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Selective feeding by Anodonta cygnea (Linnaeus, 1771): The effects of seasonal changes and nutritional demands
Limnologica 44 (2014) 18–22
Contents lists available at ScienceDirect
Limnologica journal homepage: www.elsevier.com/locate/limno
Selective feeding by Anodonta cygnea (Linnaeus, 1771): The effects of seasonal changes and nutritional demands Manuel Lopes-Lima a,b , Paula Lima a , Mariana Hinzmann a,b , António Rocha a , Jorge Machado a,b,∗ a b
ICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas, no. 289, 405-123 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 28 February 2013 Received in revised form 10 July 2013 Accepted 12 July 2013 Available online 14 August 2013 Keywords: Microalgae Stomach contents Selective feeding Gametogenesis Brooding
a b s t r a c t Many animal species, during their life cycles, can select specific food elements that meet the special and unique metabolic needs of crucial stages such as growth, gonad maturation or larvae production and brooding. The objective of this study was to analyze the seasonal phytoplankton composition in the stomach contents of the freshwater mussel Anodonta cygnea in order to determine whether it was capable of selecting food seasonally and which were its preferences. Specimens and water samples were collected monthly from Barrinha de Mira lagoon in the northwest of Portugal during one year. From the microalgae composition found in the water and stomach content samples, Chlorophyta presented a clear predominance, followed by Cryptophyta and Bacillariophyta in water samples, and Bacillariophyta and Cyanobacteria in stomach contents. Although mussels ingested algae in a pattern very similar to its abundance in the natural habitat, in some periods specific groups were preferred even if they were present in very low concentrations in the environment. Thus, these animals are capable of selecting food by its specific characteristics and this selectivity may be associated with its physiological cycle, mainly with the reproductive cycle. Namely, the large relative abundance (ratio stomach/environment) peak of blue green algae that co-occur with gamete development covering two other smaller peaks: one of Bacillariophyta that co-occurs with gametogenesis restart and the other of Chlorophyta at the end. In addition, a significant peak of Cryptophyta co-occurs with growth and glochidia brooding periods. © 2013 Elsevier GmbH. All rights reserved.
Introduction Bivalve food consists of not only a variety of suspended particles such as bacteria, phytoplankton, microzooplankton, and detritus, but also dissolved organic material (DOM) such as amino acids and sugars (Gosling 2003). Many bivalves are able to compensate for reductions in food quality, and thereby maintain energy intake, by altering filtration and ingestion rates, and sorting or absorption efficiencies (Iglesias et al. 1992; Hawkins et al. 1996). Several studies have shown that bivalve mollusks deal with particle availability and diversity of seston in a variety of ways. According to Shumway et al. (1985) these include: (a) the differential clearance due to a selective retention at the level of gills, (b) the preingestive selection in gills and palps that occur associated with the process of pseudofaeces formation, and (c) the selective digestion or differential utilization of ingested particles within the gut. Particle selection mechanism
∗ Corresponding author at: ICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal. Tel.: +351 222062294; fax: +351 222062232. E-mail address: [email protected] (J. Machado). 0075-9511/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.limno.2013.07.001
in suspension-feeders is controlled by diverse physical, chemical, and biological factors in the environment and many previous studies have shown that changes in size, density, electrostatic charges or concentration of particles can affect selection (Iglesias et al. 1996; Bougrier et al. 1997; Ward and Shumway 2004). Some studies have also demonstrated that chemical cues (e.g. extracellular phytoplankton metabolites and carbohydrates coating microalgae cell-surfaces) represent important factors mediating particle selection mechanisms in bivalves (Shumway et al. 1985; Ward and Targett 1989; Pales Espinosa et al. 2007, 2008). This selection allows them to enhance the nutritive value of consumed particles and to optimize energy gain. In unionoids, the ability of capturing particles varies among species, life-stages, and sexes but more thorough studies are needed to understand these processes. Silverman et al. (1997) found that stream-dwelling species were much better capturing bacteria than pond-dwelling species. Beck and Neves (2003) revealed a marked difference of particle selection with age and Tankersley and Dimock (1993) found that females, which use the gills for brooding, have lower flow rates than males. As other suspension feeding bivalves, Anodonta cygnea has phytoplankton as the main component of its diet, which includes a
M. Lopes-Lima et al. / Limnologica 44 (2014) 18–22
variety of species differing in cell size, shape and other structural features (Bricelj and Shumway 1991; MacDonald and Ward 1994; Shumway et al. 1987). The non-tropical unionoids have generally a clear annual physiological cycle; in A. cygnea it is shown that, across the year, there are specific periods for shell growth, reproduction and brooding (Lopes-Lima et al. 2009). These different functions need distinct metabolic responses by the organism that might imply the need for different nutrients. The ability to select different food items to fulfill their seasonal metabolic needs deserves attention and until the present this is still a neglected issue in unionoids or even in bivalves in general. Therefore, the present study compares seasonal data on phytoplankton composition of seston and stomach contents of A. cygnea mussels sampled monthly during one year in order to determine the degree of similarity between stomach contents of mussels and the microalgal composition of the water column as an index reflecting the possible occurrence of differential retention, ingestion, and selective digestion of species of phytoplankton within the feeding apparatus of these mussels.
