- Email: [email protected]
Use of an index based on the blue mussel (Mytilus edulis and Mytilus trossulus) digestive gland weight to assess the nutritional quality of mussel farm sites
Aquaculture 241 (2004) 633 – 654 www.elsevier.com/locate/aqua-online
Use of an index based on the blue mussel (Mytilus edulis and Mytilus trossulus) digestive gland weight to assess the nutritional quality of mussel farm sites S. Cartiera, J. Pellerina,*, M. Fournierb, E. Tamigneauxc, L. Giraultc, N. Lemairea a
Institut des Sciences de la Mer de Rimouski, Universite´ du Que´bec a` Rimouski, 310 alle´e des Ursulines, Rimouski, Que., Canada G5L 3A1 b INRS-Institut Armand Frappier, Universite´ du Que´bec, 245 Hymus, Pointe-Claire, Que., Canada H9R 3G6 c Centre colle´gial de transfert de technologie des peˆches, 167, la Grande-Alle´e Est, Grande-Rivie`re, Que., Canada G0C 1V0 Received 16 December 2003; received in revised form 21 July 2004; accepted 19 August 2004
Abstract An index based on the digestive gland (DGI) of transplanted (Gaspe´, Havre-St-Pierre and Grande-Rivie`re) and indigenous (Gaspe´ and Magdalen Islands) 1-year-old blue mussels (Mytilus edulis and Mytilus trossulus) from eastern Que´bec (Canada) was used to assess the nutritional quality of mussel farm sites. To understand variations of this index, the effects of temperature and phytoplankton concentrations were examined on growth, gametogenesis, and immunocompetence of transplanted (M. trossulus, Gould 1850) and cultivated mussels (M. trossulus and M. edulis, Linnaeus 1758) in Eastern Quebec mussel farm sites. DGI was influenced by the gametogenic cycle and was decreased before spawning with the utilization of the lipids and glycogen reserves stocked in the digestive gland. DGI patterns from all sites showed variations linked to food supply that contributed to the storage of energy reserves. Protein concentrations showed an inverse relationship with DGI, suggesting that structural proteins were not used as an energy source during spawning. Indigenous mussels from the Magdalen Islands site showed a stable DGI, suggesting both the
* Corresponding author. Tel.: +1 418 723 1986x1704; fax: +1 418 724 1842. E-mail address: [email protected] (J. Pellerin). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.08.015
634
S. Cartier et al. / Aquaculture 241 (2004) 633–654
utilization of energy reserves for reproduction and storage of energy reserves due to the abundant food supply in the Magdalen Islands. Transplanted mussels showed a clear seasonal pattern of the condition index closely related to the gametogenic cycle. Differences in soft tissue and shell growths were observed suggesting a nonsynchronous growth. In all sites, phytoplankton cells b2 Am were more abundant than the larger-size classes, and concentrations seemed sufficient for mussel growth. Phagocytosis varied according to gametogenesis and decreased during spawning. Due to the high energetic cost of spawning, which mainly influenced DGI, a direct influence of environmental parameters could not be clearly detected. D 2004 Elsevier B.V. All rights reserved. Keywords: Mytilus trossulus; Mytilus edulis; Digestive gland; Metabolic reserves; Immunocompetence; Aquaculture
1. Introduction Developing easy to use physiological indicators for mussel farmers that can give information about nutritional quality of their farm sites is quite challenging. If these indicators can detect effects of stress in mussels before the occurrence of mortality in the stocks, remediation could take place before the loss of production. Although mussel farm production in Eastern Quebec is increasing, severe problems of mortality are still reported every year. Mortality was observed generally in late summer in the Magdalen Islands (Que´bec) and was also reported on the Atlantic and Pacific coasts of North America (Incze et al., 1980; Emmett et al., 1987; Mallet et al., 1990). The explanation of massive mortality is not simple, and Tremblay et al. (1998) have suggested the possibility of a synergistic interaction between the lack of food, temperature, a postspawning stress, and the genetics of the stock. Many other factors can also affect mussel survival including salinity, light, pollutants, high densities, exposure to air, wave action, etc. (Harger, 1970; Bøhle, 1972; Seed, 1976; Pellerin-Massicotte, 1997). Food supply seems to be the principal factor which can limit production in suspended cultured systems (Navarro et al., 1991). It can also affect mussel survival during periods of metabolic stress like spawning (Incze et al., 1980). Freeman and Dickie (1979) and Worral and Widdows (1984) have observed higher mortality for large mussels, which is probably the result of an increase of postspawning stress. This increase is explained by the larger proportion of energy, which is invested into gamete production with increasing size (Worral and Widdows, 1984). Therefore, there is a necessity to develop tools for the aquaculture industry to understand the cause of such mortalities. Thompson et al. (1974), in a starvation experiment, observed a difference between the digestive gland index (DGI) of fed and starved mussels. The index was expressed as a ratio of dry digestive glands weight to total mantle-free dry weight. Due to the role in the storage of metabolic energy reserves, there are seasonal changes in the biochemical composition of bivalve digestive glands (Zandee et al., 1980; Barber and Blake, 1981). During physiological stress, like starvation or food shortage and/or high energy demand periods like gametogenesis, metabolic reserves stored in the digestive gland and other body tissues are mobilized for maintenance (Ansell, 1974; Zandee et al., 1980; Barber and Blake, 1981). Stress on the digestive gland will affect its biochemical composition due to
S. Cartier et al. / Aquaculture 241 (2004) 633–654
635
the utilization of the metabolic reserves during this period. Many physiological processes are closely linked and could act in synergy to further stress mussels faced with challenging conditions. It is now known that sexual maturation is closely linked with food availability in order to produce ripe gametes during gametogenesis (Seed and Suchanek, 1992). Moreover, modulation of immunocompetence by steroid hormones is recognised at least in fish, and measures of phagocytosis could be useful to understand the causes and consequences of stress in mussels (Watanuki et al., 2002). Phagocytic activity, used commonly as a biomarker for toxic exposure in the aquatic environment, is also influenced by environmental stress like temperature and salinity (Fisher and Tamplin, 1988; Chu and La Peyre, 1993; Cajaraville et al., 1996; Sauve´ et al., 2002; Luengen et al., 2004). Thus, coupling these two processes with the weight of the digestive gland could improve the assessment of food quality in a mussel farm. Finally, food quantity and quality are determining factors for growth rate and fecundity (Soniat and Ray, 1985; Fre´chette and Bourget, 1987). The food, or seston, is composed of a mixture of organic and inorganic particulate matter, which varies with temperature, water stratification, and hydrodynamics (Berg and Newell, 1986; Claereboudt et al. 1995; Abraham, 1998). Mussels can filter a variety of suspended particles like bacterioplankton, phytoplankton, organic detritus, microzooplankton, and mesozooplankton (Hawkins and Bayne, 1992; Lehane and Davenport, 2002). Phytoplankton and organically rich detrital particles generally are the main component of mussel nutrition (Bayne and Hawkins, 1992). Several studies have indicated that bivalve molluscs retain particles in the 2–20 Am range (Bayne and Newell, 1983). Cytometric studies have showed that mussels are particularly efficient at filtering particles in the 3–5 Am range (Cucci et al., 1989). This paper examined the effects of temperature and phytoplankton on growth, reproduction, and immunocompetence of transplanted (Mytilus trossulus, Gould 1850) and cultivated mussels (M. trossulus and Mytilus edulis, Linnaeus 1758) in eastern Quebec mussel farms. This study correlated the variations of the digestive gland weight, phagocytic activity, and sexual maturation stages with the food availability in each farming site studied.
2. Materials and methods 2.1. Site selection and sampling period One year old juvenile cultivated blue mussels (M. trossulus; 27 to 40 mm in shell length) were collected in May 2002 from a mussel farm located in Gaspe´ Bay (Quebec) and were transplanted to three farm sites: Havre-St-Pierre (50814VN 63836VW, north shore of the Gulf of St. Lawrence), Grande-Rivie`re (48824VN 64830VW, entrance of the Bay of Chaleur), and Gaspe´ (48846VN 64817VW, inside Gaspe´ Bay). Mussels from Gaspe´ Bay were chosen because of the good bacteriological quality of the site and lack of a toxic algal bloom in this area. One year old juvenile cultivated mussels (M. trossulus) from Gaspe´ Bay located at 5 m depth at the transplanted site and from the Magdalen Islands (M. edulis; 47825VN 61848VW, in Havre-aux-Maisons lagoon) were also sampled and surveyed throughout the experimental period. Experimental sites are illustrated in Fig. 1. Bivalve
636
S. Cartier et al. / Aquaculture 241 (2004) 633–654
.
Fig. 1. Location of the study area, Gulf of St. Lawrence, Canada, and experimental sites ( ).
translocation is commonly used to compare growth or effects of pollutant levels between sites (Skibinski and Roderick, 1989; Okumus and Stirling, 1998; Rome´o et al., 2003). Mussels were placed in three cages on each site. Cages were made of wood (1206030 cm) covered with VexarR diamond mesh sheets (HH in.). Cages were placed along submerged longlines (9- to 10-m depth) with a mean distance of 10 m between each cage. One thousand mussels were placed in each cage. Cages were cleaned at each sampling time to prevent biofouling. The experiment lasted 6 months (June to November 2002) and sampling occurred every 2 weeks. Fifty (50) mussels were randomly collected at each sampling date (N=25 for physiological analysis and N=25 for immunocompetence analysis). 2.2. Environmental factors Thermographs were installed at each site, at the same depth as the cages (9–10 m, except for Magdalen Islands lagoon, 4 m), and water temperature was measured three times per day (0800, 1600, and 2400 h). For statistical analysis, the mean of the three measurements was used. One water sample was collected at each sampling date with a 5-l Niskin bottle. Water was sampled at the same depth as the cages (9–10 m). Water was immediately transferred into a 1-l dark high-density polyethylene (HDPE) bottle, stored with ice in an insulated container and sent to the laboratory. Then, three 4.5-ml subsamples were taken from each plastic bottle. These subsamples were transferred to 5ml cryovials and fixed with 0.5 ml of a fresh paraformaldehyde solution (20% w/w, 0.2 Am prefiltered). The cryovials were frozen in liquid nitrogen and stored at 80 8C until further analysis of the samples. A Facsort (Becton-Dickinson), equipped with a 488-nm
S. Cartier et al. / Aquaculture 241 (2004) 633–654
637
laser, was used to determine phytoplankton concentrations, sizes of phytoplankton cells (b2–20 Am), and their fluorescence signature (Chlorophyll a and phycoerythrin), following the procedure of Mostajir et al. (2001). Sheath fluid used for the analysis was natural marine water prefiltered on a 0.22-Am membrane filter. Fluorescent microbeads of 2-, 10- and 20-Am diameter were mixed with 1.5 ml of each sample before analysis with the flow cytometer. The beads were used as an internal calibration standard for the determination of particle size. 2.3. Morphological measures, digestive gland, and condition indices determination For each sampling date and for each mussel, shell length was measured with a digital calliper. Total weights, as well as the weights of wet flesh and digestive gland, were recorded. Digestive gland and mantle samples were kept at 80 8C until analysis. The tissues were frozen in a HistobathR (ShandonR) containing cold acetone (50 8C) to preserve molecular and tissue integrity. A modified Thompson et al. (1974) digestive gland index was calculated as follows: DGI ¼
DG ST
DGI: digestive gland index; DG: wet digestive gland weight; ST: wet soft tissue weight. Also, a commercial condition index (modified from Hickman and Illingworth, 1980) was calculated as follows: CI ¼
ST ST þ SW
CI: condition index; ST: wet soft tissue weight; SW: shell weight. 2.4. Digestive gland metabolic reserves The analysis of the metabolic reserves was performed on 12 randomly sampled digestive glands at every sampling date and sites for transplanted mussels. Total proteins were determined according to the protein–Coomassie blue dye binding principle (Bradford, 1976) using bovine serum albumin (Sigma, fraction V) as the standard. Lipid concentration in the digestive gland was obtained by the colorimetric method based on the sulfo-phospho-vanillin reaction described by Frings et al. (1972) using olive oil as the standard. In both methods, the digestive gland was homogenized in 0.1 M phosphate buffer at pH 7. Glycogen concentration was estimated after an enzymatic digestion of the digestive gland homogenate (in a 0.1 M citrate buffer at pH 5) with amyloglucosidase and analysed according to the colorimetric method described by Carr and Neff (1984). Glycogen from oyster (Sigma, type III) was used as standard. All samples were analysed with a spectrophotometer (UV-VIS Beckman DU 640).
