984) and histochemical studies on the posterior caeca of CoroFhiurn volutator show that this tissue is not involved in digestion and absorption, and that the storage of materials is limited to glycogen. Although there is a direct connection between the lumen of the intestine and that of the posterior caeca, tracer particles in the diet do not enter the caeca. The fine structure of the epithelium in Corophium volutator and also Orchestia cavimani, a terrestrial amphipod (Graf and Michaut, 1980), has many of the characteristics of tissues that transport ions and fluids. In comparison with C. volutator, the posterior caeca of O. cavimani are much longer and are responsible for calcium metabolism (Graf and Meyran, 1983). Calcium is accumulated also in the posterior caeca and intestine of NiFhargus virei, a freshwater amphipod inhabiting subterranean pools (Graft 1968) and to a smaller extent in Gammarus pulex, a freshwater amphipod inhabiting rivers and ponds (Graf, 1968). This tink between functional morphology and the habitat has been demonstrated in other Gammaridea, where the posterior caeca is least developed in sub-littoral, marine species and most developed in terrestrial species (Nebeski, 1880; Agrawal, 1964b, 1966; Graf, 1968). They are vestigial in the sub-littoral amphipods of the Caprelli- dea (Keith, 1974) and Hyperiidea (Laval, 1980).

The crustacean midgut generally secretes a peritrophic membrane (Georgi, 1969), which packages the solid material entering the midgut from the foregut. In anaphi- pods, the membrane is produced by the anterior dorsal caeca and the anterior intestine, and in decapods, by the anterior midgut caeca and the midgut. In the posterior caeca of the amphipod Niphargus virei, a peritrophic membrane is wrapped around calcium granules secreted into the lumen (Graf, 1968), and in the hepatopancreatic caeca of the decapod Carcinus maenas this membrane surrounds vacuoles containing waste material extruded from B-cells (Hopkin and Nott, 1980). It is probable that all the tissues of the midgut can produce this membrane. In Corophium volutator, it takes the form of a mucoid substance (Icely and Nott, 1984) which might protect the epithelium from the passage of abrasive mud particles.

Ventral caeca

The ventral caeca is the principal site for secondary digestion, the production of digestive enzymes, and the absorption and storage of the products of digestion. This is reflected in the current study by the activity of non-


Table 4. Interpretation of function of hepatopancreatic caeca in amphipods from studies on fine structure. + + : high concentration; + : moderate concentration;-: not observed; *:not described

Species Location of Location of storage products Source protein production a Lipid Glycogen Metals

Corophium volutator R/F+ + B+ R/F+ + B - R/F+ + B+

Stegocephaloides R/F+ + B + R/F + + B - R/F* B + christianiensis

Orchestiaplatensis R+ F+ + B+ R+ + F+ B+ R+ + F+ B+

GammarusIoeusta R+ F + + B+ R + + F + B+ R + + F + B+

Gammarusminus R+ F+ + B+ R+ + F+ B+ R - F - ]3-

TaIitrussaltator R+ F+ + B* R+ + F+ B* R+ + F+ B*

Cu: R /F+ + B -

Fe: R / F + + B - Ca: R / F + + B -

Icely and Nott (1980, 1984)

Moore and Rainbow (1984)

Moritz et al. (1973)

Moritz et al. (I973)

Schultz (1976)

Storch and Burkhardt (1984)

Complexes of rough endoplasmic reticulum and ribosomes

specific esterases and proteases, the uptake o f tracer particles ingested with the diet and the storage of carbo- hydrate and lipid. In other studies, Agrawal (1963a, b, 1964a) identified carbohydrases, proteases, lipases and esterases in most extracts from the ventral caeca o f Corophium volutator, Marinogammarus minus and Orches- tia gammarella and cellulases in O. gammarella. Halcrow (1971) found cellulases in the caeca of Gammarus oceanicus. Other enzymes have been localised by cytochemical tests on sectioned material o f M. obtusatus by Martin (1964) and G. pulex by Mabillot (1955). Martin (1964) used iron saccharate as a tracer in the diet o f M. obtusatus, showing that absorption occurs principally in R/F-cells adjacent to the B-cells. Lipid only occurs in the R/F-cells of C, volutator and Stegocephaloides christianiensis, unlike other species wherein it also occurs in the B-cells (Table 4). Generally, glycogen is detected in all the cell types, but metals are reported only in the R/F-cells of C. volutator and of S. christianiensis (Table 4).

