Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Quantitative Investigations of Hatching in Brine Shrimp Cysts Charlie DrewesEcology, Evolution & Organismal BiologyIowa State University, Ames, IAArtemia franciscana North American brine shrimpClass Branchiopodagilled feetmeaning: “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 eggsArtemia life cyclefertilized eggs in brood sacAdult femalerelease of encysted gastrula stage embryos = ‘winter eggs’Artemia franciscana (dry, encysted ‘winter eggs’)HATCHArtemia life cycleXwinter ‘eggs’ASWAdvantages 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)‘Paint’ cysts on double-stick tape with small, soft, dry paintbrushMethod AMethod Apaint cysts on sticky circleplastic petri dishtransparency strip with grid10 mm x 10 mmstrip of double-stick tape covers grid tape holds strip to dishMethod B Cysts painted on sticky grid with brushHatching of encystedArtemia embryosA1-A2: Encysted gastrulae in ASW and stuck to tapeB: Cyst swells and wall splitsC1-C2: ‘Umbrella’ stage emerges (membrane-bound) D1-D2: Release of free-swimming nauplius larvaeA1BA2C1C2 D1 D2Each day, a pipet is used to remove and count hatched, swimming nauplius larvaeTreatment Group (control or experimental) _________________ Initial number of cysts in dish _____# of hatched nauplius larvae removedCumulative # of hatched nauplius larvae (day 1 + 2 + 3…)Cumulative hatching % (cumulative # of hatched larvae / initial number of cysts X 100)Day 0 startDay 1(24 h)Day 2(48 h)Day 3(72 h)Day 4(96 h)050CONTROL0 0%22 22 44%3911 33 6 1 4078%80%66%%HATCHTypical hatching curve:2. ambient light effects during hydration5. second hydration1. dry cysts exposed to thermal extremes3. temperature effects during hydration4. salinity effectsExamples of experimental variables that may affect hatching curves6. oxygenation effects during hydration7. environmental toxicant effects8. irradiation effects on dry or hydrating cystsMeasurements & End-points * Hatching % * hatching time-coursedevelopmental anomalieslarval motility molting & growth'N Cyst'N CystA 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.'N Cyst'N CystA 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