638
S. Cartier et al. / Aquaculture 241 (2004) 633–654
2.5. Sexual maturation stage determination Sections of 5 Am were done on mantle, frozen at 30 8C, cut with a CryotomeR 0620 (ThermoR ShandonR), then stained in a mix of methylene blue basic fuchsin. Sex and the maturation stages were identified with a light microscope (Leitz Ortholux II) according to Gauthier-Clerc et al. (2002). 2.6. Immunology 2.6.1. Collection of hemocytes and phagocytosis monitoring Hemocytes were collected every month in hemolymph, by puncture of the posterior adductor muscle, using 3-ml syringes and 23-G needles. Cells were washed by centrifugation with mussel’s hemolymph and adjusted to 1106 cells per milliliter of hemolymph. Hemocytes were mixed with yellow-green latex FluoSpheres (Molecular Probes, Eugene, OR, USA) at a ratio of 1:100 (hemocytes/beads). The cells were incubated, while being gently shaken, at 20 8C in the dark. After 18 h, an aliquot of 0.5 ml of each cell suspension was taken, layered over a 3% BSA gradient and centrifuged at 150g for 8 min at room temperature to remove free beads. The cell pellets were resuspended in 0.5 ml of 0.5% hematall solution (Fisher Scientific, Ottawa, Ont., Canada). 2.7. Flow cytometry acquisition A FACScan (Becton Dickinson, San Jose, CA, USA) with an air-cooled argon laser providing an excitation at 488 nm was used. Fluorescence emission was collected at 520 nm. Hemocyte populations were defined based on their forward and right angle scatter properties. A total of 10,000 events were acquired for each sample and stored in the list mode data format. The data were then analysed, once displayed as twoparameter complexity of the intracellular organelles development and cell size, in the process of gating and as fluorescence (FL1) frequency distribution histogram. Data collection and analysis were performed with an LYSIS-II program (Brousseau et al., 1998). 2.8. Statistical treatments A one-way repeated measures ANOVA was used to test significance of variations in phytoplankton cell abundance, digestive gland and condition indices, metabolic reserves, between each sampling date and the previous sampling. Normal distribution and homogeneity of variances were previously tested on data. Growth of soft tissue and shell length was tested using t-tests for paired samples. Digestive gland index and environmental factors were correlated using the Enter method regression. Because of the proximity between indigenous and transplanted stocks, same data for temperature and phytoplankton were used for regression. Pearson chi-square test was used to compare the partition of maturation stages between transplanted sites. One-way ANOVA
S. Cartier et al. / Aquaculture 241 (2004) 633–654
639
was used to test significance of variations of phagocytic activity followed by an All Pairwise Multiple Comparison (Tukey test) to identify differences among sampling periods. Data of phagocytosis of one bead and more were compared. Same comparisons were made for phagocytosis of three beads and more. For all statistical tests, we have used individual mussels as replicates, and results were considered significant with a probability ( p) value of b0.05. All data were statistically analyzed with SPSS 9.0 for Windows.
3. Results 3.1. Temperature During the sampling period in Gaspe´, temperature increased from 4 to 14.5 8C during August, and then decreased to near 2 8C in November. At the Havre-St-Pierre site, three peaks of temperature varying between 7 and 12 8C were observed: in July, mid-August, and end of September. In Grande-Rivie`re, the temperature pattern was similar to Gaspe´. In Magdalen Islands, temperature was near 12 8C at the beginning of sampling, reached 23 8C in mid-August, and decreased to near 4 8C in November. 3.2. Phytoplankton abundance At the Gaspe´ site (Fig. 2A), a first bloom of 10–20 Am cells (significant increase, pb0.05) was observed during the end of June. During the following months, three successive blooms of cells smaller than 2 Am were observed (three significant increases, pb0.05). In Havre-St-Pierre (Fig. 2B), the sampling was dominated by cells smaller than 2 Am. Three blooms of this size class of cells were observed at the end of June, during midAugust, and at the end of the sampling period (three significant increases, pb0.05). In Grande-Rivie`re (Fig. 2C), cells smaller than 2 Am were also predominant during all samplings. Three blooms were observed: end of May, end of July, and end of October (three significant increases, pb0.05). A bloom of 2–10 Am cells was observed in mid-July (significant increase, pb0.05). At the Magdalen Islands site (Fig. 2D), two blooms dominated by cells smaller than 2 Am were observed: at mid-August and the end of September (two significant increases, pb0.05). Number of phytoplankton cells was higher in Magdalen Islands in comparison with the transplanted sites. In transplanted sites, number of phytoplankton cells was lower in the Gaspe´ site in comparison with Havre-StPierre and Grande-Rivie`re sites. 3.3. Variation of wet soft tissue weight and shell length Variations in meat yield for all sites, expressed in wet weight, are illustrated in Fig. 3. Gaspe´ site showed a slow growth during the sampling period, but growth was not statistically significant. However, growth was statistically significant for cultivated mussels from Gaspe´ ( pb0.05) and Magdalen Islands ( pb0.05) and also transplanted stocks from Gaspe´ to Grande-Rivie`re ( pb0.05) and Havre-St-Pierre sites ( pb0.05). Shell
640
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fig. 2. Variations of the abundance of three classes of phytoplankton cells. Samplings were done from May to November 2002 in four sites: Gaspe´ (A), Havre-St-Pierre (B), Magdalen Islands (C), and Grande-Rivie`re (D).
growth was statistically significant in mussels from Havre-St-Pierre transplanted stock ( pb0.05) and both cultivated mussel stocks ( pb0.05; Fig. 4). 3.4. Proteins A similar seasonal pattern was observed in protein concentrations in the digestive gland from the three sites (Fig. 5A). The pattern was characterized by a decrease in late spring ( pb0.05), then the protein concentrations recovered and remained relatively stable for the rest of the sampling period. Significant increase in protein concentrations ( pb0.05) began in July in Havre-St-Pierre, later than the two other sites. 3.5. Total lipids Lipid concentrations in the digestive gland for the three sites (Fig. 5B) showed a seasonal pattern characterized by a significant increase ( pb0.05) in spring/summer than
S. Cartier et al. / Aquaculture 241 (2004) 633–654
641
Fig. 3. Variations of wet soft tissue weight from mussels in experimental sites (N=25) from May to November 2002. Graph (A) illustrates variations in transplanted mussels from Gaspe´ to Gaspe´, Havre-St-Pierre, and GrandeRivie`re. Graph (B) illustrates variations in indigenous cultivated mussels from Gaspe´ and Magdalen Islands. The symbol d*T indicates a significant difference between first and last sampling dates within sites.
followed by a significant decrease ( pb0.05). Grande-Rivie`re showed greater lipid storage during summer than the other sites. Utilization of lipids was slower at the Gaspe´ site. Lipids recovered during the fall with concentrations similar for the three sites. 3.6. Glycogen Glycogen concentration in the digestive gland for the three sites is illustrated in Fig. 5C. Glycogen concentrations showed a similar seasonal pattern with lipid concentrations for Havre-St-Pierre and Grande-Rivie`re, characterized by a significant increase in summer ( pb0.05) followed by a significant decrease ( pb0.05). Higher values were observed during summer in Havre-St-Pierre and Grande-Rivie`re sites. In the Gaspe´ site, the seasonal pattern was not evident. Glycogen concentrations were more stable during the sampling period, but a significant increase ( pb0.05) was observed during late fall for Gaspe´ and Grande-Rivie`re.
642
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fig. 4. Variations of shell length from mussels in experimental sites (N=25) from May to November 2002. Graph (A) illustrates variations in transplanted mussels from Gaspe´ to Gaspe´, Havre-St-Pierre, and Grande-Rivie`re. Graph (B) illustrates variations in indigenous cultivated mussels from Gaspe´ and Magdalen Islands. The symbol d*T indicates a significant difference between first and last sampling dates within sites.
3.7. Digestive gland and condition indices The variations of the digestive gland index (DGI) at the three transplanted sites (Fig. 6A) showed a similar pattern characterized by the highest value during May–June. Then, a significant decrease ( pb0.05) was observed at all three sites. DGI decreased earlier in Havre-St-Pierre. Maximal DGI value (0.235F0.079) in transplanted sites was observed in Grande-Rivie`re. Cultivated mussel DGI variations are illustrated in Fig. 6C. In commercial mussels from Gaspe´, a similar DGI pattern with Gaspe´-transplanted mussels was observed with a recovery of the DGI during September. In Magdalen Islands, DGI showed a significant decrease ( pb0.05) during summer, then a recovery was observed during fall. Transplanted mussels (Fig. 6B) showed a significant decrease of the condition index during July ( pb0.05). Cultivated mussels (Fig. 6D) from Gaspe´ showed a stable condition index through the sampling period, with a decrease during August/September, but the index
S. Cartier et al. / Aquaculture 241 (2004) 633–654
643
Fig. 5. Variations of proteins (A), lipid (B), and glycogen (C) concentrations in transplanted mussels’ digestive gland (N=12) from May to November 2002.
recovered during the fall. In Magdalen Islands, the condition index significantly decreased ( pb0.05) during July, then a significant increase ( pb0.05) during fall was observed. 3.8. Sexual maturation stages Sexual maturation (Fig. 7) was expressed in percentage of partitioning between stages. Spawning for male mussels in Gaspe´ was observed in mid-July and from September to November, while in females, spawning was continuous from mid-July to November. In
644
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fig. 6. Variations of the digestive gland and condition indices of transplanted mussels and indigenous cultivated mussels (N=25) from May to November 2002. Graphs (A) and (B) illustrate variations for DGI and condition index of transplanted mussels. Graphs (C) and (D) illustrate variations for DGI and condition index of indigenous cultivated mussels.
Havre-St-Pierre, the spawning period was similar for both sexes from mid-July to November. In Grande-Rivie`re, the spawning period was continuous from July to November for males, and from July to the end of October for females. Statistical analyses showed significantly ( pb0.05) different sexual maturation patterns among Gaspe´, HavreSt-Pierre, and Grande-Rivie`re sites for males and females. 3.9. Phagocytic activities Phagocytic activities (Fig. 8) in Gaspe´ were similar during June and July. During August, activities increased ( pb0.05) then decreased ( pb0.05) at the end of the sampling period. Transplanted mussels in Havre-St-Pierre and indigenous mussels from Gaspe´
S. Cartier et al. / Aquaculture 241 (2004) 633–654
645
Fig. 7. Gametogenesis was assessed by comparing partition of maturation stages of transplanted mussels (N=12). Stages were defined as described in Gauthier-Clerc et al. (2002). Partitioning is expressed in percent between each observed development stage from May to November 2002.
showed a similar pattern with transplanted mussels in Gaspe´. Phagocytic activities in Grande-Rivie`re showed a decline ( pb0.05) between June and July. Phagocytic activities then increased ( pb0.05) between July and August, with a decrease ( pb0.05) in the fall.
646
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fig. 8. Phagocytic activity of hemocytes from mussels’ hemolymph (N=15) in experimental sites [transplanted mussels in Gaspe´ (A), Havre-St-Pierre (B), Grande-Rivie`re (C), and indigenous cultivated mussels from Magdalen Islands (D) and Gaspe´ (E)] from May to November 2002. Data were divided into two categories. dOne bead and moreT represents hemocytes with phagocytosis of at least one bead. dThree beads and moreT represents more efficient hemocytes which engulfed three beads or more. Small letters indicate significant differences between months.
S. Cartier et al. / Aquaculture 241 (2004) 633–654
647
Table 1 Coefficients (b) of predictors and R 2 of the regression equations explaining DGI variations Sites
Temperature
b2 Am
2–10 Am
10–20 Am
R2
Gaspe´ Havre-St-Pierre Grande-Rivie`re Magdalen Islands (I) Gaspe´ (I)
0.622*** 0.030 0.308*** 0.347*** 0.590***
0.209*** 0.198* 0.113* 0.391** 0.190***
0.096 0.357*** 0.018 0.805** 0.157**
0.202*** 0.116 0.425*** 0.552** 0.238***
0.464*** 0.084*** 0.315*** 0.115*** 0.438***
(I)Eindigenous mussels (cultivated mussels followed simultaneously as transplanted mussels). * pb0.05. ** pb0.01. *** pb0.001.
This pattern was also evident at the Magdalen Islands site, which showed a similar pattern with the Grande-Rivie`re site. 3.10. Digestive gland index versus environmental parameters Regression equations showed that temperature, cells smaller than to 2 Am, and cells comprised between 10 and 20 Am were the factors which explained DGI variability in most of the experimental sites (Table 1). Environmental factors in Gaspe´ (transplanted and indigenous mussels) and Grande-Rivie`re explained more of the variability of the DGI with values of R 2 of 46.4%, 43.8%, and 31.5%, respectively. Gaspe´- and Grande-Rivie`retransplanted mussel DGIs were mainly influenced by temperature, cells smaller than 2 Am, and cells comprised between 10 and 20 Am. Havre-St-Pierre-transplanted mussel DGI was influenced only by cell size, especially cells smaller than 2 Am and cells comprised between 2 and 10 Am. Gaspe´ and Magdalen Islands indigenous mussel DGIs were influenced by temperature and all sizes of cells equally.
4. Discussion It is now well known that the digestive gland of Mytilus spp. plays three major roles. It is a site of intracellular digestion and carbon assimilation. Also, it is a storage site for metabolic reserves used during high metabolic and stress periods. Finally, the digestive gland serves as a site of transfer for metabolic reserves to other organs. In the field, cultured mussels can be stressed by many factors, such as high temperature and/or diminished food supply. Thompson et al. (1974) observed an effect of warm temperature and starvation stress on the digestive gland index in mussels kept in laboratory conditions. The digestive gland index decreased in stressful conditions, but starvation seems to affect the index on a longer period than temperature. It is well known that gametogenesis in bivalves is associated with the energy storage cycle of glycogen and lipids (Gabbott, 1976). In this study, environmental parameters (temperature, phytoplankton) explained partly the variability of the digestive gland index. The index seems to vary with other parameters. Even if the digestive gland index used in this study was a slight modification of the one used by Thompson et al. (1974), we have obtained a similar seasonal pattern.