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Microalgal species were grouped, in seven categories: Chlorophyta, Cryptophyta, Cyanobacteria, Bacillariophyta, Chrysophyta, Euglenophyta and Dinophyta. Stomach content analysis Collected mussels (n = 6) were immediately placed on ice in an insulated box and transported to the laboratory. Prior to excision, mussels were anaesthetized with a solution of ethylene glycol monophenyl ether (0.4 ml/L). The adductor muscles were cut with a scalpel and the valves were separated. Following the partial dissection of the visceral mass, the whole stomach contents were then collected using a hypodermic syringe. The stomach content weight was registered and added to 15 ml of gluteraldehyde 1%. 1 ml of the fixed content was then allowed to settle in a Sedgewick Rafter Cell counting cell chamber. Around 400 cells were analyzed under the microscope. Cell abundance was obtained by multiplying the number of cells counted in the aliquot by the volume of fixative used for the dilution. Relative abundance of a given group was defined as the fraction this group represents of the total cell number.
Materials and methods
Selectivity
Area of study
To analyze the selectivity, the Jacobs (1974) modified forage ratio index (log Q) was calculated for each month.
The Barrinha de Mira Lagoon, located in Mira, Aveiro, Portugal (40◦ 26 54 N; 8◦ 47 48 W) is a shallow eutrophic freshwater lagoon with an area of 45 ha. Its depth ranges between a few centimeters up to two meters with a mean depth of about 0.5 m. It is inserted in a mixed urban/agricultural/forest zone and it is used mainly for recreation. It is of great proximity to the sea and integrates the Natura 2000 site “Dunas de Mira, Gândara and Gafanhas”. It is connected through a series of man-made canals to the Vouga river estuary. The most significant pressures are related to agriculture, contamination by urban and industrial effluents, as well as sedimentation. The lagoon has healthy freshwater mussel populations of A. cygnea and Unio delphinus that are spread evenly throughout most of the lagoon area.
Statistical analysis A total of 6 water samples per month were used for phytoplankton composition. The same number of individuals (6) per month was also used for the stomach contents analysis. Given the differences in cell abundance between water and stomach samples, statistical comparisons were based on relative abundance values (means) to obviate the concentration factor that affects the stomach contents. Data were compared by two-way ANOVA (sample type by month) followed by post hoc test comparisons by Scheffé Test using SPSS Progress release 18 software (SPSS Inc., 2009, Chicago, IL, USA). All tests were performed at a significance level of 5%.
Sampling
Results
Six mussels and water samples were collected monthly at Barrinha de Mira Lagoon from October 2008 to September 2009, always during mid day. Six 250 ml water samples were collected at the mussel siphons level in six different spots of the mussel bed site for phytoplankton composition analysis. Total suspended solids (mg/L) were determined by standard procedures (ESS Method 340.2). Temperature (◦ C), pH, dissolved oxygen (mg/L) and conductivity (S/cm) were analyzed with a portable multiparameter probe WTW ProWLine Multi 1970i in three different spots at a depth just above the soft bottom.
The water temperature in Barrinha de Mira Lagoon ranged from 10 to 25.9 ◦ C with the highest temperatures recorded in July and the lowest in January (Table 1). Dissolved oxygen levels showed a high variation especially during summer and varied from 4.2 to 10.3 mg/L. The pH ranged from 6.6 to 7.4 being the lowest levels found during winter and the highest during summer. Conductivity ranged from a minimum value of 330 S/cm and a maximum of 490 S/cm. Total suspended solids showed the highest value during November (59.7 mg/L) and the lowest during September (6.7 mg/L). The total number of phytoplankton cells in water samples reached a peak during February (9697 cells/ml) while for stomach content the highest values were found in August and September with 6.8 × 108 cells/g and 6.3 × 108 cells/g, respectively (Fig. 1). The microalgal composition found in water and stomach content samples was much diversified being represented by 7 major groups: Chlorophyta, Cryptophyta, Cyanobacteria, Bacillariophyta, Euglenophyta, Chrysophyta, and Dinophyta. These last 3 divisions, were observed as minor groups in all samples and so were not taken in overall analysis. The results obtained for phytoplankton composition in the water of Barrinha de Mira Lagoon corroborate the importance of Chlorophyta, presenting the largest contribution all year except during March and April when Bacillariophyta was the most abundant group. Cyanobacteria were the group with lower indices in all the analyzed samples (Fig. 2).
