Consequences of keeping - Helgoland Marine Research

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hepatopancreas of the fiddler crab, []ca minax. -J. Invertebr. Pathol. ... coconut crab, Birgus latro (L.) (Crustacea, Decapoda).- Zool. Anz. 208, 115-123.
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HELGOLANDER MEERESUNTERSUCHUNGEN Helgol~inder Meeresunters. 45,445-464 (1991)

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C o n s e q u e n c e s of k e e p i n g Mytilus in the laboratory as assessed by different cellular condition indices M. P. Cajaraville, G. Diez, I. A. Marig6mez & E.

Angulo

Zitologi eta I--Iistologi Laborategia, Biologia Zelularra eta Zientzia Morfologikoen Safla, Zientzi Fakultatea; Euskal Hernko Unibertsitatea, 644 P. K., 48080-Bilbo, Spain

ABSTRACT: Mytilus galloprovincialis Lmk. were maintained in the laboratory for three months in a semicontinuous water flow system. Animals were fed a commercial filter-feeder food and sampled after 0, 21, 35, 49, 77, and 91 days. In order to establish whether laboratory conditions and the food used were deleterious to mussels, their health status was assessed by quantifying different histological parameters of the digestive gland tissue. It was concluded that mussels kept for more than 35 days under the described laboratory conditions showed signs of stress presumably caused by the reproductive state of the mussels investigated. The food used and the nutrition-related health status of the animals were adequate, as shown by transmission electron microscopical studies after the 91day maintenance period. A stress response was also evoked by a 10-day starvation period, which was reflected by an increased proportion of type I and type IV digestive tubules, and a reduced "Mean EpitheLial Thickness" (MET). Finally, the results demonstrate the sensitivity of quantitative histological diagnosis in comparison tO subjective tubule grading procedures in the assessment of the degree of stress experienced by mussels.

INTRODUCTION Mussels are used world-wide in monitoring programmes as pollution indicators (Goldberg, 1986). Mussels are ideal monitoring species as they are d o m i n a n t m e m b e r s of coastal a n d estuarine communities, accumulate different toxicants in their tissues, are responsive to m a n y e n v i r o n m e n t a l pollutants, a n d have a wide geographical distribution (Livingstone et al., 1988). This situation has created an increased interest in k e e p i n g mussel populations in the laboratory in order to monitor the effects of pollutants in partially controlled conditions (Bayne & Thompson, 1970). Livingstone et al. (1988) report that mussels do not display stress due to prolonged handling, while long-term experim e n t s carried out both in laboratory flow-through systems a n d mesocosm basins indicate the opposite (Bayne & Thompson, 1970; Bayne, 1973; Lowe, 1988). Non-stressing m a i n t e n a n c e protocols for animals of potential biomonitoring use are rare (Sindermann, 1988). Besides, the nutritional status of test animals is a factor that might modify the toxicity of w a t e r b o r n e chemicals to aquatic organisms (Lanno et al., 1989). Mussels show some physiological r e q u i r e m e n t s that make their m a i n t e n a n c e in the laboratory difficult. Microalgae are the major source of food, a n d thus, mussels are usually m a i n t a i n e d in the laboratory on monoalgal diets of different species of microalgae or on mixtures of algae, the latter often resulting in higher growth rates (Hummel, 1985). Furthermore, food has to be provided continuously at a certain concentration for long9 Biologische AnstaIt Helgoland, Hamburg