648
S. Cartier et al. / Aquaculture 241 (2004) 633–654
This pattern is characterized by an increase of the index just prior to spawning. In the three sites, spawning occurred in July, and peaks of the index were observed in May–June. This pattern was also observed with the sea scallop Placopecten magellanicus digestive gland weight in Newfoundland (Canada) (Penney and McKenzie, 1996). Therefore, the digestive gland index seems to be sensitive to gametogenesis and to the energy storage cycle. Variations of the index were similar to those of glycogen and lipid concentrations in the digestive gland. Gametogenesis in marine bivalves requires a lot of energy. This energy can be supplied by food, metabolic reserves, or a combination of both (Bayne, 1976a). Metabolic reserves are stored during periods of abundant food supply. Newell et al. (1982) have observed spawning in mussels prior to or during periods of maximum food availability. Our results support those of Newell et al. (1982). By spawning during high food abundance, it allows mussels to have sufficient nutrients to accumulate reserves during the postspawning period and to allow ample food for larvae (Bayne, 1976a; Gabbott, 1976; Starr et al., 1990; Seed and Suchanek, 1992). This storage after spawning can be clearly observed in Havre-StPierre and Grande-Rivie`re sites (Fig. 5). Therefore, high concentrations of glycogen and lipids stored in the digestive gland before spawning suggest abundant food in the environment. Lipid and glycogen reserves are used for the formation of gametes (Gabbott, 1976; Lubet et al., 1976). Lipid levels and DGI were high before spawning. Similar results were reported in months preceding spawning in a population of M. edulis from eastern Newfoundland, Canada (Thompson, 1984). A loss of lipids stored in the digestive gland before spawning was also observed in other bivalves like bay scallops (Argopecten irridians concentricus; Barber and Blake, 1981). Whole body lipids of the flat oyster (Ostrea edulis) and pacific oyster (Crassosstrea gigas) were also maximal before spawning (Ruiz et al., 1992; Kang et al., 2000). Okumus and Stirling (1998) have observed in M. edulis a sharp decline of whole body lipids in April and minimum condition index values in March–April, which coincided with spawning in sea lochs on the west coast of Scotland. In this study, condition index for transplanted mussels decreased during the spawning period. A loss of proteins in the digestive gland before spawning was also observed in mussels from the three sites. This decrease is, in part, explained by the utilization of protein to formulate oocytes because protein is the main constituent of eggs, followed by lipids and glycogen (Holland, 1978). Proteins can also serve as an energy source during gametogenesis (Mann and Glomb, 1978; Adachi, 1979; Barber and Blake, 1981); but in this study, protein concentrations followed an inverse pattern with the DGI, which could indicate that mussels had sufficient energy reserves for reproduction and did not need structural proteins to achieve gametogenesis. Mussels transplanted to GrandeRivie`re showed soft tissue growth and highest values in term of lipids and glycogen and the highest digestive gland index value. Among the transplanted sites, the number of phytoplankton cells, all classes included, was highest in Grande-Rivie`re. All these facts suggest that this site was possibly more productive than the other transplanted sites. Transplanted and indigenous mussels from Gaspe´ showed similar values of digestive gland index with Havre-St-Pierre. However, no soft tissue growth was observed in Gaspe´transplanted mussels in contrast with Gaspe´ indigenous mussels and Havre-St-Pierretransplanted mussels. This suggests an intrasite variability in terms of somatic growth. More phytoplankton cells were observed in Havre-St-Pierre than in Gaspe´, explaining in
S. Cartier et al. / Aquaculture 241 (2004) 633–654
649
part soft tissue growth. In this zone, which is a frontal region, cold waters are observed due to tidal mixing (Pingree and Griffiths, 1980). This mixing contributes to nutrient replenishment in the water column. This phenomenon was observed in this study, and it could explain the three blooms observed at this site. In Magdalen Islands, amount of phytoplankton cells was considerably higher than in all other sites with a predominance of cells smaller than 2 Am. Roy et al. (1991) have observed, in summer 1987, that small particles (2–20 Am) were more abundant in the Great Entry Lagoon (Magdalen Islands), with a second population of larger cells (50 Am) appearing in early August. Tremblay et al. (1998) also observed an increase in the unit volume of seston during August. In this study, decrease of wet soft tissue weight in Magdalen Islands was observed during August, when temperature was near 20 8C, which is considered as a stressing temperature (Incze et al., 1980). Even if larger particles (larger than 20 Am) could be possibly more abundant during this period, it was not enough to limit somatic growth. Furthermore, this site possesses a sufficient amount of food to maintain the DGI during spawning, which usually occurs in late June (Myrand, 1991). A second spawning can also be observed in late July (Myrand and Gaudreault, 1995). These mussels have possibly invested more energy in somatic growth than in reproduction. Fecundity in mussels can vary annually, suggesting an adjustment of the energy allocated to reproduction as a function of food availability (Seed and Suchanek, 1992). Our results also showed different growth patterns between soft tissues and the shell. Transplanted mussels at the Havre-St-Pierre site and cultivated mussels from Gaspe´ and Magdalen Islands sites showed both significant soft tissue and shell growth. Soft tissue growth was observed for transplanted mussels at Gaspe´ and Grande-Rivie`re sites, but no shell growth was detected statistically. These results support the fact that somatic and shell growth are not necessarily synchronous (Rodhouse et al., 1984; Hilbish, 1986). AlunnoBruscia et al. (2001) showed in a food-regulated experiment that shell growth continued, even if food was scarce, while soft tissue was affected negatively. Thus, in an aquaculture context, it is risky to assess meat yield by observations based on shell length. Even if in all sites small cells (smaller than 2 Am) were more abundant, somatic growth was not compromised in most of the sites. Even if larger cells (over 20 Am) were not considered in this study, it is well known that in Gulf of St. Lawrence and the Baie des Chaleurs, which present oligotrophic conditions, picoplanktonic cells (smaller than 5 Am) are more abundant (Legendre and Le Fe`vre, 1991; Tamigneaux et al., 1995). The transplanted sites showed mussels with a different sexual maturation pattern. This could be explained by the effect of environmental parameters. An increase of temperature above 10–12 8C is considered essential to initiate the release of gametes (Bayne, 1976b; Newell et al., 1991). In Havre-St-Pierre, water mixing was observed and could cause many spawning events during a longer period. In this study, somatic growth was detected even if spawning was observed throughout summer. In Gaspe´, where females spawned from August to November, somatic growth was not detected statistically and could have been negatively influenced by spawning. Reproduction at a site with less nutrients, could affect somatic growth and, on a larger scale, the farm production. A seasonal pattern was also noted for phagocytic activities by mussel hemocytes. This decrease was particularly evident in the Grande-Rivie`re and Magdalen Islands. Because it was occurring during spawning, this decline can possibly be driven by
650
S. Cartier et al. / Aquaculture 241 (2004) 633–654
hormonal regulation. Sex steroid hormones are known to modulate the immune system (Berczi and Nagy, 1998). In mussels, Reis-Henriques et al. (1990) have observed an increase in progesterone followed by an increase of 17h-estradiol and estrone during advanced stages of sexual development. This suggests the implication of these hormones in sexual maturation regulation. Nevertheless, in Crassostrea gigas, recent studies have shown increased phagocytic activity with gonadal development. Similar to our results, they have also shown a decrease at the spawning stage and a recovery of phagocytosis until the decrease in water temperature (Ishikawa et al., 1998). Watanuki et al. (2002) have shown that sex steroids like h-estradiol, progesterone, and 11ketotestosterone exert suppressive effects on phagocytic cells of carp head kidney in vivo. Thus, the increase during spawning of sex steroids in mussels could possibly have a suppressive effect on the phagocytic activity of hemocytes. This period of reduced phagocytic activity could possibly increase disease susceptibility. It may be important to study which mechanisms are involved in these regulatory events. Further investigation should therefore be done in vitro and in vivo. After spawning, immunocompetence recovered to similar or higher values observed at prespawning levels. Restoration of phagocytic competence, in addition to increasing immune protection, may also be involved in the process of gamete resorption. Hemocytes possibly remove the moribund cells by phagocytosis and digest these cells with lysosomal hydrolases (Moore and Lowe, 1977; Suresh and Mohandas, 1990). Finally, in fall, there is a second decrease of phagocytosis, and it could be associated with another spawning event. Decrease in temperature, which also initiated the down-regulation of metabolic activities associated with hibernation, could influence phagocytosis. Carballal et al. (1997) have observed, in an in vitro study, a depression of phagocytic activities of Mytilus galloprovincialis hemocytes at 10 8C. In summary, for the first time to our knowledge, we were able to correlate reproduction and immunocompetence in M. edulis and M. trossulus. Also, seasonal variations in energy reserves were linked to gametogenesis and spawning targeted as a main physiological event that could be stressful in sites with low nutritional levels. Variations of the DGI during the summer–fall period for all sites showed a close relationship with gametogenesis. Effects of the environmental parameters were detected but did not explain entirely the DGI variations. However, different DGI patterns were observed in transplanted and cultivated mussels suggesting different energy allocation influenced by environmental parameters in an indirect way. Phagocytic activities were also affected by reproduction. According to these results, the DGI was too sensitive to gametogenesis and metabolic reserves cycle to be used to assess nutritional quality of farm sites. However, protein reserves have been shown to follow growth closely. Therefore, it appears that a simple measure of proteins in the digestive gland could be sufficient to achieve our goals and the needs of producers.
Acknowledgements Financial support was provided by the Conseil des Recherches en Peˆches et en Agroalimentaire du Que´bec (CORPAQ), the Natural Sciences and Engineering Research
S. Cartier et al. / Aquaculture 241 (2004) 633–654
651
Council of Canada (NSERC) through the Discovery grant of Jocelyne Pellerin, the Fondation de l’UQAR, ISMER and Que´bec-Oce´an. The authors also wish to thank He´le`ne Doucet-Beaupre´, Pascal Rioux, Ste´phanie Poirault, Marle`ne Fortier, Francesca Proulx, Ian Beaudin, and Vale´rie McInnis for helpful technical assistance. The authors are also grateful to the mussel farmers involved in this study.
References Abraham, E.R., 1998. The generation of plankton patchiness by turbulent stirring. Nature 391, 577 – 580. Adachi, K., 1979. Seasonal changes of the protein level in the adductor muscle of the clam, Tapes philippinarum (Adams and Reeve) with reference to the reproductive seasons. Comp. Biochem. Physiol. 64A, 85 – 89. Alunno-Bruscia, M., Bourget, E., Fre´chette, M., 2001. Shell allometry and length–mass–density relationship for Mytilus edulis in an experimental food-regulated situation. Mar. Ecol., Prog. Ser. 219, 177 – 188. Ansell, A.D., 1974. Seasonal changes in biochemical composition of the bivalve Chlamys septemradiata from the Clyde Sea area. Mar. Biol. 25, 85 – 99. Barber, B.J., Blake, N.J., 1981. Energy storage and utilization in relation to gametogenesis in Argopecten irridians concentricus (Say). J. Exp. Mar. Biol. Ecol. 52, 121 – 134. Bayne, B.L., 1976a. Aspects of reproduction in bivalve molluscs. In: Wiley, M. (Ed.), Estuarine Processes, Uses, Stresses, and Adaptation to the Estuary, vol. 1. Academic Press, New York, NY, pp. 432 – 448. Bayne, B.L., 1976b. The biology of mussel larvae. In: Bayne, B.L. (Ed.), Marine Mussels: Their Ecology and Physiology. Cambridge University Press, New York, NY, pp. 81 – 120. Bayne, B.L., Hawkins, A.J.S., 1992. Ecological and physiological aspects of herbivory in benthic suspensionfeeding molluscs. In: John, D.M., Hawkins, S.J., Price, J.H. (Eds.), Plant–Animal Interaction in the Marine Benthos. Oxford University Press, Toronto, pp. 265 – 287. Bayne, B.L., Newell, R.C., 1983. Physiological energetics of marine molluscs. In: Saleuddin, A.S.M., Wilbur, K.M. (Eds.), The Mollusca, Physiology, Part 1 vol. 4. Academic Press, New York, NY, pp. 407 – 515. Berczi, I., Nagy, E., 1998. Hormones as immune modulating agents. In: Kresina, T.F. (Ed.), Immune Modulating Agents. Marcel Dekker, New York, NY, pp. 75 – 120. Berg, J.A., Newell, R.I.E., 1986. Temporal and spatial variation in the composition of seston available to the suspension feeder Crassostrea virginica. Estuar. Coast. Shelf Sci. 23, 375 – 386. Bbhle, B., 1972. Effects of adaptation to reduced salinity on filtration activity and growth of mussels (Mytilus edulis L.). J. Exp. Mar. Biol. Ecol. 10, 41 – 47. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilising the principle of protein dye binding. Anal. Biochem. 72, 248 – 254. Brousseau, P., Payette, Y., Blakley, B., Boermans, H., Flipo, D., Tryphonas, H., Fournier, M., 1998. Manual of Immunological Methods. CRC Press, Boston, USA. 141 pp. Cajaraville, M.P., Olabarrieta, I., Marigomez, I., 1996. In vitro activities in mussels hemocytes as biomarkers of environmental quality: a case study in the Abra estuary (Biscay Bay). Ecotoxicol. Environ. Saf. 35, 253 – 260. Carballal, M.J., Lo´pez, C., Azevedo, C., Villalba, A., 1997. In vitro study of phagocytic ability of Mytilus galloprovincialis Lmk. haemocytes. Fish Shellfish Immunol. 7, 403 – 416. Carr, R.S., Neff, J.M., 1984. Quantitative semi-automated enzymatic assay for tissue glycogen. Comp. Biochem. Physiol. 77B, 447 – 449. Chu, F.-L.E., La Peyre, J.F., 1993. Perkinsus marinus susceptibility and defense-related activities in eastern oysters Crassostrea virginica: temperature effects. Dis. Aquat. Org. 16, 223 – 234. Claereboudt, M.R., Coˆte´, J., Bonardelli, J.C., Himmelman, J.H., 1995. Seasonal variation in abundance and size structure of phytoplankton in Baie des Chaleurs, southwestern Gulf of St. Lawrence, in relation to physical oceanographic conditions. Hydrobiologia 306, 147 – 157. Cucci, T.L., Shumway, S.E., Brown, W.S., Newell, C.R., 1989. Using phytoplankton and flow cytometry to analyze grazing by marine organisms. Cytometry 10, 659 – 669. Emmett, B., Thompson, K., Popham, J.D., 1987. The reproductive and energy storage cycles of two populations of Mytilus edulis (Linne) from British Columbia. J. Shellfish Res. 6, 29 – 36.