Phytoplankton composition of water samples Water samples were immediately fixed by the addition of Lugol’s solution (Merck KGaA, Darmstadt, Germany) to give a final concentration of 1%. Quantitative analysis was made following a modification of the Uthermol sedimentation technique (Hasle 1978): 50 ml of water were allowed to settle for 36–48 h in a combined Uthermol chamber and cylinder. Identification and counting followed under an inverted microscope (Nikon, phase contrast DIAPHOT). The aliquot counted represented 5% of total chamber volume. Identification of phytoplankton divisions was performed according (Ricard 1987; Balech 1988; Tomas 1993; Van den Hoek et al. 1995; Hasle and Syvertsen 1996; Steidinger and Jangen 1996).
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M. Lopes-Lima et al. / Limnologica 44 (2014) 18–22
Table 1 Environmental data recorded during the one-year study period (from October 2008 to September 2009) in Barrinha de Mira Lagoon.
October November December January February March April May June July August September
Temperature (◦ C)
pH
Dissolved oxygen (mg/L)
Conductivity (S/cm)
TSS (mg/L)
18.9 14.2 10.2 10.0 14.3 14.0 15.0 21.0 25.0 25.9 25.2 18.9
7.0 7.0 6.8 6.7 6.9 6.7 7.4 7.4 7.3 7.3 7.0 6.6
8.8 9.7 10.1 6.9 8.7 7.1 8.8 8.4 10.3 4.2 7.4 6.3
460 350 350 480 330 490 370 430 400 430 450 420
20.3 59.7 24.3 17.9 23.3 10.2 22.0 19.7 16.3 13.8 8.9 6.7
In stomach contents, as in water samples, Chlorophyta were predominant, followed by Bacillariophyta although in a smaller percentage. This was true along the year except during August and September when Cryptophyta was the group with higher abundance. Curiously, during the rest of the year this group had a smaller contribution (Figs. 2 and 3). Overall, comparing the results for abundance of the two types of samples it was evident that these mussels tend to ingest food with a similar pattern as their availability (Fig. 4). However this was not true for Cyanobacteria and Cryptophyta. Surprisingly, the highest percentage of blue green algae in A. cygnea’s stomach was found during February and March when their abundance in the water was
Fig. 1. Variation of the total number of phytoplankton cells in water and stomach samples during the study period from October 2008 to September 2009.
the lowest. In fact, during the sampled period the mussels showed a preference for these blue green algae despite its small representation in the phytoplankton community. An opposite behavior was registered toward Cryptophyta, although its constant presence in water samples, it was almost undetected in stomach contents until summer (August and September) when it was the taxa with higher percentage showing a clear preference from the mussels. It was also true that its abundance in the water increased during late summer but this increase within stomach contents was even more pronounced. Discussion Gut content analyses revealed that phytoplankton represents an important food constituent for A. cygnea. Throughout the year, the most representative groups found in the gut contents were Chlorophyta, Cryptophyta, Cyanobacteria and Bacillariophyta. The present results showed a lower concentration of phytoplankton in the water during August and September that might be overcome with a faster filtration or enhanced efficiency, which would explain the remarkable higher number of cells found in the stomach contents during these months. Corroborating our statement, Lima et al. (2004) observed that in an eutrophic habitat there was no clear correlation between food availability and growth. In fact, filtration rates are known to be related with the concentration of active microalgae cells up to an optimum maximum level, above that level the feeding efficiency decreases mainly due to gill clogging (Morton 1971; Mathers 1974; Palmer 1980). From the main groups of microalgae studied in the present work an outstanding selectivity for Cyanobacteria was noticed mainly from January to April despite their lower presence in the water.
Fig. 2. Monthly variation of abundance of the main phytoplankton groups in water samples from Barrinha de Mira Lagoon during the study period from October 2008 to September 2009.
M. Lopes-Lima et al. / Limnologica 44 (2014) 18–22
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Fig. 3. Abundance variation of the most abundant algal taxa recorded in stomach contents of A. cygnea during the study period from October 2008 to September 2009.
Baker and Levinton (2003) found that native mussels preferentially ingest the unicellular cyanobacterium, Microcystis sp. (4 m) over most other phytoplankton species (≥6 m) due to their size. We can presume that Cyanobacteria were preferred because of their smaller size. Curiously these cells are rich in proteins and contain carotenoids, vitamins, minerals, and essential fatty acids (Aakermann et al. 1992). A. cygnea restarts gametogenesis during spring (Lima et al. 2012), and so this preference might be related to the demands of this physiological process. Gametogenesis is an energy demanding process. Hence, the energy increase obtained from food in this period maybe stored to be used in gametogenesis similarly to species regarded as “conservative” that use previously stored energy reserves for gonad development (Bayne 1976). This strategy contrast with the so-called opportunistic behavior of some species that use energy derived from food directly for gonad development (e.g. Crassostrea gigas, Bayne 1976; Kang et al. 2000).
