Contrasting responses of blue mussels to two different HAB species

Transcription

1 Contrasting responses of blue mussels to two different HAB species Galimany, E. 1,5, Sunila, I. 2, Hégaret, H. 3, Ramón, M. 4, 5, Wikfors, G. H. 6 1 IRTA, St. Carles de la Ràpita, Spain 2 State of Connecticut, Department of Agriculture, Bureau of Aquaculture, Milford, CT, USA 3 University of Connecticut, Department of Marine Sciences, Groton, CT, USA 4 IEO-Centre Oceanogràfic de Balears, Palma de Mallorca, Spain 5 ICM-CSIC, Barcelona, Spain 6 Northeast Fisheries Science Center, NOAA Fisheries Service, Milford, CT, USA 1

2 Introduction Mussels: - bivalves harvested and consumed worldwide, also considered as sentinel animals for environmental quality. - known to have unusual tolerances to microalgal biotoxins and act as vectors (Bricelj et al., 1990; Marsden and Shumway, 1991; Wooton et al., 2003). - no studies concerning physiological or immune responses of M. edulis when feeding on toxic algae. Alexandrium fundyense (HAB) Prorocentrum minimum (HAB) histopathological and hemocyte responses Rhodomonas sp. M. edulis 2

3 Experimental design Computer-automated rearing-chamber system 20 L glass aquarium 16 feedings per day 3 aquaria per treatment Mussels size (44 77 mm) 2 experiments: Alexandrium fundyense Prorocentrum minimum toxicity 4x10 3 cells/ml 1x10 4 cells/ml 100 mussel/aquarium 45 mussel/aquarium On days 0, 3, 6 or 7, and 9 10 mussels/aquarium were removed 3

4 Experimental design 1. Hemocyte parameters (flow cytometry): cell density, size and internal complexity hemocyte mortality phagocytosis respiratory burst adhering hemocytes apoptotic hemocytes 2. Stomach contents 3. Histopathology (cross-section) 4. Feces and pseudofeces 4

5 Results P. minimum Toxicity Bay-scallop bioassay (Rosetta and McManus, 2003) : Argopecten irradians irradians seed 5 bay scallops/beaker filtered seawater filtered seawater diluted 1:1 Rhodomonas sp. 1.9x10 5 cell/ml P. minimum 1.9x10 5 cell/ml 20 h after the exposure all scallops exposed to P. minimum were dead bioactive against bivalves 5

6 Results P. minimum Mussel feeding response to P. minimum ingestion 1. Stomach contents, feces and pseudofeces: -Presence of P. minimum and Rhodomonas sp. - Hemocytes in feces of P. minimum-exposed mussels forming aggregations around the toxic cells. 6

7 Results P. minimum Pathological changes 2. Histopathology: Increase in pathological changes (day 3) Rhodomonas sp. 40 Hemocytes in the alimentary canal (3 days) Bacteria in the alimentary canal (3 days) Hemocytes in connective tissue between gonadal follicles (9 days) % affected mussels P. minimum Days 6 9 7

8 Results P. minimum Hemocytes in the stomach Bacteria in the intestine Hemocytes in connective tissue between gonadal follicles 100 µm 100 µm 8

9 Results P. minimum 3. Hemocytes and immunological analysis: Photomicrograph of hemocytes Hemocyte biplot from the cytometer Internal complexity of hemocytes showed significant differences on day 6 of the experiment. The immunological functions did not show significant differences throughout the experiment. 9

10 Results P. minimum DAY 0 P. minimum ingestion MUSSELS inflammatory response hemocytes diapedesis epithelial barrier alimentary canal increase in hemocytes bacterial proliferation encapsulation of P. minimum apoptosis necrosis DAY 9 infiltration into gonads 10