652
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fisher, S.W., Tamplin, M., 1988. Environmental influence on activities and foreign-particles binding by hemocytes of American oysters, Crassostrea virginica. Can. J. Fish. Aquat. Sci. 45, 1309 – 1315. Fre´chette, M., Bourget, E., 1987. Significance of small-scale spatio-temporal heterogeneity in phytoplankton abundance for energy flow in Mytilus edulis. Mar. Biol. 94, 231 – 240. Freeman, K.R., Dickie, L.M., 1979. Growth and mortality of the blue mussel (Mytilus edulis) in relation to environmental indexing. J. Fish. Res. Board Can. 36, 1238 – 1249. Frings, C.S., Fendley, T.W., Dunn, R.T., Queen, C.A., 1972. Improved determination of total serum lipids by the sulfo-phospho-vanillin reaction. Clin. Chem. 18, 673 – 674. Gabbott, P.A., 1976. Energy metabolism. In: Bayne, B.L. (Ed.), Marine Mussels: Their Ecology and Physiology. Cambridge University Press, New York, NY, pp. 293 – 355. Gauthier-Clerc, S., Pellerin, J., Blaise, C., Gagne´, F., 2002. Delayed gametogenesis of Mya arenaria in the Saguenay fjord (Canada): a consequence of endocrine disruptors? Comp. Biochem. Physiol. 131C, 457 – 467. Harger, J.R., 1970. The effect of wave impact on some aspects of the biology of sea mussels. Veliger 12, 401 – 414. Hawkins, A.J.S., Bayne, B.L., 1992. Physiological interrelations, and the regulation of production. In: Gosling, E. (Ed.), The Mussel Mytilus: Ecology, Physiology, Genetics and Culture. Elsevier, New York, NY, pp. 171 – 222. Hickman, R.W., Illingworth, J., 1980. Condition cycle of the green-lipped mussel Perna canaliculus in New Zealand. Mar. Biol. 60, 27 – 38. Hilbish, T.J., 1986. Growth trajectories of shell and soft tissue in bivalves: seasonal variation in Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 96, 103 – 113. Holland, D.L., 1978. Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. In: Malins, D.C., Sargent, J.R. (Eds.), Biochemical and Biophysical Perspectives in Marine Biology. Academic Press, London, pp. 85 – 123. Incze, L.S., Lutz, R.A., Watling, L., 1980. Relationships between effects of environmental temperature and seston on growth and mortality of Mytilus edulis in a temperate northern estuary. Mar. Biol. 57, 147 – 156. Ishikawa, H., Takahashi, K., Osada, M., Matsutani, T., Mori, K., 1998. Effect of reproductive cycle on seasonal variation of phagocytosing activity in the hemocytes of Crassostrea gigas. Dev. Comp. Immunol. 22, 132 – 133. Kang, C.-K., Park, M.S., Lee, P.-Y., Choi, W.-J., Lee, W.-C., 2000. Seasonal variations in condition, reproduction activity, and biochemical composition of the pacific oyster, Crassostrea gigas (Thunberg), in suspended culture in two coastal bays of Korea. J. Shellfish Res. 19, 771 – 778. Legendre, L., Le Fe`vre, J., 1991. From individual plankton cells to pelagic marine ecosystems and to global biogeochemical cycles. In: Demers, S. (Ed.), Particles Analysis in Oceanography. Springer-Verlag, New York, NY, pp. 260 – 299. Lehane, C., Davenport, J., 2002. Ingestion of mesozooplankton by three species of bivalve: Mytilus edulis, Cerastoderma edule and Aequipecten opercularis. J. Mar. Biol. Assoc. U.K. 82, 615 – 619. Lubet, P., Herlin, P., Mathieu, M., Collin, F., 1976. Tissu de re´serve et cycle sexuel chez les lamellibranches. Haliotis 7, 59 – 62. Luengen, A.C., Friedman, C.S., Raimondi, P.T., Flegal, A.R., 2004. Evaluation of mussel immune responses as indicators of contamination in San Francisco Bay. Mar. Environ. Res. 57, 197 – 212. Mallet, A.L., Carver, C.E.A., Freeman, K.R., 1990. Summer mortality of the blue mussel in eastern Canada: spatial, temporal, stock and age variation. Mar. Ecol. Prog. Ser. 67, 35 – 41. Mann, R., Glomb, S.J., 1978. The effect of temperature on growth and ammonia excretion of the manila clam Tapes japonica. Estuar. Coast. Mar. Sci. 6, 335 – 339. Moore, M.N., Lowe, D.M., 1977. The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles. J. Invertebr. Pathol. 29, 18 – 30. Mostajir, B., Gosselin, M., Gratton, Y., Booth, B., Vasseur, C., Garneau, M.-E., Fouilland, E., Vidussi, F., Demers, S., 2001. Surface water distribution of pico- and nanophytoplankton in relation to two distinctive water masses in the North Water, northern Baffin Bay, during fall. Aquat. Microb. Ecol. 23, 205 – 212. Myrand, B., 1991. Conditions environnementales dans les lagunes des Iˆles-de-la-Madeleine et parame`tres biologiques de la moule bleue, Atelier de travail sur la mortalite´ estivale des moules aux Iˆles-de-la-Madeleine. MAPA-Peˆcheries, Iˆles-de-la-Madeleine, Que´bec, pp. 1 – 40.
S. Cartier et al. / Aquaculture 241 (2004) 633–654
653
Myrand, B., Gaudreault, J., 1995. Summer mortality of blue mussels (Mytilus edulis, Linnaeus 1758) in the Magdalen Islands (southern Gulf of St. Lawrence, Canada). J. Shellfish Res. 14, 395 – 404. Navarro, E., Iglesias, J.I.P., Camacho, A.P., Labarta, U., Beiras, R., 1991. The physiological energetics of mussels (Mytilus galloprovincialis Lmk) from different cultivation rafts in the Ria de Arosa (Gallicia, N.W. Spain). Aquaculture 94, 197 – 212. Newell, R.I.E., Hilbish, T.J., Koehn, R.K., Newell, C.J., 1982. Temporal variation in the reproductive cycle of Mytilus edulis L. (Bivalvia, Mytilidae) from localities on the east coast of the United States. Biol. Bull. 162, 299 – 310. Newell, C.R., Hidu, H., McAlice, B.J., Podniesinski, G., Short, F., Kindblom, L., 1991. Recruitment and commercial seed procurement of the blue mussel Mytilus edulis in Maine. J. World Aquac. Soc. 22, 134 – 152. Okumus, I., Stirling, H.P., 1998. Seasonal variations in the meat weight, condition index and biochemical composition of mussels (Mytilus edulis L.) in suspended culture in two Scottish sea lochs. Aquaculture 159, 249 – 261. Pellerin-Massicotte, J., 1997. Influence of elevated temperature and air-exposure on MDA levels and catalase activities in digestive glands of the blue mussel (Mytilus edulis L.). J. Rech. Oce´anogr. 22, 91 – 98. Penney, R.W., McKenzie, C.H., 1996. Seasonal changes in the body organs of cultured sea scallop, Placopecten magellanicus, and coincidence of spawning with water temperature, seston, and phytoplankton community dynamics. Can. Tech. Rep. Fish. Aquat. Sci., 2104. 22 pp. Pingree, R.D., Griffiths, D.K., 1980. A numerical model of the M2 tide in the Gulf of St. Lawrence. Oceanol. Acta 3, 221 – 225. Reis-Henriques, M.A., Le Guellec, D., Remy-Martin, J.P., Adessi, G.L., 1990. Studies of endogenous steroids from the marine mollusc Mytilus edulis L. by gas chromatography and mass spectrometry. Comp. Biochem. Physiol. 95B, 303 – 309. Rodhouse, P.G., Roden, C.M., Hensey, M.P., Ryan, T.H., 1984. Resource allocation in Mytilus edulis on the shore and in suspended culture. Mar. Biol. 84, 27 – 34. Rome´o, M., Hoarau, P., Garello, G., Gnassia-Barelli, M., Girard, J.P., 2003. Mussel transplantation and biomarkers as useful tools for assessing water quality in the NW Mediterranean. Environ. Pollut. 122, 369 – 378. Roy, S., Mayzaud, P., Souchu, P., 1991. Environnement physico–chimique et trophique d’un site mytilicole aux ˆIles-de-la-Madeleine (Que´bec): II—Matie`re particulaire, composition biochimique et productivite´ primaire. In: Therriault, J.-C. (Ed.), Le golfe du St-Laurent: Petit oce´an ou grand estuaire? Can. Spec. Publ. Fish. Aquat. Sci., pp. 219 – 230. Ruiz, C., Martinez, D., Mosquera, G., Abad, M., Sa´nchez, J.L., 1992. Seasonal variations in condition, reproductive activity and biochemical composition of the flat oyster, Ostrea edulis, from San Cibran (Galicia, Spain). Mar. Biol. 112, 67 – 74. Sauve´, S., Brousseau, P., Pellerin, J., Morin, Y., Sene´cal, L., Goudreau, P., Fournier, M., 2002. Phagocytic activity of marine and freshwater bivalves: in vitro exposure of hemocytes to metals (Ag, Cd, Hg and Zn). Aquat. Toxicol. 58, 189 – 200. Seed, R., 1976. Ecology. In: Bayne, B.L. (Ed.), Marine Mussels: Their Ecology and Physiology. Cambridge University Press, New York, NY, pp. 13 – 66. Seed, R., Suchanek, T.H., 1992. Population and community ecology of Mytilus. In: Gosling, E. (Ed.), The Mussel Mytilus: Ecology, Physiology, Genetics and Culture. Elsevier, New York, NY, pp. 87 – 169. Skibinski, D.O.F., Roderick, E.E., 1989. Heterozygosity and growth in transplanted mussels. Mar. Biol. 102, 73 – 84. Soniat, T.M., Ray, S.M., 1985. Relationships between possible available food and the composition, condition and reproductive state of oysters from Galveston Bay, Texas. Contrib. Mar. Sci. 28, 109 – 121. Starr, M., Himmelman, J.H., Therriault, J.-C., 1990. Marine invertebrate spawning induced by phytoplankton. Science 247, 1071 – 1074. Suresh, K., Mohandas, A., 1990. Number and types of hemocytes in Sunetta scripta and Villorita cyprinoides var. cochinensis (Bivalvia), and leucocytosis subsequent to bacterial challenge. J. Invertebr. Pathol. 55, 312 – 318. Tamigneaux, E., Vazquez, E., Mingelbier, M., Klein, B., Legendre, L., 1995. Environmental control of phytoplankton assemblages in nearshore marine waters, with special emphasis on phototrophic ultraplankton. J. Plankton Res. 17, 1421 – 1447.
654
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Thompson, R.J., 1984. The reproductive cycle and physiological ecology of the mussel Mytilus edulis in a subarctic, non-estuarine environment. Mar. Biol. 79, 277 – 288. Thompson, R.J., Ratcliffe, N.A., Bayne, B.L., 1974. Effects of starvation on structure and function in the digestive gland of the mussel (Mytilus edulis L.). J. Mar. Biol. Assoc. U.K. 54, 699 – 712. Tremblay, R., Myrand, B., Sevigny, J.-M., Blier, P., Guderley, H., 1998. Bioenergetic and genetic parameters in relation to susceptibility of blue mussels, Mytilus edulis (L.) to summer mortality. J. Exp. Mar. Biol. Ecol. 221, 27 – 58. Watanuki, H., Yamaguchi, T., Sakai, M., 2002. Suppression in function of phagocytic cells in common carp Cyprinus carpio L. injected with estradiol, progesterone or 11-ketotestosterone. Comp. Biochem. Physiol. 132C, 407 – 413. Worral, C.M., Widdows, J., 1984. Investigation of factors influencing mortality in Mytilus edulis L. Mar. Biol. Lett. 5, 85 – 97. Zandee, D.I., Kluytmans, J.H., Zurburg, W., Pieters, H., 1980. Seasonal variations in biochemical composition of Mytilus edulis with reference to energy metabolism and gametogenesis. Neth. J. Sea Res. 14, 1 – 29.