Overall, Chlorophyta were predominant in stomach contents, which can be explained by their predominance in the water samples. Parker et al. (1998) reported that gut contents of adult Amblema plicata (Say, 1817) and Quadrula pustulosa (Lea, 1831) from the Ohio River revealed ingested particles ranging from 4 to 70 m, and that the relative abundance of algal species within the gut was similar to their relative abundance in the external environment. Green algae are generally considered a kind of whole food with all the materials to support life to these species. In fact, our study corroborates this finding since Chlorophyta abundance was clearly proportional to its abundance in the major part of the year with the exception of March. Although at lower percentage than Chlorophyta, A. cygnea also ingested Bacillariophyta at a pattern proportional to their abundance in the water, except on the above mentioned peak in February. Allthough, Bacillariophyta have heavy silicate frustule protections that render them unpalatable to mussels, which may
Fig. 4. Selectivity of A. cygnea using Jacobs modified forage ratio (log Q) of algal groups in the gut vs. the environment during the study period from October 2008 to September 2009. Asterisks reveal significance (p < 0.05).
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lead to them being rejected through pseudofaeces, studies have proven that Bacillariophyta are an important food source due to their high content in polyunsaturated and unsaturated fatty acids (Gatenby et al. 2003). In the last microalgae group that was detected with relevant abundance, the Cryptophyta, our data showed that during summerautumn months it was the group with the highest percentage in the gut contents, suggesting their preference as a food source during this period. Cryptophyta are known for its high content in starch. Carbohydrates (CHO) are presumably the primary energy reserve in adult bivalves; whereas, the larvae accumulate lipids in the form of triacylglycerols as an energy source for developing organelles and metamorphosis (Epifanio 1979; Wikfors et al. 1984; Enright et al. 1986). The faster growth of A. cygnea’s young adults (Lima et al. 2004) as well as the spawning process during summer–autumn requires high nutritional demands. As mentioned above, it is precisely during this period that the highest number of total microalgae cells in A. cygnea gut content was found suggesting an intense feeding activity. In conclusion, this study showed that the comparison of the stomach and seston compositions in terms of frequency and abundance of phytoplankton species constitutes a realistic approach to this subject and reveals feeding selectivity for specific groups throughout the year. This fact is probably due to the distinct physiological functions that are active within each annual cycle that may have specific nutrient needs. However, this process should be investigated more thoroughly due to the various levels of selection interacting across the feeding processes of bivalves (Rouillon and Navarro 2003). So, more studies are needed that can follow the pathway of nutrients not only into the stomach but also to the interior of the organisms. Studies using labeled elements like carbon and nitrogen, in vitro, should be especially useful. Acknowledgments This work was supported by the Portuguese Science and Technology Foundation (FCT) under project PTDC/AACAMB/117688/2010 and grant SFRH/BD/76265/2011. The authors wish to thank Ronaldo Sousa and Elsa Froufe for their comments and help in the elaboration of the manuscript. References Aakermann, T., Skulberg, O.M., Liaaen-Jensen, S., 1992. Carotenoids of blue-green algae. 12. A comparison of the carotenoids of the strains of Oscillatoria and Spirulina (cyanobacteria). Biochem. Syst. Ecol. 20, 761–769. Balech, E., 1988. Los dinoflagelados del Atlantico Sudoccidental. Publicación especial ˜ de Oceanografía, pp. 1. del Instituto Espanol Bayne, B.L., 1976. Aspects of reproduction in bivalve molluscs. In: Wiley, M. (Ed.), Estuarine Processes 1. Academic Press, New York, pp. 432–448. Baker, M., Levinton, S., 2003. Selective feeding by three native North American freshwater mussels implies food competition with zebra mussels. Hydrobiologia 505, 97–105. Beck, K., Neves, R.J., 2003. An evaluation of selective feeding by three age groups of the rainbow mussel (Villosa iris). N. Am. J. Aquacult. 65, 203–209. Bougrier, S., Hawkins, A.J., Heral, M., 1997. Preingestive selection of different microalgal mixtures in Crassostrea gigas and Mytilus edulis analysed by flow cytometry. Aquaculture 150, 123–134. Bricelj, V.M., Shumway, S., 1991. Physiology: energy acquisition and utilization. In: Shumway, S.E. (Ed.), Scallops: Biology, Ecology and Aquaculture. Elsevier, Amsterdam, pp. 305–346. Enright, C.T., Newkirk, C.F., Craigie, J.S., Castell, J.D., 1986. Evaluation of phytoplankton as diet for juvenile Ostrea edulis L. J. Exp. Mar. Biol. Ecol. 96, 1–13. Epifanio, C.E., 1979. Growth in bivalve molluscs: nutritional effects of two or more species of algae in diets fed to the American oyster Crassostrea virginica (Gmelin) and the hard clam Mercenaria mercenaria. Aquaculture 18, 1–12. Gatenby, C.M., Orcutt, D.M., Kreeger, D.A., Parker, B.C., Jones, V.A., Neves, R.J., 2003. Biochemical composition of three algal species proposed as food for captive freshwater mussels. J. Appl. Phycol. 15, 1–11.
