p1: The European amphioxus species we are working with, Branchiostoma lanceolatum
p1: lives in the sand of shallow waters.
p1: The population density is high at around 5metres deep at the Racou Beach near the
p1: city of Argelès-sur-mer, located in the southwestern part of the French Riviera.
p1: To collect ripe animals, the sailors of the Observatoire use the Nereis boat from
p1: the lab, and sail from the Banyuls- sur-mer harbor -where the observatoire
p1: is located- to the Racou beach once a week during the breeding season.
p1: Using a dredge attached to a cable that they drag several meters the sailors recover
p1: the first centimeters of sand from the seabed.
p1: The collected sand is then transferred into containers and the sailors quickly
p1: check for the presence of amphioxus.
p1: If there are amphioxus, they bring the sand back to Banyuls.
p1: Once they are back, they deposit the containers on the port and we use sieves
p1: and containers filled with sea water to recover the animals buried in the sand.
p1: We transfer the adult amphioxus into aquariums, and once all the sand has
p1: been sieved, we bring the animals to the amphioxus facilities.
p1: The amphioxus facility is a dark room with an air conditioning system to maintain the
p1: temperature at around 18 to 20°C.
p1: In this room, several cabinets are installed.
p1: In each of them, the light is controlled
p1: and the aquariums are maintained in
p1: a water bath, the temperature of which is controlled by the use of a
p1: heater and a cooler.
p1: The seawater of each aquarium is changed several times per day
p1: by using sea water stored in a tank above the cabinet
p1: and its temperature is controlled in the same way.
p1: We separate the adult amphioxus collected from the sand
p1: and place around 30 animals in each small aquarium.
p1: In the aquarium we add a little bit of sand coming from the same place
p1: the animals were collected so that they can burrow into it.
p1: The aquariums are labelled and installed in the cabinet
p1: in which temperature is maintained at 17° with a light-dark cycle
p1: of 14 hours, 10 hours, the light period starting at 7am and ending at 9pm.
p1: Every day a member of the team comes to verify that there are no dead animals
p1: in any tanks and the water changes are working correctly.
p1: The day before the day we want spawning, animals that were kept for at least 2 weeks
p1: in the facility at 17° are taken out of the sand of the aquarium
p1: and placed in an aquarium only filled with sea water.
p1: This tank is deposited in an incubator in which a temperature is maintained at 23°
p1: and in which a light-dark cycle similar to the one of the cabinets is applied.
p1: The number of animals we use depends on the number of gametes
p1: needed for experimentation.
p1: The amphioxus spawn naturally at sunset.
p1: During the afternoon on the spawning day we take the animals out of the facility
p1: and move them to a dark room with the light on close to our lab space.
p1: We place each animal into a plastic cup filled with a small volume of
p1: filtered seawater
p1: and the cups are positioned above a black surface that allows for
p1: egg visualization after spawning.
p1: At 9pm, we turn off the light in the room and we leave the animals for one hour.
p1: Then by entering the room was a red light so as not to disturb the animals
p1: we check for spawning.
p1: If the animal in the cup is a male,
p1: the water becomes milky when the sperm is released.
p1: If the animal is a female, the small eggs are visible at the bottom of the cup
p1: thanks to the black covering of the bench.
p1: We then bring the animals that spawned to the lab space.
p1: If we want to use the eggs for microinjection, they are placed in
p1: small Petri dishes and kept at 4° where they can be stored several hours.
p1: If we want to directly work with embryos, eggs are transferred in bigger Petri dishes
p1: filled with filtered seawater.
p1: We mix the sea water with the sperm of all the males that spawned
p1: and we keep it on ice.
p1: When we want to fertilise, we add a few drops of the sperm solution in the
p1: Petri dishes with eggs.
p1: To test if fertilization occurs normally, we check under the microscope.
p1: Just after fertilization, the chorion or fertilization membrane should swell up.
p1: If it is the case, we are ready to work with amphioxus embryos!