11 Results A. fundyense Toxicity HPLC analysis of the algal culture (units are pg cell -1 ) GTX1, ± 0.02 NEO 0.70 ± 0.07 GTX2, ± 0.01 B ± 0.00 STX 0.16 ± 0.00 C1, ± 0.06 total toxicity = 0,43 pg STXeq cell -1 Parkhill and Cembella (1999) Mussel feeding response to A. fundyense ingestion 1. Stomach contents, feces and pseudofeces: - Presence of A. fundyense and Rhodomonas sp. - A. fundyense was found mostly as temporary cysts in all cases. 11

12 Results A. fundyense 2. Histopathology: Increase in pathological changes (day 3) Blue-mucus secretory cells in gills (3 days) Hemocytes in the alimentary canal (7 days) Hemocytes in connective tissue between gonadal follicles (9 days) Ceroidosis (9 days) Trematodes (9 days) % affected mussels Pathological changes Rhodomonas sp A. fundyense 0 3 Days

13 Results A. fundyense Blue-mucus secretory cells in gills Ceroidosis Trematode 13

14 Results A. fundyense 3. Hemocytes and immunological analysis: Rhodomonas sp. A. fundyense Size basophilic * ± ± 50 Size eosinophilic ± ± 61 Size aggregates ± ± 149 Number basophilic 441 ± ± 58 Number eosinophilic * ± ± 258 Number aggregates 100 ± ± 11 Complexity basophilic * 208 ± ± 6 Complexity eosinophilic * 612 ± ± 33 Complexity aggregates * ± ±

15 Results A. fundyense Immunological functions: mussels exposed to A. fundyense had fewer, less-complex hemocytes than Rhodomonas-exposed mussels cells/ml Total number of circulating hemocytes Rhodomonas sp A. fundyense Day

16 Results A. fundyense DAY 0 A. fundyense ingestion MUSSELS inflammatory response hemocytes circulating hemocytes degranulation diapedesis decrease in size, number and granules pathological changes detoxification process compromised immune system DAY 9 infiltration into gonads lipofuchsin increase in parasite infestation blue, mucus-secretory cells in gills 16

17 Conclusions This work combined immuno- and histopathological methods to study effects of two HAB on mussels M. edulis Algal toxicity: both algae proved to be bioactive against mussels Mussel responses to ingestion P. minimum A. fundyense Stomach contents, feces and pseudofeces Present Present Histopathology Increase in pathologies after day 3 Hemocytes in the alimentary canal Hemocytes in the connective tissue between gonadal follicles Bacteria in the alimentary canal Increase in trematodes Blue-mucus secretory cells in gills Ceroidosis 17

18 Conclusions Mussel feeding response to the toxic alga ingestion P. minimum A. fundyense Hemocytes - Immunology Internal complexity Size Total number Detoxification Encapsulation Lipid peroxidation M. edulis exposed to P. minimum and A. fundyense are themselves affected by the biotoxins. Effects are more severe with longer exposure to both HAB species tested. Different harmful algal species can stimulate very different immunological and pathophysiological pathways in a single bivalve species, thus, it is necessary to study the effects and detoxification pathways for specific bivalve/algal interactions to project risk of economic losses in marine fisheries and aquaculture to HABs. 18

19 Thanks to: Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Milford Laboratory staff: Jennifer H. Alix, April N. Croxton, Mark S. Dixon, Diane Kapareiko, Yaqin Li, Shannon L. Meseck, and Barry C. Smith. Bureau of Aquaculture staff: John Karolus and Joseph DeCrescenzo IRTA staff: Pablo de la Iglesia and Jorge Diogène More information: Galimany, E., Sunila, I., Hégaret, H., Ramón, M., Wikfors, G. H. (2008) Experimental exposure of the blue mussel (Mytilus edulis, L.) to the toxic dinoflagellate Alexandrium fundyense: histopathology, immune responses, and recovery. Harmful Algae 7, Galimany, E., Sunila, I., Hégaret, H., Ramón, M., Wikfors, G. H. (2008) Pathology and immune response of the blue mussel (Mytilus edulis L.) after an exposure to the harmful dinoflagellate Prorocentrum minimum. Harmful Algae 7,