Use of an index based on the blue mussel (Mytilus edulis and Mytilus trossulus) digestive gland weight to assess the nutritional quality of mussel farm sites S. Cartiera, J. Pellerina,*, M. Fournierb, E. Tamigneauxc, L. Giraultc, N. Lemairea a
Institut des Sciences de la Mer de Rimouski, Universite´ du Que´bec a` Rimouski, 310 alle´e des Ursulines, Rimouski, Que., Canada G5L 3A1 b INRS-Institut Armand Frappier, Universite´ du Que´bec, 245 Hymus, Pointe-Claire, Que., Canada H9R 3G6 c Centre colle´gial de transfert de technologie des peˆches, 167, la Grande-Alle´e Est, Grande-Rivie`re, Que., Canada G0C 1V0 Received 16 December 2003; received in revised form 21 July 2004; accepted 19 August 2004
Abstract An index based on the digestive gland (DGI) of transplanted (Gaspe´, Havre-St-Pierre and Grande-Rivie`re) and indigenous (Gaspe´ and Magdalen Islands) 1-year-old blue mussels (Mytilus edulis and Mytilus trossulus) from eastern Que´bec (Canada) was used to assess the nutritional quality of mussel farm sites. To understand variations of this index, the effects of temperature and phytoplankton concentrations were examined on growth, gametogenesis, and immunocompetence of transplanted (M. trossulus, Gould 1850) and cultivated mussels (M. trossulus and M. edulis, Linnaeus 1758) in Eastern Quebec mussel farm sites. DGI was influenced by the gametogenic cycle and was decreased before spawning with the utilization of the lipids and glycogen reserves stocked in the digestive gland. DGI patterns from all sites showed variations linked to food supply that contributed to the storage of energy reserves. Protein concentrations showed an inverse relationship with DGI, suggesting that structural proteins were not used as an energy source during spawning. Indigenous mussels from the Magdalen Islands site showed a stable DGI, suggesting both the
* Corresponding author. Tel.: +1 418 723 1986x1704; fax: +1 418 724 1842. E-mail address: [email protected] (J. Pellerin). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.08.015
634
S. Cartier et al. / Aquaculture 241 (2004) 633–654
utilization of energy reserves for reproduction and storage of energy reserves due to the abundant food supply in the Magdalen Islands. Transplanted mussels showed a clear seasonal pattern of the condition index closely related to the gametogenic cycle. Differences in soft tissue and shell growths were observed suggesting a nonsynchronous growth. In all sites, phytoplankton cells b2 Am were more abundant than the larger-size classes, and concentrations seemed sufficient for mussel growth. Phagocytosis varied according to gametogenesis and decreased during spawning. Due to the high energetic cost of spawning, which mainly influenced DGI, a direct influence of environmental parameters could not be clearly detected. D 2004 Elsevier B.V. All rights reserved. Keywords: Mytilus trossulus; Mytilus edulis; Digestive gland; Metabolic reserves; Immunocompetence; Aquaculture
1. Introduction Developing easy to use physiological indicators for mussel farmers that can give information about nutritional quality of their farm sites is quite challenging. If these indicators can detect effects of stress in mussels before the occurrence of mortality in the stocks, remediation could take place before the loss of production. Although mussel farm production in Eastern Quebec is increasing, severe problems of mortality are still reported every year. Mortality was observed generally in late summer in the Magdalen Islands (Que´bec) and was also reported on the Atlantic and Pacific coasts of North America (Incze et al., 1980; Emmett et al., 1987; Mallet et al., 1990). The explanation of massive mortality is not simple, and Tremblay et al. (1998) have suggested the possibility of a synergistic interaction between the lack of food, temperature, a postspawning stress, and the genetics of the stock. Many other factors can also affect mussel survival including salinity, light, pollutants, high densities, exposure to air, wave action, etc. (Harger, 1970; Bøhle, 1972; Seed, 1976; Pellerin-Massicotte, 1997). Food supply seems to be the principal factor which can limit production in suspended cultured systems (Navarro et al., 1991). It can also affect mussel survival during periods of metabolic stress like spawning (Incze et al., 1980). Freeman and Dickie (1979) and Worral and Widdows (1984) have observed higher mortality for large mussels, which is probably the result of an increase of postspawning stress. This increase is explained by the larger proportion of energy, which is invested into gamete production with increasing size (Worral and Widdows, 1984). Therefore, there is a necessity to develop tools for the aquaculture industry to understand the cause of such mortalities. Thompson et al. (1974), in a starvation experiment, observed a difference between the digestive gland index (DGI) of fed and starved mussels. The index was expressed as a ratio of dry digestive glands weight to total mantle-free dry weight. Due to the role in the storage of metabolic energy reserves, there are seasonal changes in the biochemical composition of bivalve digestive glands (Zandee et al., 1980; Barber and Blake, 1981). During physiological stress, like starvation or food shortage and/or high energy demand periods like gametogenesis, metabolic reserves stored in the digestive gland and other body tissues are mobilized for maintenance (Ansell, 1974; Zandee et al., 1980; Barber and Blake, 1981). Stress on the digestive gland will affect its biochemical composition due to
S. Cartier et al. / Aquaculture 241 (2004) 633–654
635
the utilization of the metabolic reserves during this period. Many physiological processes are closely linked and could act in synergy to further stress mussels faced with challenging conditions. It is now known that sexual maturation is closely linked with food availability in order to produce ripe gametes during gametogenesis (Seed and Suchanek, 1992). Moreover, modulation of immunocompetence by steroid hormones is recognised at least in fish, and measures of phagocytosis could be useful to understand the causes and consequences of stress in mussels (Watanuki et al., 2002). Phagocytic activity, used commonly as a biomarker for toxic exposure in the aquatic environment, is also influenced by environmental stress like temperature and salinity (Fisher and Tamplin, 1988; Chu and La Peyre, 1993; Cajaraville et al., 1996; Sauve´ et al., 2002; Luengen et al., 2004). Thus, coupling these two processes with the weight of the digestive gland could improve the assessment of food quality in a mussel farm. Finally, food quantity and quality are determining factors for growth rate and fecundity (Soniat and Ray, 1985; Fre´chette and Bourget, 1987). The food, or seston, is composed of a mixture of organic and inorganic particulate matter, which varies with temperature, water stratification, and hydrodynamics (Berg and Newell, 1986; Claereboudt et al. 1995; Abraham, 1998). Mussels can filter a variety of suspended particles like bacterioplankton, phytoplankton, organic detritus, microzooplankton, and mesozooplankton (Hawkins and Bayne, 1992; Lehane and Davenport, 2002). Phytoplankton and organically rich detrital particles generally are the main component of mussel nutrition (Bayne and Hawkins, 1992). Several studies have indicated that bivalve molluscs retain particles in the 2–20 Am range (Bayne and Newell, 1983). Cytometric studies have showed that mussels are particularly efficient at filtering particles in the 3–5 Am range (Cucci et al., 1989). This paper examined the effects of temperature and phytoplankton on growth, reproduction, and immunocompetence of transplanted (Mytilus trossulus, Gould 1850) and cultivated mussels (M. trossulus and Mytilus edulis, Linnaeus 1758) in eastern Quebec mussel farms. This study correlated the variations of the digestive gland weight, phagocytic activity, and sexual maturation stages with the food availability in each farming site studied.
2. Materials and methods 2.1. Site selection and sampling period One year old juvenile cultivated blue mussels (M. trossulus; 27 to 40 mm in shell length) were collected in May 2002 from a mussel farm located in Gaspe´ Bay (Quebec) and were transplanted to three farm sites: Havre-St-Pierre (50814VN 63836VW, north shore of the Gulf of St. Lawrence), Grande-Rivie`re (48824VN 64830VW, entrance of the Bay of Chaleur), and Gaspe´ (48846VN 64817VW, inside Gaspe´ Bay). Mussels from Gaspe´ Bay were chosen because of the good bacteriological quality of the site and lack of a toxic algal bloom in this area. One year old juvenile cultivated mussels (M. trossulus) from Gaspe´ Bay located at 5 m depth at the transplanted site and from the Magdalen Islands (M. edulis; 47825VN 61848VW, in Havre-aux-Maisons lagoon) were also sampled and surveyed throughout the experimental period. Experimental sites are illustrated in Fig. 1. Bivalve
636
S. Cartier et al. / Aquaculture 241 (2004) 633–654
.
Fig. 1. Location of the study area, Gulf of St. Lawrence, Canada, and experimental sites ( ).
translocation is commonly used to compare growth or effects of pollutant levels between sites (Skibinski and Roderick, 1989; Okumus and Stirling, 1998; Rome´o et al., 2003). Mussels were placed in three cages on each site. Cages were made of wood (1206030 cm) covered with VexarR diamond mesh sheets (HH in.). Cages were placed along submerged longlines (9- to 10-m depth) with a mean distance of 10 m between each cage. One thousand mussels were placed in each cage. Cages were cleaned at each sampling time to prevent biofouling. The experiment lasted 6 months (June to November 2002) and sampling occurred every 2 weeks. Fifty (50) mussels were randomly collected at each sampling date (N=25 for physiological analysis and N=25 for immunocompetence analysis). 2.2. Environmental factors Thermographs were installed at each site, at the same depth as the cages (9–10 m, except for Magdalen Islands lagoon, 4 m), and water temperature was measured three times per day (0800, 1600, and 2400 h). For statistical analysis, the mean of the three measurements was used. One water sample was collected at each sampling date with a 5-l Niskin bottle. Water was sampled at the same depth as the cages (9–10 m). Water was immediately transferred into a 1-l dark high-density polyethylene (HDPE) bottle, stored with ice in an insulated container and sent to the laboratory. Then, three 4.5-ml subsamples were taken from each plastic bottle. These subsamples were transferred to 5ml cryovials and fixed with 0.5 ml of a fresh paraformaldehyde solution (20% w/w, 0.2 Am prefiltered). The cryovials were frozen in liquid nitrogen and stored at 80 8C until further analysis of the samples. A Facsort (Becton-Dickinson), equipped with a 488-nm
S. Cartier et al. / Aquaculture 241 (2004) 633–654
637
laser, was used to determine phytoplankton concentrations, sizes of phytoplankton cells (b2–20 Am), and their fluorescence signature (Chlorophyll a and phycoerythrin), following the procedure of Mostajir et al. (2001). Sheath fluid used for the analysis was natural marine water prefiltered on a 0.22-Am membrane filter. Fluorescent microbeads of 2-, 10- and 20-Am diameter were mixed with 1.5 ml of each sample before analysis with the flow cytometer. The beads were used as an internal calibration standard for the determination of particle size. 2.3. Morphological measures, digestive gland, and condition indices determination For each sampling date and for each mussel, shell length was measured with a digital calliper. Total weights, as well as the weights of wet flesh and digestive gland, were recorded. Digestive gland and mantle samples were kept at 80 8C until analysis. The tissues were frozen in a HistobathR (ShandonR) containing cold acetone (50 8C) to preserve molecular and tissue integrity. A modified Thompson et al. (1974) digestive gland index was calculated as follows: DGI ¼
DG ST
DGI: digestive gland index; DG: wet digestive gland weight; ST: wet soft tissue weight. Also, a commercial condition index (modified from Hickman and Illingworth, 1980) was calculated as follows: CI ¼
ST ST þ SW
CI: condition index; ST: wet soft tissue weight; SW: shell weight. 2.4. Digestive gland metabolic reserves The analysis of the metabolic reserves was performed on 12 randomly sampled digestive glands at every sampling date and sites for transplanted mussels. Total proteins were determined according to the protein–Coomassie blue dye binding principle (Bradford, 1976) using bovine serum albumin (Sigma, fraction V) as the standard. Lipid concentration in the digestive gland was obtained by the colorimetric method based on the sulfo-phospho-vanillin reaction described by Frings et al. (1972) using olive oil as the standard. In both methods, the digestive gland was homogenized in 0.1 M phosphate buffer at pH 7. Glycogen concentration was estimated after an enzymatic digestion of the digestive gland homogenate (in a 0.1 M citrate buffer at pH 5) with amyloglucosidase and analysed according to the colorimetric method described by Carr and Neff (1984). Glycogen from oyster (Sigma, type III) was used as standard. All samples were analysed with a spectrophotometer (UV-VIS Beckman DU 640).