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M. P. Cajaraville, G. Diez, I. A. M a r i g 6 m e z & E. A n g u l o

term experiments. A l g a l cultures of considerable v o l u m e have to b e g r o w n for this purpose. T h e s e features r e q u i r e t i m e - c o n s u m i n g care a n d an infrastructure not always available in r e s e a r c h laboratories. For these reasons w e d e c i d e d to test an artificial commercial diet for f e e d i n g mussels. The p r e s e n t w o r k on the m u s s e l Mytilus galloprovincialis Link., m a i n t a i n e d u n d e r constant conditions of immersion, reports quantitative c h a n g e s in histology of the digestive g l a n d monitored over a t h r e e - m o n t h period. Histological diagnosis at the electron microscope level is a h i g h l y sensitive m e t h o d of d e t e c t i n g early r e s p o n s e s to starvation, as d e m o n s t r a t e d in the i n v e s t i g a t i o n s on h e p a t o p a n c r e a t i c R-cells of crustaceans (Storch et al., 1982; Storch & Burkhardt, 1984). S e v e r a l p a p e r s h a v e shown that the ingestion of i n a p p r o p r i a t e food also l e a d s to histopathological c h a n g e s (Storch & Burkhardt, 1984; V o g t et al., 1986; S e g n e r et al., 1987) that are distinguishable from c h a n g e s i n d u c e d b y starvation ( S e g n e r et al., 1987). Histological diagnosis at the light a n d electron microscope level is also of g r e a t help in monitoring the effects of pollutants such as pesticides (Vogt, 1987), a r o m a t i c hydrocarbons (Robinson & Dillaman, 1985; Cajaraville et al., 1990b, c) a n d metals ( P a p a t h a n a s s i o u & King, 1986; M a r i g 6 m e z et al., 1990; 1991) in a variety of invertebrates. As in crustaceans, the digestive g l a n d (or h e p a t o p a n c r e a s ) of molluscs shows a g r e a t n a t u r a l plasticity, a n d the m o r p h o l o g y of the digestive tubules c h a n g e s c o n s i d e r a b l y with alterations of e n v i r o n m e n t a l conditions (Thompson et al., 1974; Lowe et al., 1981; Couch, 1984; A x i a k et al., 1988; V e g a et al., 1989). In the p r e s e n t investigation, the digestive tubule structure was m e a s u r e d in terms of m e a n epithelial thickness, m e a n diverticular radius and m e a n luminal radius b y using a planimetric p r o c e d u r e (Vega et al., 1989). A subjective tubule g r a d i n g m e t h o d was also u s e d to estimate the proportions of tubules at different digestive stages (Langton, 1975; Robinson & Langton, 1980). The v o l u m e density of basophilic cells w a s d e t e r m i n e d in paraffin sections b y a p o i n t - c o u n t i n g m e t h o d (Cajaraville et al., 1990a). T h e s e quantitative p a r a m e t e r s can s e r v e as a basis to e v a l u a t e the d e g r e e of stress in natural a n d cultivated p o p u l a t i o n s of mussels. Transmission electron m i c r o s c o p y was also u s e d to provide a further analysis of t h e condition of the digestive a n d basophilic cells after the t h r e e - m o n t h m a i n t e n a n c e period. T h e g o n a d i n d e x (Seed, 1969) was d e t e r m i n e d to assess the reproductive state of mussels. The objective of the p r e s e n t study was thus twofold: first, to establish w h e t h e r the laboratory conditions u s e d w e r e t h e m s e l v e s deleterious to mussels, a n d second, to d e t e r m i n e the sensitivity of the different histological stress p a r a m e t e r s d e s c r i b e d a b o v e in the detection of subtle c h a n g e s in mussel condition. Special attention w a s also p a i d to the a s s e s s m e n t of the nutritive value of the food used. MATERIALS A N D M E T H O D S Experimental procedure Mussels, collected from Mefiakoz, Biscay (43 ~ 24' N, 2~ ' W) in M a r c h 1988, w e r e transferred to the laboratory. 50 individuals (2.5-3.5 cm shell length) w e r e d i s t r i b u t e d in 25-1 p o l y e t h y l e n e covered t a n k s in a t h e r m o s t a t i z e d semicontinuous w a t e r flow system using natural s e a w a t e r (Zierbena, Biscay) filtered t h r o u g h activated c h a r c o a l a n d glasswool, the w a t e r b e i n g r e p l a c e d every 2 days. Mussels w e r e m a i n t a i n e d u n f e d at 15-16 ~ C for 10 days to provide acclimatization to l a b o r a t o r y conditions. Thereafter, i n d i v i d u a l s