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Contents lists available at ScienceDirect
Limnologica journal homepage: www.elsevier.com/locate/limno
Selective feeding by Anodonta cygnea (Linnaeus, 1771): The effects of seasonal changes and nutritional demands Manuel Lopes-Lima a,b , Paula Lima a , Mariana Hinzmann a,b , António Rocha a , Jorge Machado a,b,∗ a b
ICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal CIIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas, no. 289, 405-123 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 28 February 2013 Received in revised form 10 July 2013 Accepted 12 July 2013 Available online 14 August 2013 Keywords: Microalgae Stomach contents Selective feeding Gametogenesis Brooding
a b s t r a c t Many animal species, during their life cycles, can select specific food elements that meet the special and unique metabolic needs of crucial stages such as growth, gonad maturation or larvae production and brooding. The objective of this study was to analyze the seasonal phytoplankton composition in the stomach contents of the freshwater mussel Anodonta cygnea in order to determine whether it was capable of selecting food seasonally and which were its preferences. Specimens and water samples were collected monthly from Barrinha de Mira lagoon in the northwest of Portugal during one year. From the microalgae composition found in the water and stomach content samples, Chlorophyta presented a clear predominance, followed by Cryptophyta and Bacillariophyta in water samples, and Bacillariophyta and Cyanobacteria in stomach contents. Although mussels ingested algae in a pattern very similar to its abundance in the natural habitat, in some periods specific groups were preferred even if they were present in very low concentrations in the environment. Thus, these animals are capable of selecting food by its specific characteristics and this selectivity may be associated with its physiological cycle, mainly with the reproductive cycle. Namely, the large relative abundance (ratio stomach/environment) peak of blue green algae that co-occur with gamete development covering two other smaller peaks: one of Bacillariophyta that co-occurs with gametogenesis restart and the other of Chlorophyta at the end. In addition, a significant peak of Cryptophyta co-occurs with growth and glochidia brooding periods. © 2013 Elsevier GmbH. All rights reserved.
Introduction Bivalve food consists of not only a variety of suspended particles such as bacteria, phytoplankton, microzooplankton, and detritus, but also dissolved organic material (DOM) such as amino acids and sugars (Gosling 2003). Many bivalves are able to compensate for reductions in food quality, and thereby maintain energy intake, by altering filtration and ingestion rates, and sorting or absorption efficiencies (Iglesias et al. 1992; Hawkins et al. 1996). Several studies have shown that bivalve mollusks deal with particle availability and diversity of seston in a variety of ways. According to Shumway et al. (1985) these include: (a) the differential clearance due to a selective retention at the level of gills, (b) the preingestive selection in gills and palps that occur associated with the process of pseudofaeces formation, and (c) the selective digestion or differential utilization of ingested particles within the gut. Particle selection mechanism
∗ Corresponding author at: ICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal. Tel.: +351 222062294; fax: +351 222062232. E-mail address: [email protected] (J. Machado). 0075-9511/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.limno.2013.07.001
in suspension-feeders is controlled by diverse physical, chemical, and biological factors in the environment and many previous studies have shown that changes in size, density, electrostatic charges or concentration of particles can affect selection (Iglesias et al. 1996; Bougrier et al. 1997; Ward and Shumway 2004). Some studies have also demonstrated that chemical cues (e.g. extracellular phytoplankton metabolites and carbohydrates coating microalgae cell-surfaces) represent important factors mediating particle selection mechanisms in bivalves (Shumway et al. 1985; Ward and Targett 1989; Pales Espinosa et al. 2007, 2008). This selection allows them to enhance the nutritive value of consumed particles and to optimize energy gain. In unionoids, the ability of capturing particles varies among species, life-stages, and sexes but more thorough studies are needed to understand these processes. Silverman et al. (1997) found that stream-dwelling species were much better capturing bacteria than pond-dwelling species. Beck and Neves (2003) revealed a marked difference of particle selection with age and Tankersley and Dimock (1993) found that females, which use the gills for brooding, have lower flow rates than males. As other suspension feeding bivalves, Anodonta cygnea has phytoplankton as the main component of its diet, which includes a
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variety of species differing in cell size, shape and other structural features (Bricelj and Shumway 1991; MacDonald and Ward 1994; Shumway et al. 1987). The non-tropical unionoids have generally a clear annual physiological cycle; in A. cygnea it is shown that, across the year, there are specific periods for shell growth, reproduction and brooding (Lopes-Lima et al. 2009). These different functions need distinct metabolic responses by the organism that might imply the need for different nutrients. The ability to select different food items to fulfill their seasonal metabolic needs deserves attention and until the present this is still a neglected issue in unionoids or even in bivalves in general. Therefore, the present study compares seasonal data on phytoplankton composition of seston and stomach contents of A. cygnea mussels sampled monthly during one year in order to determine the degree of similarity between stomach contents of mussels and the microalgal composition of the water column as an index reflecting the possible occurrence of differential retention, ingestion, and selective digestion of species of phytoplankton within the feeding apparatus of these mussels.