The structure of the R/F-cells, and therefore their function, changes along the length of the caeca in Coro- phium volutator. The cells in the distal region contain more rough endoplasmic reticulum and, therefore, must produce more protein than the cells in the proximaI region which contain most o f the storage products.

"l-he B-cells are sites of digestive enzyme activity which occurs primarily in the "apical complex". It may occur in the fluid of the vacuole, but this is probably extracted by the histochemical procedures. Indeed, the substrate-film technique (see "Materials and methods - Histochemical tests - Non-specific esterases") which avoids extraction of material from the tissues produces evidence for digestive enzymes in the vacuole. In the gut of copepods, the hydrolytic enzymes, acid phosphatase and aryl sulphatase are confined to the B-cells, where they occur in the vacuolar system, dictyosomes and Golgi vesicles (Arnaud et al., 1984). In Corophium volutator, particulate material occurs in the "apical complex" and this indicates that the products of primary digestion from the foregut can be absorbed from the lumen of the ventral caecum by B-cells. Finally, the contents of the B-cells are extruded into the

lumen of the caecum by apocrine (Schultz, 1976) or holocrine secretion (Shyamasundari and Varghese, 1973). Martin (1964) has seen dark-staining bodies in the vacuoles of discharged B-cells in Marinogammarus obtusatus, and he suggests that these are excretory products. This process should be regulated by the digestive cycle and, indeed, in recently moulted C. volutator the B-cells are not discharged (holocrine) until the amphipods have been feeding for some time (Icely, 1981).

Hindgut

As in the foregut, there is no evidence for the absorption of tracer particles or for the localisation of lipid and glycogen by the hindgut o f Corophium volutator; minimal non-specific esterase has been detected below the cuticle. Fox (1952) has observed that water can be swallowed into the hindgut by rhythmic contractions of the muscles in the anus (see Fig. 4f, g in Icely and Nott, 1984), and similar observations have been made in the present work. It is possible that this process may be linked with the activities of the "ion pumps" described by Icely and Nott (1984) in the hindgut of C. volutator.

Literature cited

Agrawal, V. P.: A study of the physiology of digestion in Marino- gammarus marinus Leach. J. mar. biol. Ass. India 5, 62-67 (1963 a)

Agrawal, V, P.: Studies on the physiology of digestion in Coro- phium volutator. J. mar. biol. Ass. U.K. 43, 125-128 (1963b)

Agrawal, V. P.: Studies on the physiology of digestion in Orchestia gammarella. Proc. zool. Soc. Lond. I43, 133-141 (1964a)

Agrawal, V. P.: The digestive system of some British amphipods. III. The alimentary canal. Proc. natn. Acad. Sci. India 34B, 429-458 (1964 b)

Agrawal, V. P.: The digestive system of some British amphipods. IV. The alimentary canal. Proc. natn. Acad. Sci. India 36B, 457-483 (1966)

A1-Mohanna0 S. Y.: The structure and function of the hepatopancreas of Penaeus semisulcatus De Haan (Crustacea: Deca- poda) during the digestive and moult cycles, 391 pp. Ph.D. thesis, University of Wales 1983


Arnaud, J., M. Brunet and J. Mazza: Cytochemical detection of phosphatase and arylsulphatase activities in the midgut of Centropages typicus (copepod, calanoid). Bas. appl. Histo- chem. 28, 399-412 (1984)

