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in alternated and endogenously synchronized phases of i food filtration, ii extracellu- lar digestion in the stomach and, iii intracellular digestion within the gland. These
studies brought great information regarding anatomy, morphology and histochemistry of the digestive system but they fail to provide information on the physiological control of
the digestive function since none of them analyzed the changes of the parameters characterizing intra- or extracellular digestive activity in bivalves fed different foods.
The aim of this study was to identify the cellular mechanisms involved in the acute and acclimated changes of the functional gut capacity of cockles treated to different food
qualities and quantities. We performed a histological study quantifying the volume density of lysosomes, digestive and basophilic cells, the thickness of digestive epithelia
and the radium of digestive tubules together with the determination of the digestive enzyme activities of cockles fed four diets two different qualities supplied at two
different rations for 3 and 11 days. Details on the characteristics of the diets and recorded feeding rates, absorption efficiencies and absorption rates of total organic
matter and biochemical components have been reported in the preceding publication I. Feeding and absorption.
2. Materials and methods
2.1. Experimental set-up and manipulation of cockles A total of 140 cockles ranging in size 24–28 mm were collected from an intertidal
mud-flat in the Mundaka estuary Biscay, N. Spain during February 1993. In the laboratory, 120 cockles were divided in four groups, each fed one of the four diets
described in the experimental protocol Ll, Lh, Hl and Hh, and the remaining 20 cockles were submitted to starvation S. Further experimental details on experimental
set-up were reported in the preceding contribution. After 3 days and 11 days of exposure to the diets, digestive gland and crystalline styles of 15 cockles from each experimental
condition were excised. The enzyme activities of the digestive gland were determined in 10 individuals, whereas the remaining five individuals were used for histological
measurements.
2.2. Histological measurements 2.2.1. Preparation of digestive glands
The digestive glands were excised and divided in two portions. One portion was fixed in Bouin’s fluid 32–36 h, dehydrated, embedded in paraffin and sectioned at 9 mm.
Sections were stained with haematoxylin-eosin Pearse, 1976 to determine: i stereological measurements, ii planimetric measurements and iii digestive phases of
the digestive tubules. The second portion was frozen with Bright Cryo-Spray dichloro- fluoromethane, 2 50 558C, embedded in Bright Cryo M-Bed and cut in a Bright’s
cryostat at 2 28 308C in sections of 9 mm and stored at 2 708C until used to determine stereological measurements of the lysosomes.
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. Ibarrola et al. J. Exp. Mar. Biol. Ecol. 252 2000 199 –219
2.2.2. Stereology of the digestive tubes A Weibel reticule Multipurpose test system M-168 was used to calculate the
volumetric fractions of the different cell types in the digestive tubes. Two sections cut from different layers, and five randomly selected areas per section were counted for each
individual i.e. 10 measurements per individual. The volumetric fractions of digestive VF and basophilic VF cells were determined. In addition, we determined the
D B
volumetric fraction corresponding to apical cell fragments detached from digestive cells of disintegrating tubules VF
.
CF
2.2.3. Planimetric determinations For each individual digestive gland, a total of 50 tubule sections were drawn with the
aid of a drawing tube coupled to a Nikon Optiphot microscope. We used an objective lens of 3 100. Five sections were randomly selected from each 10 paraffin section
belonging to two different portions of the gland 50 mm apart. The profiles of the 50 sections were recorded with a Watanabe DT1000 digitizer and the resulting planimetric
measurements were calculated with a personal computer following the method of
´ Marigomez 1989 which is based in the geometrical transformation of the tubule
section shapes. Two parameters are obtained: mean diverticular radius MDR and mean epithelial thickness MET.
Simultaneously, the tubules were classified according to their morphological appear- ance. Four different tubule types were distinguished following the standards of Morton
1983: holding H, absorptive A, disintegrating D and reconstituting R. 2.2.4. Stereology of the lysosomal system
Lysosomes were stained following the method of Moore 1976 as described in Cajaraville et al. 1991 which is based on the cytochemical reaction for b-glucuronid-
ase. The enzymatic reaction yields a purple-colored product. Light microscopic images were then obtained by coupling an image processing system comprising a BW-CCD
TV camera, a HR-TV colour screen and a personal computer AT-PC, to the Leitz light microscope and using an objective lens of 3 100. The area corresponding to the
lysosomes in purple colour and the rest of cytoplasmic matter of the digestive cells were manually segmented by assigning them different colours on the monitor. Computerized
measurements of the areas corresponding to different colours allows computation of the following parameters Lowe et al., 1981: lysosomal volume density VD, lysosomal
volume cytoplasmic volume, lysosomal numerical density ND, number of lysosomes cytoplasmic volume, lysosomal surface density SD, lysosome surface cytoplasmic
volume and the surface to volume ratio of the lysosomes S V.
2.3. Digestive enzyme activities Amylase, cellulase, laminarinase and not-specific protease activities of the digestive
gland were determined according to the procedures described elsewhere Ibarrola et al., 1996, 1998a,b, 1999 and expressed as specific activity mg of end product released per
mg protein in the digestive extract per hour and total activity mg of end product released per individual digestive gland per hour.
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2.4. Statistical procedures Significant effects promoted by food quality ql and quantity qn on histological
measurements and enzyme activities in the acute and acclimated responses of cockles were analyzed by performing two factor analysis of variance Zar, 1984. The arcosin
transformation was used to normalize the data corresponding to histological parameters which are expressed as percentage. The effect promoted by acclimation to the different
diets was analyzed by testing for significant differences t-test between the mean values obtained for the acute and acclimated responses.
Possible functional relationship between enzyme activities and digestive structures was analyzed by calculation of the correlation indexes between specific activities and
volume densities of the different digestive structures.
3. Results