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Microalgal species were grouped, in seven categories: Chlorophyta, Cryptophyta, Cyanobacteria, Bacillariophyta, Chrysophyta, Euglenophyta and Dinophyta. Stomach content analysis Collected mussels (n = 6) were immediately placed on ice in an insulated box and transported to the laboratory. Prior to excision, mussels were anaesthetized with a solution of ethylene glycol monophenyl ether (0.4 ml/L). The adductor muscles were cut with a scalpel and the valves were separated. Following the partial dissection of the visceral mass, the whole stomach contents were then collected using a hypodermic syringe. The stomach content weight was registered and added to 15 ml of gluteraldehyde 1%. 1 ml of the fixed content was then allowed to settle in a Sedgewick Rafter Cell counting cell chamber. Around 400 cells were analyzed under the microscope. Cell abundance was obtained by multiplying the number of cells counted in the aliquot by the volume of fixative used for the dilution. Relative abundance of a given group was defined as the fraction this group represents of the total cell number.
Materials and methods
Selectivity
Area of study
To analyze the selectivity, the Jacobs (1974) modified forage ratio index (log Q) was calculated for each month.
The Barrinha de Mira Lagoon, located in Mira, Aveiro, Portugal (40◦ 26 54 N; 8◦ 47 48 W) is a shallow eutrophic freshwater lagoon with an area of 45 ha. Its depth ranges between a few centimeters up to two meters with a mean depth of about 0.5 m. It is inserted in a mixed urban/agricultural/forest zone and it is used mainly for recreation. It is of great proximity to the sea and integrates the Natura 2000 site “Dunas de Mira, Gândara and Gafanhas”. It is connected through a series of man-made canals to the Vouga river estuary. The most significant pressures are related to agriculture, contamination by urban and industrial effluents, as well as sedimentation. The lagoon has healthy freshwater mussel populations of A. cygnea and Unio delphinus that are spread evenly throughout most of the lagoon area.
Statistical analysis A total of 6 water samples per month were used for phytoplankton composition. The same number of individuals (6) per month was also used for the stomach contents analysis. Given the differences in cell abundance between water and stomach samples, statistical comparisons were based on relative abundance values (means) to obviate the concentration factor that affects the stomach contents. Data were compared by two-way ANOVA (sample type by month) followed by post hoc test comparisons by Scheffé Test using SPSS Progress release 18 software (SPSS Inc., 2009, Chicago, IL, USA). All tests were performed at a significance level of 5%.
Sampling
Results
Six mussels and water samples were collected monthly at Barrinha de Mira Lagoon from October 2008 to September 2009, always during mid day. Six 250 ml water samples were collected at the mussel siphons level in six different spots of the mussel bed site for phytoplankton composition analysis. Total suspended solids (mg/L) were determined by standard procedures (ESS Method 340.2). Temperature (◦ C), pH, dissolved oxygen (mg/L) and conductivity (S/cm) were analyzed with a portable multiparameter probe WTW ProWLine Multi 1970i in three different spots at a depth just above the soft bottom.
The water temperature in Barrinha de Mira Lagoon ranged from 10 to 25.9 ◦ C with the highest temperatures recorded in July and the lowest in January (Table 1). Dissolved oxygen levels showed a high variation especially during summer and varied from 4.2 to 10.3 mg/L. The pH ranged from 6.6 to 7.4 being the lowest levels found during winter and the highest during summer. Conductivity ranged from a minimum value of 330 S/cm and a maximum of 490 S/cm. Total suspended solids showed the highest value during November (59.7 mg/L) and the lowest during September (6.7 mg/L). The total number of phytoplankton cells in water samples reached a peak during February (9697 cells/ml) while for stomach content the highest values were found in August and September with 6.8 × 108 cells/g and 6.3 × 108 cells/g, respectively (Fig. 1). The microalgal composition found in water and stomach content samples was much diversified being represented by 7 major groups: Chlorophyta, Cryptophyta, Cyanobacteria, Bacillariophyta, Euglenophyta, Chrysophyta, and Dinophyta. These last 3 divisions, were observed as minor groups in all samples and so were not taken in overall analysis. The results obtained for phytoplankton composition in the water of Barrinha de Mira Lagoon corroborate the importance of Chlorophyta, presenting the largest contribution all year except during March and April when Bacillariophyta was the most abundant group. Cyanobacteria were the group with lower indices in all the analyzed samples (Fig. 2).