Doyle, R. W.: Ingestion rate of a selective deposit feeder in a complex mixture of particles: testing the energy-optimisation hypothesis. Limnol. Oceanogr. 24, 867-876 (1979)

Fenchel, T.: Aspects of decomposer food chains in marine benthos. Verh. dt. zool. Ges. 65, 14-23 (1972)

Fenchel, T., L. H. Kofoed and A. Lappalainen: Particle size- selection of two deposit feeders: the amphipod Corophium volutator and the prosobranch Hydrobia ulvae. Mar. Biol. 30, 119-128 (1975)

Fox, H. M.: Anal and oral intake of water by Crustacea. J. exp. Biol. 29, 583-599 (1952)

Fratello, B.: Enhanced interpretation of tissue protease activity by use of photographic color film as a substrate. Stain Technol. 43, 125-128 (1968)

Georgi, R.: Fine structure of the peritrophic membranes in Crustacea. Z. Morph. Okol. Tiere 65, 225-273 (1969)

Gibson, R. and P. L. Barker: The decapod hepatopancreas. Oceanogr. mar. Biol. A. Rev. 17, 285-346 (1979)

Gomori, G.: Histochemistry of esterases. Int. Rev. Cytol. 1, 323-335 (1952)

Graf, F.: Le stockage de calcium avant la mue Ies Crustacts Amphipodes Orchestia (Talitrudt) et Niphargus (Gammarid6 hypogt), 124 pp. Th+se, Facult6 des Sciences de l'universit6 de Dijon 1968

Graf, F. and J.-C. Meyran: Premolt calcium secretion in midgnt posterior caeca of the crustacean Orchestia: ultrastructure of the epithelium. J. Morph. 177, 1-23 (1983)

Graf, F. and P. Michaut: Fine structure of the midgut posterior caeca in the crustacean Orchestia in intermolt: recognition of two distinct segments. J. Morph. 165, 261-284 (1980)

Halcrow, K.: Cellulase activity in Gammarus oceanicus Segerstrgde (Amphipoda). Crustaceana 20, 121-124 (1971)

Hargrave, B. T.: Prediction of egestion by the deposit-feeding amphipod Hyalella azteca. Oikos 23, 116-124 (1972)

Hart, T. J.: Preliminary notes on the bionomics of the amphipod Corophium volutator (Pallas). J. mar. biol. Ass. U.K. 16, 761-789 (1930)

Hopkin, S. P.: The structure and function of the hepatopancreas of the shore crab Carcinus maenas (L.): a study by electron microscopy and X-ray microanalysis, 209 pp. Ph.D. thesis, University of Wales 1980

Hopkin, S. P. and J. A. Nott: Studies on the digestive cycle of the shore crab Carcinus maenas (L.) with special reference to the B-cells in the hepatopancreas. J. mar. biol. Ass. U.K. 60, 891-907 (1980)

Icety, J. D.: A study of digestion and excretion in Corophium volutator (Pallas), with some reference to the metabolism of heavy metals, 152 pp. Ph.D thesis, University of Wales 1981

Icely, J. D. and J. A. Nott: Accumulation of copper within the "hepatopancreatic" caeca of Corophium volutator (Crustacea: Amphipoda). Mar. Biol. 57, 193-199 (1980)

Icely, J. D. and J. A. Nott: On the morphology and fine structure of the alimentary canal of Corophium volutator (Pallas) (Crus- tacea: Amphipoda). Phil. Trans. R. Soc. (Set. B) 306, 4%78 (1984)

Keith, D. E.: A comparative study of the digestive tracts of Caprella equilibria Say and Cyamus boopis Latken (Amphi- poda, Caprellidea). Crustaceana 26, 127-132 (1974)

Laval, P.: Hyperiid amphipods as crustacean parasitoids asso- ciated with gelatinous zooplankton. Oceanogr. mar. Biol. A. Rev. 18, 11-56 (1980)