638
S. Cartier et al. / Aquaculture 241 (2004) 633–654
2.5. Sexual maturation stage determination Sections of 5 Am were done on mantle, frozen at 30 8C, cut with a CryotomeR 0620 (ThermoR ShandonR), then stained in a mix of methylene blue basic fuchsin. Sex and the maturation stages were identified with a light microscope (Leitz Ortholux II) according to Gauthier-Clerc et al. (2002). 2.6. Immunology 2.6.1. Collection of hemocytes and phagocytosis monitoring Hemocytes were collected every month in hemolymph, by puncture of the posterior adductor muscle, using 3-ml syringes and 23-G needles. Cells were washed by centrifugation with mussel’s hemolymph and adjusted to 1106 cells per milliliter of hemolymph. Hemocytes were mixed with yellow-green latex FluoSpheres (Molecular Probes, Eugene, OR, USA) at a ratio of 1:100 (hemocytes/beads). The cells were incubated, while being gently shaken, at 20 8C in the dark. After 18 h, an aliquot of 0.5 ml of each cell suspension was taken, layered over a 3% BSA gradient and centrifuged at 150g for 8 min at room temperature to remove free beads. The cell pellets were resuspended in 0.5 ml of 0.5% hematall solution (Fisher Scientific, Ottawa, Ont., Canada). 2.7. Flow cytometry acquisition A FACScan (Becton Dickinson, San Jose, CA, USA) with an air-cooled argon laser providing an excitation at 488 nm was used. Fluorescence emission was collected at 520 nm. Hemocyte populations were defined based on their forward and right angle scatter properties. A total of 10,000 events were acquired for each sample and stored in the list mode data format. The data were then analysed, once displayed as twoparameter complexity of the intracellular organelles development and cell size, in the process of gating and as fluorescence (FL1) frequency distribution histogram. Data collection and analysis were performed with an LYSIS-II program (Brousseau et al., 1998). 2.8. Statistical treatments A one-way repeated measures ANOVA was used to test significance of variations in phytoplankton cell abundance, digestive gland and condition indices, metabolic reserves, between each sampling date and the previous sampling. Normal distribution and homogeneity of variances were previously tested on data. Growth of soft tissue and shell length was tested using t-tests for paired samples. Digestive gland index and environmental factors were correlated using the Enter method regression. Because of the proximity between indigenous and transplanted stocks, same data for temperature and phytoplankton were used for regression. Pearson chi-square test was used to compare the partition of maturation stages between transplanted sites. One-way ANOVA
S. Cartier et al. / Aquaculture 241 (2004) 633–654
639
was used to test significance of variations of phagocytic activity followed by an All Pairwise Multiple Comparison (Tukey test) to identify differences among sampling periods. Data of phagocytosis of one bead and more were compared. Same comparisons were made for phagocytosis of three beads and more. For all statistical tests, we have used individual mussels as replicates, and results were considered significant with a probability ( p) value of b0.05. All data were statistically analyzed with SPSS 9.0 for Windows.
3. Results 3.1. Temperature During the sampling period in Gaspe´, temperature increased from 4 to 14.5 8C during August, and then decreased to near 2 8C in November. At the Havre-St-Pierre site, three peaks of temperature varying between 7 and 12 8C were observed: in July, mid-August, and end of September. In Grande-Rivie`re, the temperature pattern was similar to Gaspe´. In Magdalen Islands, temperature was near 12 8C at the beginning of sampling, reached 23 8C in mid-August, and decreased to near 4 8C in November. 3.2. Phytoplankton abundance At the Gaspe´ site (Fig. 2A), a first bloom of 10–20 Am cells (significant increase, pb0.05) was observed during the end of June. During the following months, three successive blooms of cells smaller than 2 Am were observed (three significant increases, pb0.05). In Havre-St-Pierre (Fig. 2B), the sampling was dominated by cells smaller than 2 Am. Three blooms of this size class of cells were observed at the end of June, during midAugust, and at the end of the sampling period (three significant increases, pb0.05). In Grande-Rivie`re (Fig. 2C), cells smaller than 2 Am were also predominant during all samplings. Three blooms were observed: end of May, end of July, and end of October (three significant increases, pb0.05). A bloom of 2–10 Am cells was observed in mid-July (significant increase, pb0.05). At the Magdalen Islands site (Fig. 2D), two blooms dominated by cells smaller than 2 Am were observed: at mid-August and the end of September (two significant increases, pb0.05). Number of phytoplankton cells was higher in Magdalen Islands in comparison with the transplanted sites. In transplanted sites, number of phytoplankton cells was lower in the Gaspe´ site in comparison with Havre-StPierre and Grande-Rivie`re sites. 3.3. Variation of wet soft tissue weight and shell length Variations in meat yield for all sites, expressed in wet weight, are illustrated in Fig. 3. Gaspe´ site showed a slow growth during the sampling period, but growth was not statistically significant. However, growth was statistically significant for cultivated mussels from Gaspe´ ( pb0.05) and Magdalen Islands ( pb0.05) and also transplanted stocks from Gaspe´ to Grande-Rivie`re ( pb0.05) and Havre-St-Pierre sites ( pb0.05). Shell
640
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fig. 2. Variations of the abundance of three classes of phytoplankton cells. Samplings were done from May to November 2002 in four sites: Gaspe´ (A), Havre-St-Pierre (B), Magdalen Islands (C), and Grande-Rivie`re (D).
growth was statistically significant in mussels from Havre-St-Pierre transplanted stock ( pb0.05) and both cultivated mussel stocks ( pb0.05; Fig. 4). 3.4. Proteins A similar seasonal pattern was observed in protein concentrations in the digestive gland from the three sites (Fig. 5A). The pattern was characterized by a decrease in late spring ( pb0.05), then the protein concentrations recovered and remained relatively stable for the rest of the sampling period. Significant increase in protein concentrations ( pb0.05) began in July in Havre-St-Pierre, later than the two other sites. 3.5. Total lipids Lipid concentrations in the digestive gland for the three sites (Fig. 5B) showed a seasonal pattern characterized by a significant increase ( pb0.05) in spring/summer than
S. Cartier et al. / Aquaculture 241 (2004) 633–654
641
Fig. 3. Variations of wet soft tissue weight from mussels in experimental sites (N=25) from May to November 2002. Graph (A) illustrates variations in transplanted mussels from Gaspe´ to Gaspe´, Havre-St-Pierre, and GrandeRivie`re. Graph (B) illustrates variations in indigenous cultivated mussels from Gaspe´ and Magdalen Islands. The symbol d*T indicates a significant difference between first and last sampling dates within sites.
followed by a significant decrease ( pb0.05). Grande-Rivie`re showed greater lipid storage during summer than the other sites. Utilization of lipids was slower at the Gaspe´ site. Lipids recovered during the fall with concentrations similar for the three sites. 3.6. Glycogen Glycogen concentration in the digestive gland for the three sites is illustrated in Fig. 5C. Glycogen concentrations showed a similar seasonal pattern with lipid concentrations for Havre-St-Pierre and Grande-Rivie`re, characterized by a significant increase in summer ( pb0.05) followed by a significant decrease ( pb0.05). Higher values were observed during summer in Havre-St-Pierre and Grande-Rivie`re sites. In the Gaspe´ site, the seasonal pattern was not evident. Glycogen concentrations were more stable during the sampling period, but a significant increase ( pb0.05) was observed during late fall for Gaspe´ and Grande-Rivie`re.
642
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fig. 4. Variations of shell length from mussels in experimental sites (N=25) from May to November 2002. Graph (A) illustrates variations in transplanted mussels from Gaspe´ to Gaspe´, Havre-St-Pierre, and Grande-Rivie`re. Graph (B) illustrates variations in indigenous cultivated mussels from Gaspe´ and Magdalen Islands. The symbol d*T indicates a significant difference between first and last sampling dates within sites.
3.7. Digestive gland and condition indices The variations of the digestive gland index (DGI) at the three transplanted sites (Fig. 6A) showed a similar pattern characterized by the highest value during May–June. Then, a significant decrease ( pb0.05) was observed at all three sites. DGI decreased earlier in Havre-St-Pierre. Maximal DGI value (0.235F0.079) in transplanted sites was observed in Grande-Rivie`re. Cultivated mussel DGI variations are illustrated in Fig. 6C. In commercial mussels from Gaspe´, a similar DGI pattern with Gaspe´-transplanted mussels was observed with a recovery of the DGI during September. In Magdalen Islands, DGI showed a significant decrease ( pb0.05) during summer, then a recovery was observed during fall. Transplanted mussels (Fig. 6B) showed a significant decrease of the condition index during July ( pb0.05). Cultivated mussels (Fig. 6D) from Gaspe´ showed a stable condition index through the sampling period, with a decrease during August/September, but the index
S. Cartier et al. / Aquaculture 241 (2004) 633–654
643
Fig. 5. Variations of proteins (A), lipid (B), and glycogen (C) concentrations in transplanted mussels’ digestive gland (N=12) from May to November 2002.
recovered during the fall. In Magdalen Islands, the condition index significantly decreased ( pb0.05) during July, then a significant increase ( pb0.05) during fall was observed. 3.8. Sexual maturation stages Sexual maturation (Fig. 7) was expressed in percentage of partitioning between stages. Spawning for male mussels in Gaspe´ was observed in mid-July and from September to November, while in females, spawning was continuous from mid-July to November. In
644
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fig. 6. Variations of the digestive gland and condition indices of transplanted mussels and indigenous cultivated mussels (N=25) from May to November 2002. Graphs (A) and (B) illustrate variations for DGI and condition index of transplanted mussels. Graphs (C) and (D) illustrate variations for DGI and condition index of indigenous cultivated mussels.
Havre-St-Pierre, the spawning period was similar for both sexes from mid-July to November. In Grande-Rivie`re, the spawning period was continuous from July to November for males, and from July to the end of October for females. Statistical analyses showed significantly ( pb0.05) different sexual maturation patterns among Gaspe´, HavreSt-Pierre, and Grande-Rivie`re sites for males and females. 3.9. Phagocytic activities Phagocytic activities (Fig. 8) in Gaspe´ were similar during June and July. During August, activities increased ( pb0.05) then decreased ( pb0.05) at the end of the sampling period. Transplanted mussels in Havre-St-Pierre and indigenous mussels from Gaspe´
S. Cartier et al. / Aquaculture 241 (2004) 633–654
645
Fig. 7. Gametogenesis was assessed by comparing partition of maturation stages of transplanted mussels (N=12). Stages were defined as described in Gauthier-Clerc et al. (2002). Partitioning is expressed in percent between each observed development stage from May to November 2002.
showed a similar pattern with transplanted mussels in Gaspe´. Phagocytic activities in Grande-Rivie`re showed a decline ( pb0.05) between June and July. Phagocytic activities then increased ( pb0.05) between July and August, with a decrease ( pb0.05) in the fall.
646
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fig. 8. Phagocytic activity of hemocytes from mussels’ hemolymph (N=15) in experimental sites [transplanted mussels in Gaspe´ (A), Havre-St-Pierre (B), Grande-Rivie`re (C), and indigenous cultivated mussels from Magdalen Islands (D) and Gaspe´ (E)] from May to November 2002. Data were divided into two categories. dOne bead and moreT represents hemocytes with phagocytosis of at least one bead. dThree beads and moreT represents more efficient hemocytes which engulfed three beads or more. Small letters indicate significant differences between months.
S. Cartier et al. / Aquaculture 241 (2004) 633–654
647
Table 1 Coefficients (b) of predictors and R 2 of the regression equations explaining DGI variations Sites
Temperature
b2 Am
2–10 Am
10–20 Am
R2
Gaspe´ Havre-St-Pierre Grande-Rivie`re Magdalen Islands (I) Gaspe´ (I)
0.622*** 0.030 0.308*** 0.347*** 0.590***
0.209*** 0.198* 0.113* 0.391** 0.190***
0.096 0.357*** 0.018 0.805** 0.157**
0.202*** 0.116 0.425*** 0.552** 0.238***
0.464*** 0.084*** 0.315*** 0.115*** 0.438***
(I)Eindigenous mussels (cultivated mussels followed simultaneously as transplanted mussels). * pb0.05. ** pb0.01. *** pb0.001.
This pattern was also evident at the Magdalen Islands site, which showed a similar pattern with the Grande-Rivie`re site. 3.10. Digestive gland index versus environmental parameters Regression equations showed that temperature, cells smaller than to 2 Am, and cells comprised between 10 and 20 Am were the factors which explained DGI variability in most of the experimental sites (Table 1). Environmental factors in Gaspe´ (transplanted and indigenous mussels) and Grande-Rivie`re explained more of the variability of the DGI with values of R 2 of 46.4%, 43.8%, and 31.5%, respectively. Gaspe´- and Grande-Rivie`retransplanted mussel DGIs were mainly influenced by temperature, cells smaller than 2 Am, and cells comprised between 10 and 20 Am. Havre-St-Pierre-transplanted mussel DGI was influenced only by cell size, especially cells smaller than 2 Am and cells comprised between 2 and 10 Am. Gaspe´ and Magdalen Islands indigenous mussel DGIs were influenced by temperature and all sizes of cells equally.
4. Discussion It is now well known that the digestive gland of Mytilus spp. plays three major roles. It is a site of intracellular digestion and carbon assimilation. Also, it is a storage site for metabolic reserves used during high metabolic and stress periods. Finally, the digestive gland serves as a site of transfer for metabolic reserves to other organs. In the field, cultured mussels can be stressed by many factors, such as high temperature and/or diminished food supply. Thompson et al. (1974) observed an effect of warm temperature and starvation stress on the digestive gland index in mussels kept in laboratory conditions. The digestive gland index decreased in stressful conditions, but starvation seems to affect the index on a longer period than temperature. It is well known that gametogenesis in bivalves is associated with the energy storage cycle of glycogen and lipids (Gabbott, 1976). In this study, environmental parameters (temperature, phytoplankton) explained partly the variability of the digestive gland index. The index seems to vary with other parameters. Even if the digestive gland index used in this study was a slight modification of the one used by Thompson et al. (1974), we have obtained a similar seasonal pattern.