Phytoplankton composition of water samples Water samples were immediately fixed by the addition of Lugol’s solution (Merck KGaA, Darmstadt, Germany) to give a final concentration of 1%. Quantitative analysis was made following a modification of the Uthermol sedimentation technique (Hasle 1978): 50 ml of water were allowed to settle for 36–48 h in a combined Uthermol chamber and cylinder. Identification and counting followed under an inverted microscope (Nikon, phase contrast DIAPHOT). The aliquot counted represented 5% of total chamber volume. Identification of phytoplankton divisions was performed according (Ricard 1987; Balech 1988; Tomas 1993; Van den Hoek et al. 1995; Hasle and Syvertsen 1996; Steidinger and Jangen 1996).
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Table 1 Environmental data recorded during the one-year study period (from October 2008 to September 2009) in Barrinha de Mira Lagoon.
October November December January February March April May June July August September
Temperature (◦ C)
pH
Dissolved oxygen (mg/L)
Conductivity (S/cm)
TSS (mg/L)
18.9 14.2 10.2 10.0 14.3 14.0 15.0 21.0 25.0 25.9 25.2 18.9
7.0 7.0 6.8 6.7 6.9 6.7 7.4 7.4 7.3 7.3 7.0 6.6
8.8 9.7 10.1 6.9 8.7 7.1 8.8 8.4 10.3 4.2 7.4 6.3
460 350 350 480 330 490 370 430 400 430 450 420
20.3 59.7 24.3 17.9 23.3 10.2 22.0 19.7 16.3 13.8 8.9 6.7
In stomach contents, as in water samples, Chlorophyta were predominant, followed by Bacillariophyta although in a smaller percentage. This was true along the year except during August and September when Cryptophyta was the group with higher abundance. Curiously, during the rest of the year this group had a smaller contribution (Figs. 2 and 3). Overall, comparing the results for abundance of the two types of samples it was evident that these mussels tend to ingest food with a similar pattern as their availability (Fig. 4). However this was not true for Cyanobacteria and Cryptophyta. Surprisingly, the highest percentage of blue green algae in A. cygnea’s stomach was found during February and March when their abundance in the water was
Fig. 1. Variation of the total number of phytoplankton cells in water and stomach samples during the study period from October 2008 to September 2009.
the lowest. In fact, during the sampled period the mussels showed a preference for these blue green algae despite its small representation in the phytoplankton community. An opposite behavior was registered toward Cryptophyta, although its constant presence in water samples, it was almost undetected in stomach contents until summer (August and September) when it was the taxa with higher percentage showing a clear preference from the mussels. It was also true that its abundance in the water increased during late summer but this increase within stomach contents was even more pronounced. Discussion Gut content analyses revealed that phytoplankton represents an important food constituent for A. cygnea. Throughout the year, the most representative groups found in the gut contents were Chlorophyta, Cryptophyta, Cyanobacteria and Bacillariophyta. The present results showed a lower concentration of phytoplankton in the water during August and September that might be overcome with a faster filtration or enhanced efficiency, which would explain the remarkable higher number of cells found in the stomach contents during these months. Corroborating our statement, Lima et al. (2004) observed that in an eutrophic habitat there was no clear correlation between food availability and growth. In fact, filtration rates are known to be related with the concentration of active microalgae cells up to an optimum maximum level, above that level the feeding efficiency decreases mainly due to gill clogging (Morton 1971; Mathers 1974; Palmer 1980). From the main groups of microalgae studied in the present work an outstanding selectivity for Cyanobacteria was noticed mainly from January to April despite their lower presence in the water.
Fig. 2. Monthly variation of abundance of the main phytoplankton groups in water samples from Barrinha de Mira Lagoon during the study period from October 2008 to September 2009.
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Fig. 3. Abundance variation of the most abundant algal taxa recorded in stomach contents of A. cygnea during the study period from October 2008 to September 2009.
Baker and Levinton (2003) found that native mussels preferentially ingest the unicellular cyanobacterium, Microcystis sp. (4 m) over most other phytoplankton species (≥6 m) due to their size. We can presume that Cyanobacteria were preferred because of their smaller size. Curiously these cells are rich in proteins and contain carotenoids, vitamins, minerals, and essential fatty acids (Aakermann et al. 1992). A. cygnea restarts gametogenesis during spring (Lima et al. 2012), and so this preference might be related to the demands of this physiological process. Gametogenesis is an energy demanding process. Hence, the energy increase obtained from food in this period maybe stored to be used in gametogenesis similarly to species regarded as “conservative” that use previously stored energy reserves for gonad development (Bayne 1976). This strategy contrast with the so-called opportunistic behavior of some species that use energy derived from food directly for gonad development (e.g. Crassostrea gigas, Bayne 1976; Kang et al. 2000).