Histological c o n s e q u e n c e s of k e e p i n g mussels in the l a b o r a t o r y

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w e r e k e p t in replicate series for three months u n d e r natural p h o t o p e r i o d s a n d fed with a commercial filter-feeder food (Marine Invertebrate Diet, H a w a i i a n M a r i n e Imports Inc., distributed b y Carolina Ltd.). The food was a mixture of s e l e c t e d organic particles a n d amino acids (ingredients: casein, dried yeast, thiamine, riboflavin, niacin, s o d i u m benzoate, ascorbic acid a n d sodium chloride). A p p r o x i m a t e l y two drops w e r e a d d e d p e r t a n k every day. F a e c e s w e r e m e c h a n i c a l l y r e m o v e d from the tanks once a d a y before the addition of fresh food. The tanks w e r e continuously aerated. W a t e r - t e m p e r a t u r e was r e c o r d e d every w o r k i n g day; it a v e r a g e d 15.25 _ 1.01 ~ in April, 16.17 • 0.77 ~ in May, a n d 16.47 • 1.20 ~ in June. P l a n i m e t r i c a n a l y s i s of d i g e s t i v e t u b u l e s a n d t u b u l e g r a d i n g m e t h o d Mussels w e r e s a m p l e d after 0, 21, 35, 49, 77, a n d 91 days. Five a n i m a l s w e r e r e m o v e d from e a c h replicate e x p e r i m e n t a l group at each s a m p l i n g period, e x c e p t in the s a m p l e 0 ( 1 0 - d a y starved animals), w h e r e a total of 44 animals w a s used. After the animals h a d b e e n r e m o v e d from the shell, a portion of the m a n t l e - d i g e s t i v e g l a n d tissue was carefully e x c i s e d a n d fixed in Bouin's fluid. Following d e h y d r a t i o n t h r o u g h an a s c e n d i n g e t h a n o l series, the tissues w e r e c l e a r e d in m e t h y l b e n z o a t e a n d e m b e d d e d in paraffin. Sections (9 ~m) w e r e cut a n d stained with h a e m a t o x y h n - e o s i n (H/E). O b s e r v a tions on the p r e s e n c e / a b s e n c e of the crystalline style w e r e carried out for e a c h animal, a n d although no a t t e m p t was m a d e to m e a s u r e style length, its size was subjectively g r a d e d into small, i n t e r m e d i a t e a n d large. In order to quantify the m o r p h o l o g i c a l structure of the digestive tubules, a p l a n i m e t tic p r o c e d u r e (based on the g e o m e t r i c a l transformation of a t u b u l e section into a r e g u l a r geometrical figure) was a p p h e d (Recio et al., 1988). Five tubule sections w e r e s e l e c t e d in each of the five fields of two different sections of the digestive g l a n d (45-180 ~m from one another). The selection of fields within a given section was m a d e b y a l w a y s starting at the top-right a n g l e of the tissue. Further fields w e r e s e l e c t e d at given intervals across the tissue section, the direction of m o v e m e n t always having the s a m e z i g - z a g pattern. Thus, 50 tubule sections w e r e d r a w n p e r mussel (50 • 10 = 500 tubule sections p e r e x p e r i m e n tal group) with the aid of a d r a w i n g - t u b e a t t a c h m e n t to a N i k o n " O p t i p h o t " microscope (total magnification 670 x). W h e n necrotic tubules (see below) w e r e present, only the outer profile of the t u b u l e section could b e drawn. In cases w h e r e a part of the digestive tubule e p i t h e l i u m still s h o w e d structural integrity, both i n n e r a n d outer profiles of this part of the e p i t h e l i u m w e r e drawn. Section profiles w e r e r e c o r d e d b y m e a n s of a W a t a n a b e DT1000 digitizer, a n d planimetric m e a s u r e s c a l c u l a t e d b y an Ohvetti M240 p e r s o n a l c o m p u t e r following the p r o c e d u r e of V e g a et al. (1989). Five p a r a m e t e r s w e r e obtained: M e a n Epithelial Thickness (MET), M e a n Diverticular Radius (MDR), M e a n Luminal Radius (MLR), M E T / M D R ratio a n d MLR/MET ratio. The three a b s o l u t e p a r a meters are c a l c u l a t e d as follows: (1) MET = according to Recio et al. (1988); (2) MDR = AV~-0/-~; a n d (3) MLR = V'-~-~; w h e r e Ao a n d Ai are the a r e a s e n c l o s e d b y the outer a n d the inner s h a p e s of the tubule section, respectively (Vega et al., 1989). Besides, the digestive tubules u s e d for the calculation of the p l a n i m e t r i c p a r a m e t e r s w e r e also a n a l y s e d using a subjective tubule g r a d i n g method. The calculation of proportions a n d v a r i a n c e s of e a c h tubule type p e r animal was b a s e d on the classification of 50 tubules. Each t u b u l e was classified according to the following types: hoIding (type I),

