Impact of the toxic dinoflagellateAlexandrium catenellaon Pacific oyster reproductive output: application of flow cytometry assays on spermatozoa

Abstract

-The toxic dinoflagellate Alexandrium catenella recurrently blooms on the coasts of France and produces Paralytic Shellfish Toxins (PSTs) that accumulate in bivalves. These toxins can affect various physiological functions including reproduction. The present study aims to validate measurements of sperm viability, DNA content and mitochondrial membrane potential in Pacific oyster Crassostrea gigas using flow cytometry coupled with fluorescent markers, and to use these measurements to assess the cellular parameters of sperm from Pacific oysters exposed to A. catenella. These parameters may influence fertilization, embryogenesis and larval development in free-spawning shellfish. Sperm viability and DNA content estimation were assessed using SYBR-14, which only penetrates cells with intact membranes. Cell mortality was measured with propidium iodide (PI), which penetrates cells with membrane damage. Mitochondrial membrane potential, used as an estimate of mitochondrial function, was measured using JC-1 dye, which selectively enters into mitochondria and reversibly changes colour from green to orange as the membrane potential increases. To assess the effect of toxic algae on oyster sperm, broodstock (ripe oysters) were fed toxic (A. catenella) or non toxic (Heterocapsa triquetra) dinoflagellates at 250 cell ml −1 for 9 days. After this exposure period, mature oysters were stripped and cellular responses of sperm analysed. Average DNA staining, as measured by SYBR-14, appeared lower and more variable in gametes from A. catenella-exposed oysters than in those from control oysters fed H. triquetra. Additionally, mitochondrial membrane potential of sperm from A. catenella-exposed oysters was significantly higher (1.5 fold) than that of sperm produced by oysters fed H. triquetra. Both the increase of mitochondrial membrane potential and the modification of DNA structure can be expected to impact spermatozoa ability to fertilize oocytes and could thus impact related reproductive processes.

The dinoflagellate Alexandrium minutum affects development of the oyster Crassostrea gigas, through parental or direct exposure

“…Short-term exposure of mature Pacific oysters to Alexandrium spp. affected spermatozoa quality, and in vitro exposure of oocytes to A. minutum increased reactive oxygen species production in oocytes (Haberkorn et al, 2010b;Le Goïc et al, 2013). Knowledge regarding potential effects of adult oyster exposure to ecological concentrations of HAB species on the next generation remains scarce (Rolton et al, 2018(Rolton et al, , 2016Vasconcelos et al, 2010).…”

ATP content and viability of spermatozoa drive variability of fertilization success in the Pacific oyster (Crassostrea gigas)

“…The resulting sperm suspensions were sieved through 60 µm mesh. Concentrations of spermatozoa were determined in duplicates by flow cytometry according to Le Goïc et al (2013), and were adjusted to 7 x 10 8 spermatozoa mL -1 by further dilution in FSW, in 20 mL plastic vials.…”

“…Flow cytometric (FCM) analyses of spermatozoa relative size and complexity, viability, MMP, and ROS production were performed according to Le Goïc et al (2013) using an EasyCyte Plus cytometer (Guava Millipore) equipped with standard optics and a 488 nm argon laser.…”

Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas

“…affect bivalve biology by modifying valve opening behaviour, filtration processes, nutrition, and cardiac activity (Gainey and Shumway, 1988a,b;Shumway, 1990;Shumway and Gainey, 1992;Lassus et al, 1999;Tran et al, 2010). Dinoflagellates also may interfere with reproduction of bivalves, altering spermatozoa quality (Haberkorn et al, 2010a;Le Goïc et al, 2013) or larval survival (Leverone et al, 2006;Shumway et al, 2006) and inducing inflammatory responses in digestive gland and gills of mussels and oysters (Galimany et al, 2008; Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish.…”