Overall, Chlorophyta were predominant in stomach contents, which can be explained by their predominance in the water samples. Parker et al. (1998) reported that gut contents of adult Amblema plicata (Say, 1817) and Quadrula pustulosa (Lea, 1831) from the Ohio River revealed ingested particles ranging from 4 to 70 m, and that the relative abundance of algal species within the gut was similar to their relative abundance in the external environment. Green algae are generally considered a kind of whole food with all the materials to support life to these species. In fact, our study corroborates this finding since Chlorophyta abundance was clearly proportional to its abundance in the major part of the year with the exception of March. Although at lower percentage than Chlorophyta, A. cygnea also ingested Bacillariophyta at a pattern proportional to their abundance in the water, except on the above mentioned peak in February. Allthough, Bacillariophyta have heavy silicate frustule protections that render them unpalatable to mussels, which may
Fig. 4. Selectivity of A. cygnea using Jacobs modified forage ratio (log Q) of algal groups in the gut vs. the environment during the study period from October 2008 to September 2009. Asterisks reveal significance (p < 0.05).
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lead to them being rejected through pseudofaeces, studies have proven that Bacillariophyta are an important food source due to their high content in polyunsaturated and unsaturated fatty acids (Gatenby et al. 2003). In the last microalgae group that was detected with relevant abundance, the Cryptophyta, our data showed that during summerautumn months it was the group with the highest percentage in the gut contents, suggesting their preference as a food source during this period. Cryptophyta are known for its high content in starch. Carbohydrates (CHO) are presumably the primary energy reserve in adult bivalves; whereas, the larvae accumulate lipids in the form of triacylglycerols as an energy source for developing organelles and metamorphosis (Epifanio 1979; Wikfors et al. 1984; Enright et al. 1986). The faster growth of A. cygnea’s young adults (Lima et al. 2004) as well as the spawning process during summer–autumn requires high nutritional demands. As mentioned above, it is precisely during this period that the highest number of total microalgae cells in A. cygnea gut content was found suggesting an intense feeding activity. In conclusion, this study showed that the comparison of the stomach and seston compositions in terms of frequency and abundance of phytoplankton species constitutes a realistic approach to this subject and reveals feeding selectivity for specific groups throughout the year. This fact is probably due to the distinct physiological functions that are active within each annual cycle that may have specific nutrient needs. However, this process should be investigated more thoroughly due to the various levels of selection interacting across the feeding processes of bivalves (Rouillon and Navarro 2003). So, more studies are needed that can follow the pathway of nutrients not only into the stomach but also to the interior of the organisms. Studies using labeled elements like carbon and nitrogen, in vitro, should be especially useful. Acknowledgments This work was supported by the Portuguese Science and Technology Foundation (FCT) under project PTDC/AACAMB/117688/2010 and grant SFRH/BD/76265/2011. The authors wish to thank Ronaldo Sousa and Elsa Froufe for their comments and help in the elaboration of the manuscript. References Aakermann, T., Skulberg, O.M., Liaaen-Jensen, S., 1992. Carotenoids of blue-green algae. 12. A comparison of the carotenoids of the strains of Oscillatoria and Spirulina (cyanobacteria). Biochem. Syst. Ecol. 20, 761–769. Balech, E., 1988. Los dinoflagelados del Atlantico Sudoccidental. Publicación especial ˜ de Oceanografía, pp. 1. del Instituto Espanol Bayne, B.L., 1976. Aspects of reproduction in bivalve molluscs. In: Wiley, M. (Ed.), Estuarine Processes 1. Academic Press, New York, pp. 432–448. Baker, M., Levinton, S., 2003. Selective feeding by three native North American freshwater mussels implies food competition with zebra mussels. Hydrobiologia 505, 97–105. Beck, K., Neves, R.J., 2003. An evaluation of selective feeding by three age groups of the rainbow mussel (Villosa iris). N. Am. J. Aquacult. 65, 203–209. Bougrier, S., Hawkins, A.J., Heral, M., 1997. Preingestive selection of different microalgal mixtures in Crassostrea gigas and Mytilus edulis analysed by flow cytometry. Aquaculture 150, 123–134. Bricelj, V.M., Shumway, S., 1991. Physiology: energy acquisition and utilization. In: Shumway, S.E. (Ed.), Scallops: Biology, Ecology and Aquaculture. Elsevier, Amsterdam, pp. 305–346. Enright, C.T., Newkirk, C.F., Craigie, J.S., Castell, J.D., 1986. Evaluation of phytoplankton as diet for juvenile Ostrea edulis L. J. Exp. Mar. Biol. Ecol. 96, 1–13. Epifanio, C.E., 1979. Growth in bivalve molluscs: nutritional effects of two or more species of algae in diets fed to the American oyster Crassostrea virginica (Gmelin) and the hard clam Mercenaria mercenaria. Aquaculture 18, 1–12. Gatenby, C.M., Orcutt, D.M., Kreeger, D.A., Parker, B.C., Jones, V.A., Neves, R.J., 2003. Biochemical composition of three algal species proposed as food for captive freshwater mussels. J. Appl. Phycol. 15, 1–11.
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