648
S. Cartier et al. / Aquaculture 241 (2004) 633–654
This pattern is characterized by an increase of the index just prior to spawning. In the three sites, spawning occurred in July, and peaks of the index were observed in May–June. This pattern was also observed with the sea scallop Placopecten magellanicus digestive gland weight in Newfoundland (Canada) (Penney and McKenzie, 1996). Therefore, the digestive gland index seems to be sensitive to gametogenesis and to the energy storage cycle. Variations of the index were similar to those of glycogen and lipid concentrations in the digestive gland. Gametogenesis in marine bivalves requires a lot of energy. This energy can be supplied by food, metabolic reserves, or a combination of both (Bayne, 1976a). Metabolic reserves are stored during periods of abundant food supply. Newell et al. (1982) have observed spawning in mussels prior to or during periods of maximum food availability. Our results support those of Newell et al. (1982). By spawning during high food abundance, it allows mussels to have sufficient nutrients to accumulate reserves during the postspawning period and to allow ample food for larvae (Bayne, 1976a; Gabbott, 1976; Starr et al., 1990; Seed and Suchanek, 1992). This storage after spawning can be clearly observed in Havre-StPierre and Grande-Rivie`re sites (Fig. 5). Therefore, high concentrations of glycogen and lipids stored in the digestive gland before spawning suggest abundant food in the environment. Lipid and glycogen reserves are used for the formation of gametes (Gabbott, 1976; Lubet et al., 1976). Lipid levels and DGI were high before spawning. Similar results were reported in months preceding spawning in a population of M. edulis from eastern Newfoundland, Canada (Thompson, 1984). A loss of lipids stored in the digestive gland before spawning was also observed in other bivalves like bay scallops (Argopecten irridians concentricus; Barber and Blake, 1981). Whole body lipids of the flat oyster (Ostrea edulis) and pacific oyster (Crassosstrea gigas) were also maximal before spawning (Ruiz et al., 1992; Kang et al., 2000). Okumus and Stirling (1998) have observed in M. edulis a sharp decline of whole body lipids in April and minimum condition index values in March–April, which coincided with spawning in sea lochs on the west coast of Scotland. In this study, condition index for transplanted mussels decreased during the spawning period. A loss of proteins in the digestive gland before spawning was also observed in mussels from the three sites. This decrease is, in part, explained by the utilization of protein to formulate oocytes because protein is the main constituent of eggs, followed by lipids and glycogen (Holland, 1978). Proteins can also serve as an energy source during gametogenesis (Mann and Glomb, 1978; Adachi, 1979; Barber and Blake, 1981); but in this study, protein concentrations followed an inverse pattern with the DGI, which could indicate that mussels had sufficient energy reserves for reproduction and did not need structural proteins to achieve gametogenesis. Mussels transplanted to GrandeRivie`re showed soft tissue growth and highest values in term of lipids and glycogen and the highest digestive gland index value. Among the transplanted sites, the number of phytoplankton cells, all classes included, was highest in Grande-Rivie`re. All these facts suggest that this site was possibly more productive than the other transplanted sites. Transplanted and indigenous mussels from Gaspe´ showed similar values of digestive gland index with Havre-St-Pierre. However, no soft tissue growth was observed in Gaspe´transplanted mussels in contrast with Gaspe´ indigenous mussels and Havre-St-Pierretransplanted mussels. This suggests an intrasite variability in terms of somatic growth. More phytoplankton cells were observed in Havre-St-Pierre than in Gaspe´, explaining in
S. Cartier et al. / Aquaculture 241 (2004) 633–654
649
part soft tissue growth. In this zone, which is a frontal region, cold waters are observed due to tidal mixing (Pingree and Griffiths, 1980). This mixing contributes to nutrient replenishment in the water column. This phenomenon was observed in this study, and it could explain the three blooms observed at this site. In Magdalen Islands, amount of phytoplankton cells was considerably higher than in all other sites with a predominance of cells smaller than 2 Am. Roy et al. (1991) have observed, in summer 1987, that small particles (2–20 Am) were more abundant in the Great Entry Lagoon (Magdalen Islands), with a second population of larger cells (50 Am) appearing in early August. Tremblay et al. (1998) also observed an increase in the unit volume of seston during August. In this study, decrease of wet soft tissue weight in Magdalen Islands was observed during August, when temperature was near 20 8C, which is considered as a stressing temperature (Incze et al., 1980). Even if larger particles (larger than 20 Am) could be possibly more abundant during this period, it was not enough to limit somatic growth. Furthermore, this site possesses a sufficient amount of food to maintain the DGI during spawning, which usually occurs in late June (Myrand, 1991). A second spawning can also be observed in late July (Myrand and Gaudreault, 1995). These mussels have possibly invested more energy in somatic growth than in reproduction. Fecundity in mussels can vary annually, suggesting an adjustment of the energy allocated to reproduction as a function of food availability (Seed and Suchanek, 1992). Our results also showed different growth patterns between soft tissues and the shell. Transplanted mussels at the Havre-St-Pierre site and cultivated mussels from Gaspe´ and Magdalen Islands sites showed both significant soft tissue and shell growth. Soft tissue growth was observed for transplanted mussels at Gaspe´ and Grande-Rivie`re sites, but no shell growth was detected statistically. These results support the fact that somatic and shell growth are not necessarily synchronous (Rodhouse et al., 1984; Hilbish, 1986). AlunnoBruscia et al. (2001) showed in a food-regulated experiment that shell growth continued, even if food was scarce, while soft tissue was affected negatively. Thus, in an aquaculture context, it is risky to assess meat yield by observations based on shell length. Even if in all sites small cells (smaller than 2 Am) were more abundant, somatic growth was not compromised in most of the sites. Even if larger cells (over 20 Am) were not considered in this study, it is well known that in Gulf of St. Lawrence and the Baie des Chaleurs, which present oligotrophic conditions, picoplanktonic cells (smaller than 5 Am) are more abundant (Legendre and Le Fe`vre, 1991; Tamigneaux et al., 1995). The transplanted sites showed mussels with a different sexual maturation pattern. This could be explained by the effect of environmental parameters. An increase of temperature above 10–12 8C is considered essential to initiate the release of gametes (Bayne, 1976b; Newell et al., 1991). In Havre-St-Pierre, water mixing was observed and could cause many spawning events during a longer period. In this study, somatic growth was detected even if spawning was observed throughout summer. In Gaspe´, where females spawned from August to November, somatic growth was not detected statistically and could have been negatively influenced by spawning. Reproduction at a site with less nutrients, could affect somatic growth and, on a larger scale, the farm production. A seasonal pattern was also noted for phagocytic activities by mussel hemocytes. This decrease was particularly evident in the Grande-Rivie`re and Magdalen Islands. Because it was occurring during spawning, this decline can possibly be driven by
650
S. Cartier et al. / Aquaculture 241 (2004) 633–654
hormonal regulation. Sex steroid hormones are known to modulate the immune system (Berczi and Nagy, 1998). In mussels, Reis-Henriques et al. (1990) have observed an increase in progesterone followed by an increase of 17h-estradiol and estrone during advanced stages of sexual development. This suggests the implication of these hormones in sexual maturation regulation. Nevertheless, in Crassostrea gigas, recent studies have shown increased phagocytic activity with gonadal development. Similar to our results, they have also shown a decrease at the spawning stage and a recovery of phagocytosis until the decrease in water temperature (Ishikawa et al., 1998). Watanuki et al. (2002) have shown that sex steroids like h-estradiol, progesterone, and 11ketotestosterone exert suppressive effects on phagocytic cells of carp head kidney in vivo. Thus, the increase during spawning of sex steroids in mussels could possibly have a suppressive effect on the phagocytic activity of hemocytes. This period of reduced phagocytic activity could possibly increase disease susceptibility. It may be important to study which mechanisms are involved in these regulatory events. Further investigation should therefore be done in vitro and in vivo. After spawning, immunocompetence recovered to similar or higher values observed at prespawning levels. Restoration of phagocytic competence, in addition to increasing immune protection, may also be involved in the process of gamete resorption. Hemocytes possibly remove the moribund cells by phagocytosis and digest these cells with lysosomal hydrolases (Moore and Lowe, 1977; Suresh and Mohandas, 1990). Finally, in fall, there is a second decrease of phagocytosis, and it could be associated with another spawning event. Decrease in temperature, which also initiated the down-regulation of metabolic activities associated with hibernation, could influence phagocytosis. Carballal et al. (1997) have observed, in an in vitro study, a depression of phagocytic activities of Mytilus galloprovincialis hemocytes at 10 8C. In summary, for the first time to our knowledge, we were able to correlate reproduction and immunocompetence in M. edulis and M. trossulus. Also, seasonal variations in energy reserves were linked to gametogenesis and spawning targeted as a main physiological event that could be stressful in sites with low nutritional levels. Variations of the DGI during the summer–fall period for all sites showed a close relationship with gametogenesis. Effects of the environmental parameters were detected but did not explain entirely the DGI variations. However, different DGI patterns were observed in transplanted and cultivated mussels suggesting different energy allocation influenced by environmental parameters in an indirect way. Phagocytic activities were also affected by reproduction. According to these results, the DGI was too sensitive to gametogenesis and metabolic reserves cycle to be used to assess nutritional quality of farm sites. However, protein reserves have been shown to follow growth closely. Therefore, it appears that a simple measure of proteins in the digestive gland could be sufficient to achieve our goals and the needs of producers.
Acknowledgements Financial support was provided by the Conseil des Recherches en Peˆches et en Agroalimentaire du Que´bec (CORPAQ), the Natural Sciences and Engineering Research
S. Cartier et al. / Aquaculture 241 (2004) 633–654
651
Council of Canada (NSERC) through the Discovery grant of Jocelyne Pellerin, the Fondation de l’UQAR, ISMER and Que´bec-Oce´an. The authors also wish to thank He´le`ne Doucet-Beaupre´, Pascal Rioux, Ste´phanie Poirault, Marle`ne Fortier, Francesca Proulx, Ian Beaudin, and Vale´rie McInnis for helpful technical assistance. The authors are also grateful to the mussel farmers involved in this study.
References Abraham, E.R., 1998. The generation of plankton patchiness by turbulent stirring. Nature 391, 577 – 580. Adachi, K., 1979. Seasonal changes of the protein level in the adductor muscle of the clam, Tapes philippinarum (Adams and Reeve) with reference to the reproductive seasons. Comp. Biochem. Physiol. 64A, 85 – 89. Alunno-Bruscia, M., Bourget, E., Fre´chette, M., 2001. Shell allometry and length–mass–density relationship for Mytilus edulis in an experimental food-regulated situation. Mar. Ecol., Prog. Ser. 219, 177 – 188. Ansell, A.D., 1974. Seasonal changes in biochemical composition of the bivalve Chlamys septemradiata from the Clyde Sea area. Mar. Biol. 25, 85 – 99. Barber, B.J., Blake, N.J., 1981. Energy storage and utilization in relation to gametogenesis in Argopecten irridians concentricus (Say). J. Exp. Mar. Biol. Ecol. 52, 121 – 134. Bayne, B.L., 1976a. Aspects of reproduction in bivalve molluscs. In: Wiley, M. (Ed.), Estuarine Processes, Uses, Stresses, and Adaptation to the Estuary, vol. 1. Academic Press, New York, NY, pp. 432 – 448. Bayne, B.L., 1976b. The biology of mussel larvae. In: Bayne, B.L. (Ed.), Marine Mussels: Their Ecology and Physiology. Cambridge University Press, New York, NY, pp. 81 – 120. Bayne, B.L., Hawkins, A.J.S., 1992. Ecological and physiological aspects of herbivory in benthic suspensionfeeding molluscs. In: John, D.M., Hawkins, S.J., Price, J.H. (Eds.), Plant–Animal Interaction in the Marine Benthos. Oxford University Press, Toronto, pp. 265 – 287. Bayne, B.L., Newell, R.C., 1983. Physiological energetics of marine molluscs. In: Saleuddin, A.S.M., Wilbur, K.M. (Eds.), The Mollusca, Physiology, Part 1 vol. 4. Academic Press, New York, NY, pp. 407 – 515. Berczi, I., Nagy, E., 1998. Hormones as immune modulating agents. In: Kresina, T.F. (Ed.), Immune Modulating Agents. Marcel Dekker, New York, NY, pp. 75 – 120. Berg, J.A., Newell, R.I.E., 1986. Temporal and spatial variation in the composition of seston available to the suspension feeder Crassostrea virginica. Estuar. Coast. Shelf Sci. 23, 375 – 386. Bbhle, B., 1972. Effects of adaptation to reduced salinity on filtration activity and growth of mussels (Mytilus edulis L.). J. Exp. Mar. Biol. Ecol. 10, 41 – 47. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilising the principle of protein dye binding. Anal. Biochem. 72, 248 – 254. Brousseau, P., Payette, Y., Blakley, B., Boermans, H., Flipo, D., Tryphonas, H., Fournier, M., 1998. Manual of Immunological Methods. CRC Press, Boston, USA. 141 pp. Cajaraville, M.P., Olabarrieta, I., Marigomez, I., 1996. In vitro activities in mussels hemocytes as biomarkers of environmental quality: a case study in the Abra estuary (Biscay Bay). Ecotoxicol. Environ. Saf. 35, 253 – 260. Carballal, M.J., Lo´pez, C., Azevedo, C., Villalba, A., 1997. In vitro study of phagocytic ability of Mytilus galloprovincialis Lmk. haemocytes. Fish Shellfish Immunol. 7, 403 – 416. Carr, R.S., Neff, J.M., 1984. Quantitative semi-automated enzymatic assay for tissue glycogen. Comp. Biochem. Physiol. 77B, 447 – 449. Chu, F.-L.E., La Peyre, J.F., 1993. Perkinsus marinus susceptibility and defense-related activities in eastern oysters Crassostrea virginica: temperature effects. Dis. Aquat. Org. 16, 223 – 234. Claereboudt, M.R., Coˆte´, J., Bonardelli, J.C., Himmelman, J.H., 1995. Seasonal variation in abundance and size structure of phytoplankton in Baie des Chaleurs, southwestern Gulf of St. Lawrence, in relation to physical oceanographic conditions. Hydrobiologia 306, 147 – 157. Cucci, T.L., Shumway, S.E., Brown, W.S., Newell, C.R., 1989. Using phytoplankton and flow cytometry to analyze grazing by marine organisms. Cytometry 10, 659 – 669. Emmett, B., Thompson, K., Popham, J.D., 1987. The reproductive and energy storage cycles of two populations of Mytilus edulis (Linne) from British Columbia. J. Shellfish Res. 6, 29 – 36.
