In Vitro Fertilization of B floridae Amphioxus

Several species of amphioxus can be found in temperate and tropical seas; the protocol given is designed for B. floridae. These can be collected in large numbers from Old Tampa Bay, Florida, or other sandy bays around the Gulf of Mexico. They can only be spawned between July and September; even then, successful spawning is obtained only once every 10-14 d.

1. Locate a suitable population of adult amphioxus, by sieving sand from subtidal regions. Sites around Old Tampa Bay include south of the Courtney Campbell Causeway and St. Petersburg beach—in each case, in around 1 m of water.

2. After 4 pm, collect several hundred amphioxus by digging sand and sieving. Transfer animals to clean seawater for transport and keep in the shade. Collect seawater from the same site.

3. Transfer ripe animals into beakers containing 50 mL seawater (up to 5 animals/ beaker). Keep males and females separate; the serially repeated gonads are white in ripe males, and yellow in females. Keep in the light at 25°C.

4. Attempts to collect eggs and sperm should be made between 9 pm and 1 am. Either place beakers of animals in the dark, or (more reliably) use a stimulator to give a brief (2-s) nonlethal shock of direct current (50 V in 10-ms pulses) to a beaker of animals.

5. If gametes are released from the gonopore, they should be transferred immediately to Petri dishes (eggs) or microfuge tubes (sperm) using clean Pasteur pipets. Use separate pipets for eggs and sperm, and keep sperm as concentrated as possible. Not every animal will spawn, even on a successful spawning night.

6. If no gametes are obtained within 30 min after electrical stimulation of 50-100 animals, it is likely that spawning cannot be induced on that night. In this case, return animals to the collecting site on the next day, and repeat steps 1-5.

7. In vitro fertilization should be set up with freshly obtained eggs and sperm. Add one drop of sperm suspension to several hundred eggs in a 9-cm Petri dish of seawater filtered through Whatman No. 1 paper. After 5 min, observation under a dissecting microscope should reveal the presence of an elevated membrane around each fertilized egg.

Change the water twice to flush away excess sperm.

8. At 25°C, first cleavage occurs after 45 min, second cleavage after 75 min, and gastrulation after 5 h.

9. Hatching from the fertilization membrane occurs at 10-12 h; at this stage, the embryo is a bean-shaped neurula actively swimming using epidermal cilia. Pour hatched cultures into 50-mL centrifuge tubes and direct an angle-poise lamp at the water surface for 20 min to attract swimming neurula away from debris. Transfer active neurulae from the top 10 mL water to fresh Petri dishes, using a P1000 Gilson pipetman (Gilson Co., Worthington, OH) fitted with a cut-off disposable tip.

10. Embryos are readily raised from 12 h (5 somite stage neurula) to 60 h in Petri dishes of filtered seawater, with little attention.

11. At 36 h, the larval mouth opens; feeding commences around 60 h. Beyond this stage, culture is considerably more difficult; ref. 2 should be consulted.

12. If embryos are to be fixed, e.g., for in situ hybridization, the embryo cultures may need to be concentrated prior to fixation. Pour cultures into 15-mL centrifuge tubes, spin at low speed (2000g) for 5 min, and dispose of all except the bottom 1 mL of water. The embryos in 1 mL seawater are then transferred to a microfuge tube and concentrated further by centrifugation at 6000g.

Was this article helpful?

0 0

Post a comment