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absorptive (type II), disintegrating (type III) and reconstituting (type IV) (Langton, 1975; Robinson & Langton, 1980). A fifth tubule type was considered to b e necrotic, as previously d e s c r i b e d b y Cajaraville et al. (1989). Necrotic tubules are c h a r a c t e r i z e d b y the a b s e n c e of a w e l l - d e l i m i t e d i n n e r b o r d e r l i n e of the tubule section as the l u m e n of the t u b u l e is c o m p l e t e l y filled with cell debris a n d with cells u n d e r g o i n g autolysis. Thus, this tubule t y p e is defined b y a MET value close to 0 ~m. S t e r e o l o g i c a l a n a l y s i s of b a s o p h i l i c c e l l s A stereological p r o c e d u r e was a p p h e d in order to quantify the v o l u m e density of basophilic ceils. Counts w e r e m a d e in e a c h of the five fields of the two different sections of the digestive g l a n d (45-180 ~m from one another). The selection of fields w i t h i n a given section was m a d e as d e s c r i b e d a b o v e for the planimetric analysis of d i g e s t i v e tubules. Thus, cell counts w e r e m a d e in 10 fields p e r mussel (10x 1 0 - - 1 0 0 c o u n t i n g s p e r e x p e r i m e n t a l group). A W e i b e l graticule (multipurpose test system M - 1 6 8 ) w a s u s e d (Weibel, 1979), a n d hits on basophilic cells a n d on the r e m a i n i n g d i g e s t i v e tissue w e r e recorded. From these values, the v o l u m e density (VD) of basophilic cells w a s d e t e r m i n e d using the following equation: VD =

Xl+X2

+... mxn

+Xn

w h e r e : X: n u m b e r of s e g m e n t e d g e s falling on basophilic cells, m: total n u m b e r of s e g m e n t e d g e s falling on digestive tissue, n: n u m b e r of counts m a d e (10 for e a c h mussel). H i s t o l o g i c a l s t u d i e s of g o n a d d e v e l o p m e n t The arbitrary s c h e m e of classification of the g o n a d condition r e p o r t e d b y S e e d (1969) for 5/Iytilus edulis was used. Four m a i n s t a g e s w e r e thus r e c o g n i z e d (developing, ripe, s p a w n i n g a n d spent), a n d the m e a n g o n a d i n d e x for each s a m p l e was d e t e r m i n e d as in S e e d (1969). Each s a m p l e c o m p r i s e d 10 individuals, e x c e p t on the first s a m p l i n g d a y (0) w h e n 44 animals w e r e g r a d e d . The i n d e x varies from zero w h e n all i n d i v i d u a l s in the s a m p l e are in the s p e n t condition, to five w h e n all individuals are fully m a t u r e . Transmission electron microscopy For electron microscopy, s a m p l e s of animals w e r e t a k e n after 91 days. Tissue blocks from the digestive g l a n d w e r e fixed in 2.5 % g l u t a r a l d e h y d e in p h o s p h a t e buffer (+ 2.5 % NaC1) at p H 7.4 for 2 h at 4 ~ C. The s a m p l e s w e r e r i n s e d in p h o s p h a t e - b u f f e r e d sucrose (1:3 sucrose 0.38 M) a n d post-fixed in 1 % osmium tetroxide in p h o s p h a t e buffer at p H 7.4 for 1 h. After d e h y d r a t i o n t h r o u g h an a s c e n d i n g ethanol series into p r o p y l e n e oxide, s p e c i m e n s w e r e e m b e d d e d in D u r c u p a n resin (ACM Fluka). Blocks w e r e cut on a n LKB ultramicrotome. Semithin sections w e r e s t a i n e d with toluidine blue a n d e x a m i n e d b y h g h t microscopy. Ultrathin sections w e r e collected on c o p p e r grids a n d s t a i n e d with uranyl acetate a n d l e a d citrate. O b s e r v a t i o n s w e r e m a d e in a JEOL (JEM 100 SX) electron microscope at 60 kV.

