1. Artemia franciscana North American brine shrimp (Crustacea, Anostraca)

2. HATCH Artemia life cycle fertilized eggs in brood sac Adult female winter ‘eggs’

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

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

5. Method A paint cysts on sticky circle

6. 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

7.  Cysts painted on sticky grid with brush

8. STUCK-on ARTEMIA Technical Advantages       easy handling & transfer of cysts  uses small, quantifiable number of cysts  follow development of individual cysts  cysts stay in single focal plane  use dissecting or compound microscope  manipulation of many relevant variables

9. Hatching of encysted Artemia embryos A1-A2: Encysted late gastrula stage (immersed in sea water and stuck to tape) B: Cyst hydrates and swells; cyst wall splits C1-C2: “Umbrella” stage emerges (membrane-bound) D1-D2: Nauplius larvae are free swimming A1 B A2 C1 C2 D1 D2

10. Each day, use pipet to remove and count hatched, swimming nauplius larvae 

11. 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%

12. %HATCH%HATCH Typical hatching curve:

13. 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

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

15. '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.