Lewis, P. R. and D. P. Knight: Staining methods for sectioned material. In: Practical methods in electron microscopy, Vol. 5. pp 1-31 I. Ed. by A. M. Glauert. Amsterdam: North-Holland Publishing Company 1977

Lincoln, R. J.: British marine Amphipoda: Gammaridea. vi, 658 pp. London: British Museum (Natural History) 1979

Mabillot, S.: Contribution a l 'ttude histophysiologique de l'appareil digestif de Gammaruspu[ex L Archs Zool. exp. gtr~. 92, 20-38 (1955)

Martin, A. L.: The alimentary canal of Marinogarnmarus obtusatus (Crustacea, Amphipoda). Proc. zool. Soc. Lond. 143, 525-544 (1964)

Martin, A. L.: Histochemistry of the moulting cycle in Gammarus pulex (Crustacea, Ampbipoda). J. Zool., Lond, 147, 185-200 (1965)

Martin, A. L.: Feeding and digestion in two intertidal gammarids: Marinogammarus obtusatus and M. pirloti. J. Zool. Lond. 148, 515-525 (1966)

Meadows, P. S. and A. Reid: The behaviour of Corophium volutator (Crustacea: Amphipoda). J. Zool., Lond. 150, 387-399 (1966)

Miller, D. C.: Mechanical post-capture particle selection by suspension- and deposit-feeding Corophium. J. exp. mar. Biol. Ecol. 82, 59-72 (1984)

Moore, P. G. and P. S. Rainbow: Ferritin crystals in the gut caeca of Stegocephaloides christianiensis Boeck and other Stego- cephalidae (Amphipoda: Gammaridea): a functional interpretation. Phil. Trans. R. Soc. (Ser. B) 306, 219-245 (1984)

Moritz, K., V. Storch und W. Buchheim: Zur Feinstruktur der Mitteldarmanh~inge yon Peracarida (Mysidacea, Amphipoda, Isopoda). Cytobiol. 8, 39-54 (1973)

Nebeski, O.: Beitfitge zur Kenntnis der Amphipoden der Adria. Arb, zool. Inst. Univ. Wien 3, 111-162 (1880)

Nielsen, M. V. and L. H. Kofoed: Selective feeding and epipsam- mic browsing by the deposit-feeding amphipod Corophium volutator. Mar. EcoL Prog. Ser. 10, 81-88 (1982)

Pearse, A. G. E.: Histochemistry, theoretical and applied, 2. 3rd ed. 757 pp. London: Churchill-Livingstone 1972

Schultz, T. W.: The ultrastructure of the hepatopancreatic caeca ~l" Gammarus minus (Crustacea, Amphipoda). J. Morph. 149, 383-400 (1976)

Sheader, M. and F. Evans: Feeding and gut structure of Para- themisto gaudichaudi (Guerin) (Amphipoda, Hyperiidea). J. mar. biol. Ass. U.K. 55, 641-656 (1975)

Shyamasundari, K. and S. Varghese: Holocrine secretion in the hepatopancreas ofamphipods. Curr. Sci. 42, 310-321 (1973)

Storch, V. and E Burkhardt: Influence of nutritional stress on the hepatopancreas of Talitrus saltator (Peracarida, Amphipoda). Helgol/inder wiss. Meeresunters. 38, 65-73 (1984)

Taghon, G. L.: Optimal foraging by deposit feeding invertebrates: roles of particle size and organic coating. Oecologia (Berlin) 52, 295-304 (1982)

Thiery, J.-P.: Mise en 6vidence des polysaccharides sur coupes fines en microscopie 61ectroniqne. J. Microscopie 6, 987-1018 (1967)

Date offinat manuscript acceptance: June 7, 1985. Communicated by J. Mauchline, Oban

feeding and digestion in corophium volutator (crustacea: amphipoda)

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