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Statistics Fiducial intervals were calculated using Student's t-test, and significant differences between means were established at p < 0 . 0 5 level (Sokal & Rob_if, 1979). One-way analysis of variance (ANOVA) was used to test significant differences between the sampling periods for each parameter (significance level p < 0.05). Correlation and regression analyses were carried out to establish significant relationships among the studied parameters. For these analyses the statistical package SPSS/PC+ (SPSS Inc., Microsoft Co.) was used in an AT personal computer (Atlas 286). Linear regression analyses were also performed to test the dependence of changes in GI with time.

RESULTS No mortality was recorded in one of the tanks during the whole experiment. However, mussels from the other aquarium started to die by day 65 in a massive fashion, and no animals survived by day 77. This mortality was accompanied by a cloudy appearance of the water at day 65 and was probably due to an accidental overuse of food affecting water quality. Crystalline styles were rarely lacking in animals sampled at any time through the assay (Fig. la). Microscopical observation of the style sac region showed that crystalline styles of different sizes were present in 71%, I00 %, 80 %, 63 %, i00 % and 80 % of the animals sampled after 0, 21, 35, 49, 77 and 91 days, respectively. There was no apparent relationship between style size and maintenance conditions. The total GI showed a continuous decrease over the experimental period (Fig. 2). GI values at the start of the experiment indicated that mussels were already spawning on March 29. Nevertheless, total GI values did not appear to reflect accurately the reproductive condition of the mussels. Consequently, GI was calculated separately for both males and females; and animals that could not be sexed (graded as 0) were added in equal shares to males and females, The general trend in female mussels was a lowering of the GI, with a mean GI value of 0 attained at the end of the experiment. A gradual decrease of GI was also observed in male mussels, although GI values of males were consistently higher than those of females. Regression analyses indicated a significant linear relationship between each total GI, female GI and male GI and time (Table l). The slopes of the three curves were very similar but the Y intercept value was higher in males than in females. Abnormal conditions, such as hemocytic infiltration of follicles containing mature gametes (Bayne et al., 1978), were very rarely observed (3 cases). Mussels in the spent or resting phase (predominant in the 77- and 91-day samples) always showed follicles empty of gametes and frequently filled with hemocytes and/or brown cells. Results of the planimetric analysis of the digestive gland tubules are given in Figure 3. The analysis of variance (Table 2) indicates that the five planimetric parameters differ significantly between samples. MET values increased steadily from day 0 to day 35 while values obtained in later samplings were markedly lower (Fig. 3a). Two different groups were also distinguished when MET/MDR values were considered (Fig. 3d). Similarly, mussels sampled until day 35 showed low values of MLRIMET while those sampled later showed nigh MLR/MET mean values (Fig. 3e). Digestive tubule size (MDR) increased from day 0 to day 35 and showed little further change (Fig. 3b). Digestive tubule lumen

