1. Quantitative Investigations of Hatching in Brine Shrimp Cysts Charlie Drewes Ecology, Evolution & Organismal Biology Iowa State University, Ames, IA

2. Artemia franciscana North American brine shrimp

3. Class Branchiopoda gilled feet meaning: “gilled feet” ● crustacean subgroup ● almost all freshwater ● use leaf-like appendages to feed, swim & respire ● e.g. fairy shrimp, brine shrimp, clam shrimp, tadpole shrimp & daphnids ● often produce anabiotic eggs

4. Artemia life cycle fertilized eggs in brood sac Adult female release of encysted gastrula stage embryos = ‘winter eggs’

5. Artemia franciscana (dry, encysted ‘winter eggs’)

6. HATCH Artemia life cycle X winter ‘eggs’ ASW

7. Advantages of “stuck-on Artemia” > easy transfer & handling of cysts > quantitate starting # of cysts and # hatched > follow development & hatching of single cysts > view hatching cysts in a single focal plane > high rates of hatching & survival > conservative use of cysts > no aeration required > open-ended investigations (many variables)

8. ‘Paint’ cysts on double-stick tape with small, soft, dry paintbrush

9. Method A Method A paint cysts on sticky circle

10. plastic petri dish transparency strip with grid 10 mm x 10 mm strip of double-stick tape covers grid tape holds strip to dish Method B

11.  Cysts painted on sticky grid with brush

12. Hatching of encysted Artemia embryos A1-A2: Encysted gastrulae in ASW and stuck to tape B: Cyst swells and wall splits C1-C2: ‘Umbrella’ stage emerges (membrane-bound) D1-D2: Release of free-swimming nauplius larvae A1 B A2 C1 C2 D1 D2

13. Each day, a pipet is used to remove and count hatched, swimming nauplius larvae

14. Treatment Group (control or experimental) _________________ Initial number of cysts in dish _____ # of hatched nauplius larvae removed Cumulative # of hatched nauplius larvae (day 1 + 2 + 3…) Cumulative hatching % (cumulative # of hatched larvae / initial number of cysts X 100) Day 0 start Day 1 (24 h) Day 2 (48 h) Day 3 (72 h) Day 4 (96 h) 0 50 CONTROL 00% 22 44% 39 1133 6 140 78% 80% 66%

15. %HATCH%HATCH Typical hatching curve:

16. 2. ambient light effects during hydration 5. second hydration 1. dry cysts exposed to thermal extremes 3. temperature effects during hydration 4. salinity effects Examples of experimental variables that may affect hatching curves 6. oxygenation effects during hydration 7. environmental toxicant effects 8. irradiation effects on dry or hydrating cysts

17. Measurements & End-points * Hatching % * hatching time-course developmental anomalies larval motility molting & growth

18. 'N Cyst A sea monkey's no monkey or chimp. It's crustacean - better known as brine shrimp. This genus Artemia Can deal with extremia. As a cyst stage, its hardly a wimp.

19. 'N Cyst A sea monkey's no monkey or chimp. It's crustacean - better known as brine shrimp. This genus Artemia Can deal with extremia. As a cyst stage, its hardly a wimp.