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.