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Fig. l a - d . Mytilus galloprov~ncialis. Light microscopy; cross-sections t h r o u g h the digestive gland, a: Stomach of a mussel sampled at day 0, after 10 days starvation, showing the crystalline style (asterisk); x 180. b: Digestive gland tubules of a 10-day starved mussel. Note that most tubules are of the reconstituting type (or type IV). S = sperm; x 95. c: General morphology of necrotic tubules (arrows) in a mussel sampled at day 35; x 190, d: Digestive gland tubules of a mussel sampled at day 35. Note that type I tubules (arrows) are relatively small, while type III tubules (asterisks) are relatively large; • 180

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Fig. 2. Values of the Gonad Index (GI) for females, males and for the total experimental population at the sampling periods size (MLR) also increased over the e x p e r i m e n t a l period (Fig. 3c), m a x i m u m values b e i n g o b t a i n e d on days 77 a n d 91. Data c o n c e r n i n g changes in the p e r c e n t a g e s of the different tubule types over the experimental period are shown in Figure 4 a n d Table 2. The p e r c e n t a g e of type I t u b u l e s showed some time-related changes, although not statistically significant (Fig. 4a, Table 2), a n d most of the tubules were at stages II a n d HI on every sampling day. No timed e p e n d e n t coherent trend was observed in the percentages of these two t u b u l e types (Figs 4b a n d 4c), although type II tubule p e r c e n t a g e was markedly decreased on day 91 correlating with a n increased p e r c e n t a g e of type III tubules. The p e r c e n t a g e of type IV tubules was very low on day 21 a n d gradually increased through the e x p e r i m e n t a l period (Fig. 4d). The p e r c e n t a g e of the latter type of tubule was high in the group of starved mussels (Fig. lb) where type I t u b u l e s were also relatively a b u n d a n t . Necrotic tubules (Fig. lc) were always a very low p e r c e n t a g e of the total tubule population a n d their quantity did not c h a n g e significantly during the assay (Fig. 4e, Table 2). The results of the stereological analysis of basophilic cells are given in Figure 5. The basophilic cell volume density (VD) of mussels m a i n t a i n e d without food was low a n d Table 1. Regression equations of female, male and total Gonad Index (GI) against time; t: time; r: correlation coefficient; P: signification of r GI~ = Glm = GIt =

-0.0202 t + 2.1673; r = -0.0250 t + 3.2497; r = -0.0226 t + 2.7085; r =

-0.8321 (P = 0.0399) -0.8838 (P = 0.0195) -0.7603 (P = 0.0041)

M. P. C a j a r a v i l l e , G. Dfez, I. A. M a r i g 6 m e z & E. A n g u l o

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Fig. 3a--e. Results of t h e ptanimetric analysis of the digestive gland tubules. Vertical segments are 95 % confidence intervals, a: MET; b: MDR; c: MLR; d: MET/MDR; e: MLR/MET

Histological consequences of keeping mussels in the laboratory so

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Fig. 4a-e. Results of the subjective tubule grading analysis of the digestive gland tubules, Vertical segments are 95 % confidence intervals, a: Type I tubules; b: Type II tubules; c: Type III tubules; d: type IV tubules; e: necrotic tubules

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M . P . C a j a r a v i l l e , G. Diez, I. A. M a r i g 6 m e z & E. A n g u l o

Table 2. One-way ANOVA to test significant differences b e t w e e n s a m p h n g periods. F: F ratio; P (F): probability of F; DFB: degrees of freedom b e t w e e n groups; DFW: degrees of freedom within groups Parameter

F

P (P)

DFB

DFW

MET MDR MLR MET/MDR MLR/MET

2.7457 4.7814 3.9419 2.9003 3.2114

0.0322" 0.0017' * 0.0055 * * 0.0255" 0.0160"

5 5 5 5 5

39 39 39 39 39

Type 1 % Type II % Type III % Type IV % Necrotic %

1.8624 5.7987 6.5740 2.6187 0.7193

0.1233 0.0004 * * 0.0002** 0.0391' 0.6129

5 5 5 5 5

39 39 39 39 39

Bas VD

8.1693