Improvement of insect ecdysome on the hatching rate of Artemia eggs

1. Improvement of insect ecdysome on the hatching rate of Artemia eggs Jia Qinxian1 Chen Lijing2 Zhen Mianping1 1 Open Laboratory of Saline Lake Resources and Environment, The Chinese Academy of Geological Sciences, Beijing, China 2 Shanghai Fisheries University, Shanghai, China

2. Content • 1. Introduction • 2.Treatment and Establish normal criterion method • 3. Results • 4. Conclusion

3. 1.IntroductionConcept：Diapause and Dormancy Diapause-----生长的停滞期，间歇期 Dormancy----睡眠，冬眠，隐匿

4. 1. Introduction:Understanding of Artemia eggs structure Outer membrane Outer lay shuck Inner lay shuck Embryon Embryonic membrane Inner membrane

5. 1. Introduction:Understanding of Artemia eggs structure Embryon Embryonic membrane

6. 1. Introduction:Study progress of Artemia eggs diapause (1)Structure Outer membrane Diapause Activation Morris and Afzelius (1967): Outer membrane broken Anderson et al. (1970) : shuck thinned

7. 1.Introduction:Study progress of Artemia eggs diapause (2)Structure New productive eggs Decapsulation but no activation Diapause Diapause Lavens and Sorgeloos (1987)

8. 1.Introduction:Study progress of Artemia eggs diapause (3)pHi pHi>7.9 diapause eggs pHi pHi Diapause Activation Some scholars find that the eggs was diapause at pHi over7.9 and guess the diapause eggs will activation at pHi reduced to acidity

9. 1. Introduction:Study progress of Artemia eggs diapause (4)Biotic active matter Dutrieu (1960) Nelis et al. (1984 a,b; 1987) Konrad Dabrowski (19991) Biotic active matter Biotic active mater have important regulation for diapause eggs activition

10. 1. Introduction:Study progress of Artemia eggs diapause (5)problem The Artemia eggs diapause mechanism not be complete comprehended The methods of activation of diapause eggs was not always efficiency for all strains Up to now, there are a great lot of low hatching quality eggs produced in Asia ,Europe and other places. Which should be improvement.

11. 1. Introduction:Some study progress of diapause in insects and other animal 郭郛等，1976， 昆虫的激素（insects hormone） 伊淑霞等，1991， 天蚕卵滞育的解除 姜在阶，1987，蜱类的滞育现象 王宗舜，1985，-蜕皮激素对蓖麻蚕环核苷酸水平的影响 魏定义、郭郛，1985，外源蜕皮激素对 蓖麻蚕蛹发育的效应 The diapause eggs become active eggs when treatment by insect hormone

12. 2. MaterialsArtemia strains

13. Chemical method: • 3% H2O2 5min 10% NaClO 5min • 5% NH4OH 5min 5% MgCl2 60min • 5% CH3OH 30min 1% NaOH 5min • 1M HCl 60min 5%CH2O 5min • Physical method : • -25℃freeze 4 to 20 weeks • re-dehydrate 3 times • Hormone: • Insect ecdysome [2b, 3b, 14a, 22(R), 25- • Pentahydroxy -7- cholesten-6-one]

14. 2.Methods(1) Hormone treatment • Concentration • 0~90ppb，5~25ppm • Dry Dipping Hormone Hatching • eggs 180 min treatment • 30~150 min • time 24 h and 48h hatching rate Fixing Fixing

15. Dry eggs Fixing Dehydration Paraffine embed Slice up Observation under microscope Dyeing Observation

16. 2. Methods(2) Observation :dye cells/not dye cells by Gimmsa biotic pigment cell no division (not dye cell) cell division (dye cell) Aim: Establish normal criterion for more times examining and could be calculated easily

17. 3.Results -- (1) Establish normal criterionComparison of dye/not dye cells of dry eggs* *Collected and experimental time of Artemia eggs: 1995~1996

18. (1) Establish normal criterionComparison of dye/not dye cells of wet eggs* *Collected and experimental time of Artemia eggs: 1995~1996

19. (1) Establish normal criterionHatching rate (%) and Dye cells/not Dye cells 1.4 1.2 1.0 Dye cells / not dye cells Hatching rate (%) 0.8 0.6 0.4 0.2 Strains (Collected and experimental time of Artemia eggs: 1995~1996)

20. (1) Establish normal criterionQuality grading criterion Hatching rate The rate of dye cells / not dye cells

21. 3. Results (2) Hormone treatmentSHY strain(from Haiyang of Shangdong, China) The rate of dye cells / not dye cells 30 60 90 120 150 min Treatment time (Collected and experimental time of Artemia eggs: 1999~2000)

22. (2) Hormone treatmentSHY strain(Haiyang of Shangdong, China) The rate of dye cells / not dye cells 5 15 25 35 45 ppb Treatment dosage (Collected and experimental time of Artemia eggs: 1999~2000)

23. (2) Hormone treatmentRUS strain(Russia) The rate of dye cells / not dye cells 30 60 90 120 150 min Treatment time (Collected and experimental time of Artemia eggs: 2000~2001)

24. (2) Hormone treatmentRUS strain (Russia) The rate of dye cells / not dye cells 5 15 35 45 ppb 25 Treatment dosage (Collected and experimental time of Artemia eggs: 2000~2001)

25. (2) Hormone treatmentQGH strain(fromGahai of Qinghai,China ) The rate of dye cells / not dye cells 30 60 90 120 150 min Treatment time (Collected and experimental time of Artemia eggs: 1999~2001)

26. (2) Hormone treatmentQGH strain(fromGahai of Qinghai,China ) The rate of dye cells / not dye cells 5 15 25 35 45 ppb Treatment dosage (Collected and experimental time of Artemia eggs: 1999~2001)

27. (2) Hormone treatmentKSK strain(from Kazakhstan) The rate of dye cells / not dye cells 30 60 90 120 150 min Treatment time (Collected and experimental time of Artemia eggs: 2000~2001)

28. (2) Hormone treatmentKSK strain (from Kazakhstan) The rate of dye cells / not dye cells 5 15 25 35 45 ppb Treatment dosage (Collected and experimental time of Artemia eggs: 2000~2001)

29. Efficiency compare of different treatment method Hatching rate

30. 4.Conclusion • The superexcellent concentration and fitness treatment time of each strain is difference. • The raise of the hatching quality is very obvious at its fit concentration and time. • The shortage concentration of hormone will no obvious effect on most strains

31. 4.Conclusion • More over concentration of hormone will obvious decrease the hatching rate. When the concentration over 35 ppb the hatching rate was maybe littler than that of no treatment • At the ecdysome concentration range 10 to 20 ppb and the treatment time range 30 to 60 min, the hatching rate of most strains was greatly improved.

32. 4.Conclusion • At superexcellent concentration and fitness treatment time, the hormone treatment was higher than the treatment by chemical reagent and physical method (-25℃freeze 4 to 20 weeks). • The highest hatching of Haiyang strain was over 57.1% than no treatment, over 10.5% than treatment by H2O2 and freeze.

33. Thank you very much！ END