652
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Fisher, S.W., Tamplin, M., 1988. Environmental influence on activities and foreign-particles binding by hemocytes of American oysters, Crassostrea virginica. Can. J. Fish. Aquat. Sci. 45, 1309 – 1315. Fre´chette, M., Bourget, E., 1987. Significance of small-scale spatio-temporal heterogeneity in phytoplankton abundance for energy flow in Mytilus edulis. Mar. Biol. 94, 231 – 240. Freeman, K.R., Dickie, L.M., 1979. Growth and mortality of the blue mussel (Mytilus edulis) in relation to environmental indexing. J. Fish. Res. Board Can. 36, 1238 – 1249. Frings, C.S., Fendley, T.W., Dunn, R.T., Queen, C.A., 1972. Improved determination of total serum lipids by the sulfo-phospho-vanillin reaction. Clin. Chem. 18, 673 – 674. Gabbott, P.A., 1976. Energy metabolism. In: Bayne, B.L. (Ed.), Marine Mussels: Their Ecology and Physiology. Cambridge University Press, New York, NY, pp. 293 – 355. Gauthier-Clerc, S., Pellerin, J., Blaise, C., Gagne´, F., 2002. Delayed gametogenesis of Mya arenaria in the Saguenay fjord (Canada): a consequence of endocrine disruptors? Comp. Biochem. Physiol. 131C, 457 – 467. Harger, J.R., 1970. The effect of wave impact on some aspects of the biology of sea mussels. Veliger 12, 401 – 414. Hawkins, A.J.S., Bayne, B.L., 1992. Physiological interrelations, and the regulation of production. In: Gosling, E. (Ed.), The Mussel Mytilus: Ecology, Physiology, Genetics and Culture. Elsevier, New York, NY, pp. 171 – 222. Hickman, R.W., Illingworth, J., 1980. Condition cycle of the green-lipped mussel Perna canaliculus in New Zealand. Mar. Biol. 60, 27 – 38. Hilbish, T.J., 1986. Growth trajectories of shell and soft tissue in bivalves: seasonal variation in Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 96, 103 – 113. Holland, D.L., 1978. Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. In: Malins, D.C., Sargent, J.R. (Eds.), Biochemical and Biophysical Perspectives in Marine Biology. Academic Press, London, pp. 85 – 123. Incze, L.S., Lutz, R.A., Watling, L., 1980. Relationships between effects of environmental temperature and seston on growth and mortality of Mytilus edulis in a temperate northern estuary. Mar. Biol. 57, 147 – 156. Ishikawa, H., Takahashi, K., Osada, M., Matsutani, T., Mori, K., 1998. Effect of reproductive cycle on seasonal variation of phagocytosing activity in the hemocytes of Crassostrea gigas. Dev. Comp. Immunol. 22, 132 – 133. Kang, C.-K., Park, M.S., Lee, P.-Y., Choi, W.-J., Lee, W.-C., 2000. Seasonal variations in condition, reproduction activity, and biochemical composition of the pacific oyster, Crassostrea gigas (Thunberg), in suspended culture in two coastal bays of Korea. J. Shellfish Res. 19, 771 – 778. Legendre, L., Le Fe`vre, J., 1991. From individual plankton cells to pelagic marine ecosystems and to global biogeochemical cycles. In: Demers, S. (Ed.), Particles Analysis in Oceanography. Springer-Verlag, New York, NY, pp. 260 – 299. Lehane, C., Davenport, J., 2002. Ingestion of mesozooplankton by three species of bivalve: Mytilus edulis, Cerastoderma edule and Aequipecten opercularis. J. Mar. Biol. Assoc. U.K. 82, 615 – 619. Lubet, P., Herlin, P., Mathieu, M., Collin, F., 1976. Tissu de re´serve et cycle sexuel chez les lamellibranches. Haliotis 7, 59 – 62. Luengen, A.C., Friedman, C.S., Raimondi, P.T., Flegal, A.R., 2004. Evaluation of mussel immune responses as indicators of contamination in San Francisco Bay. Mar. Environ. Res. 57, 197 – 212. Mallet, A.L., Carver, C.E.A., Freeman, K.R., 1990. Summer mortality of the blue mussel in eastern Canada: spatial, temporal, stock and age variation. Mar. Ecol. Prog. Ser. 67, 35 – 41. Mann, R., Glomb, S.J., 1978. The effect of temperature on growth and ammonia excretion of the manila clam Tapes japonica. Estuar. Coast. Mar. Sci. 6, 335 – 339. Moore, M.N., Lowe, D.M., 1977. The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles. J. Invertebr. Pathol. 29, 18 – 30. Mostajir, B., Gosselin, M., Gratton, Y., Booth, B., Vasseur, C., Garneau, M.-E., Fouilland, E., Vidussi, F., Demers, S., 2001. Surface water distribution of pico- and nanophytoplankton in relation to two distinctive water masses in the North Water, northern Baffin Bay, during fall. Aquat. Microb. Ecol. 23, 205 – 212. Myrand, B., 1991. Conditions environnementales dans les lagunes des Iˆles-de-la-Madeleine et parame`tres biologiques de la moule bleue, Atelier de travail sur la mortalite´ estivale des moules aux Iˆles-de-la-Madeleine. MAPA-Peˆcheries, Iˆles-de-la-Madeleine, Que´bec, pp. 1 – 40.
S. Cartier et al. / Aquaculture 241 (2004) 633–654
653
Myrand, B., Gaudreault, J., 1995. Summer mortality of blue mussels (Mytilus edulis, Linnaeus 1758) in the Magdalen Islands (southern Gulf of St. Lawrence, Canada). J. Shellfish Res. 14, 395 – 404. Navarro, E., Iglesias, J.I.P., Camacho, A.P., Labarta, U., Beiras, R., 1991. The physiological energetics of mussels (Mytilus galloprovincialis Lmk) from different cultivation rafts in the Ria de Arosa (Gallicia, N.W. Spain). Aquaculture 94, 197 – 212. Newell, R.I.E., Hilbish, T.J., Koehn, R.K., Newell, C.J., 1982. Temporal variation in the reproductive cycle of Mytilus edulis L. (Bivalvia, Mytilidae) from localities on the east coast of the United States. Biol. Bull. 162, 299 – 310. Newell, C.R., Hidu, H., McAlice, B.J., Podniesinski, G., Short, F., Kindblom, L., 1991. Recruitment and commercial seed procurement of the blue mussel Mytilus edulis in Maine. J. World Aquac. Soc. 22, 134 – 152. Okumus, I., Stirling, H.P., 1998. Seasonal variations in the meat weight, condition index and biochemical composition of mussels (Mytilus edulis L.) in suspended culture in two Scottish sea lochs. Aquaculture 159, 249 – 261. Pellerin-Massicotte, J., 1997. Influence of elevated temperature and air-exposure on MDA levels and catalase activities in digestive glands of the blue mussel (Mytilus edulis L.). J. Rech. Oce´anogr. 22, 91 – 98. Penney, R.W., McKenzie, C.H., 1996. Seasonal changes in the body organs of cultured sea scallop, Placopecten magellanicus, and coincidence of spawning with water temperature, seston, and phytoplankton community dynamics. Can. Tech. Rep. Fish. Aquat. Sci., 2104. 22 pp. Pingree, R.D., Griffiths, D.K., 1980. A numerical model of the M2 tide in the Gulf of St. Lawrence. Oceanol. Acta 3, 221 – 225. Reis-Henriques, M.A., Le Guellec, D., Remy-Martin, J.P., Adessi, G.L., 1990. Studies of endogenous steroids from the marine mollusc Mytilus edulis L. by gas chromatography and mass spectrometry. Comp. Biochem. Physiol. 95B, 303 – 309. Rodhouse, P.G., Roden, C.M., Hensey, M.P., Ryan, T.H., 1984. Resource allocation in Mytilus edulis on the shore and in suspended culture. Mar. Biol. 84, 27 – 34. Rome´o, M., Hoarau, P., Garello, G., Gnassia-Barelli, M., Girard, J.P., 2003. Mussel transplantation and biomarkers as useful tools for assessing water quality in the NW Mediterranean. Environ. Pollut. 122, 369 – 378. Roy, S., Mayzaud, P., Souchu, P., 1991. Environnement physico–chimique et trophique d’un site mytilicole aux ˆIles-de-la-Madeleine (Que´bec): II—Matie`re particulaire, composition biochimique et productivite´ primaire. In: Therriault, J.-C. (Ed.), Le golfe du St-Laurent: Petit oce´an ou grand estuaire? Can. Spec. Publ. Fish. Aquat. Sci., pp. 219 – 230. Ruiz, C., Martinez, D., Mosquera, G., Abad, M., Sa´nchez, J.L., 1992. Seasonal variations in condition, reproductive activity and biochemical composition of the flat oyster, Ostrea edulis, from San Cibran (Galicia, Spain). Mar. Biol. 112, 67 – 74. Sauve´, S., Brousseau, P., Pellerin, J., Morin, Y., Sene´cal, L., Goudreau, P., Fournier, M., 2002. Phagocytic activity of marine and freshwater bivalves: in vitro exposure of hemocytes to metals (Ag, Cd, Hg and Zn). Aquat. Toxicol. 58, 189 – 200. Seed, R., 1976. Ecology. In: Bayne, B.L. (Ed.), Marine Mussels: Their Ecology and Physiology. Cambridge University Press, New York, NY, pp. 13 – 66. Seed, R., Suchanek, T.H., 1992. Population and community ecology of Mytilus. In: Gosling, E. (Ed.), The Mussel Mytilus: Ecology, Physiology, Genetics and Culture. Elsevier, New York, NY, pp. 87 – 169. Skibinski, D.O.F., Roderick, E.E., 1989. Heterozygosity and growth in transplanted mussels. Mar. Biol. 102, 73 – 84. Soniat, T.M., Ray, S.M., 1985. Relationships between possible available food and the composition, condition and reproductive state of oysters from Galveston Bay, Texas. Contrib. Mar. Sci. 28, 109 – 121. Starr, M., Himmelman, J.H., Therriault, J.-C., 1990. Marine invertebrate spawning induced by phytoplankton. Science 247, 1071 – 1074. Suresh, K., Mohandas, A., 1990. Number and types of hemocytes in Sunetta scripta and Villorita cyprinoides var. cochinensis (Bivalvia), and leucocytosis subsequent to bacterial challenge. J. Invertebr. Pathol. 55, 312 – 318. Tamigneaux, E., Vazquez, E., Mingelbier, M., Klein, B., Legendre, L., 1995. Environmental control of phytoplankton assemblages in nearshore marine waters, with special emphasis on phototrophic ultraplankton. J. Plankton Res. 17, 1421 – 1447.
654
S. Cartier et al. / Aquaculture 241 (2004) 633–654
Thompson, R.J., 1984. The reproductive cycle and physiological ecology of the mussel Mytilus edulis in a subarctic, non-estuarine environment. Mar. Biol. 79, 277 – 288. Thompson, R.J., Ratcliffe, N.A., Bayne, B.L., 1974. Effects of starvation on structure and function in the digestive gland of the mussel (Mytilus edulis L.). J. Mar. Biol. Assoc. U.K. 54, 699 – 712. Tremblay, R., Myrand, B., Sevigny, J.-M., Blier, P., Guderley, H., 1998. Bioenergetic and genetic parameters in relation to susceptibility of blue mussels, Mytilus edulis (L.) to summer mortality. J. Exp. Mar. Biol. Ecol. 221, 27 – 58. Watanuki, H., Yamaguchi, T., Sakai, M., 2002. Suppression in function of phagocytic cells in common carp Cyprinus carpio L. injected with estradiol, progesterone or 11-ketotestosterone. Comp. Biochem. Physiol. 132C, 407 – 413. Worral, C.M., Widdows, J., 1984. Investigation of factors influencing mortality in Mytilus edulis L. Mar. Biol. Lett. 5, 85 – 97. Zandee, D.I., Kluytmans, J.H., Zurburg, W., Pieters, H., 1980. Seasonal variations in biochemical composition of Mytilus edulis with reference to energy metabolism and gametogenesis. Neth. J. Sea Res. 14, 1 – 29.