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

Content

1. Introduction 2.Treatment and Establish normal

criterion method 3. Results 4. Conclusion

1.Introduction

Concept ： Diapause and Dormancy

DDIIAAPPAAUUSSEE DDOORRMMAANNCCYY

Conditionunsuitability

No developing No developing

Condition suitability No developing Developing

Regulation Physiological Circumstance

Control mechanism Nerve system －hormone－body fluid－target cells

Conditionreflection

Diapause----- 生长的停滞期，间歇期Dormancy---- 睡眠，冬眠，隐匿

1. Introduction:Understanding of Artemia eggs structure

Outer membrane

Outer lay shuck

Inner lay shuckEmbryonEmbryonic membrane

Inner membrane

1. Introduction:Understanding of Artemia eggs structure

Embryon

Embryonic membrane

1. Introduction:Study progress of Artemia eggs diapause (1)

Structure

Outer membrane

Diapause Activation

Morris and Afzelius (1967): Outer membrane brokenAnderson et al. (1970) : shuck thinned

1.Introduction:Study progress of Artemia eggs diapause (2)

Structure

Diapause

Lavens and Sorgeloos (1987)

Diapause

New productive eggsDecapsulation

but no activation

1.Introduction:Study progress of Artemia eggs diapause (3)

pHi

pHi

pHi>7.9 diapause eggs

Diapause Activation

pHi

Some scholars find that the eggs was diapause at pHi over7.9 and guess the diapause eggs will activation at pHi reduced to acidity

1. Introduction:Study progress of Artemia eggs diapause (4)

Biotic active matter

Biotic activematter

Dutrieu (1960)Nelis et al. (1984 a,b; 1987)Konrad Dabrowski (19991)

Biotic active mater have important regulation for diapause eggs activition

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 notalways 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.

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

2. Materials Artemia strains

SIGN PRODUCING AREA REPRODUCTIONMODE

JGDSHYSYK

Guandong of Jiangshu, ChinaHaiyang of Shangdong, ChinaYangkou of Shangdong, China

CCC

ParthenogenesisParthenogenesisParthenogenesis

QGHSYC

Gahai of Qinghai, ChinaYanchi of Shanxi, China

II

ParthenogenesisBisexual

GSLKZKRUSSLPSNYTXC

Great Salt Lake, USAKazakhstanRussiaSan Luis Potos, MexicoSonora Yavaros, MexicoTexcoco, Mexico

IIIIII

BisexualBisexualBisexualBisexualBisexualBisexual

I-- inland saltlake C--coastal saltern

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 [2, 3, 14, 22(R), 25- Pentahydroxy -7- cholesten-6-one]

2.Methods(1) Hormone treatment

Concentration 0~90ppb ， 5~25ppm

Dry Dipping Hormone Hatching eggs 180 min treatment

30~150 min

time

Fixing

24 h and 48hhatching rateFixing

Dehydration Paraffine embedFixing

Slice up

DyeingObservation under microscope

Dry eggs

Observation

2. Methods(2) Observation :dye cells/not dye cells by

Gimmsa biotic pigment

cell division(dye cell)

cell no division(not dye cell)

Aim: Establish normal criterion for more times examining and could be calculated easily

3.Results -- (1) Establish normal criterion Comparison of dye/not dye cells of dry eggs*

repetitionStrains 1 2 3 4 5 6 7 8 Average

GSLJGDKZKQGHRUSSHYSLPSNYSYCSYKTXC

0.620.520.260.510.510.450.360.210.330.470.61

0.630.530.230.470.510.470.350.220.310.440.61

0.610.510.270.490.480.470.360.280.330.450.65

0.670.540.300.530.470.450.370.250.380.440.62

0.580.510.330.510.440.440.330.200.250.450.58

0.650.490.250.510.510.470.360.200.270.470.60

0.650.520.260.510.490.420.370.280.330.430.62

0.610.520.250.450.480.440.370.280.340.460.60

0.630.520.270.460.490.450.360.240.320.450.61

*Collected and experimental time of Artemia eggs: 1995~1996

(1) Establish normal criterion Comparison of dye/not dye cells of wet eggs*

RepetitionStrains 1 2 3 4 5 6 7 8 Average

GSLJGDKZKQGHRUSSHYSLPSNYSYCSYKTXC

1. 520. 770.400. 740. 760. 580. 410. 330. 520. 610.97

1. 410. 700.430. 740.770.620.500.450.530.640.80

1. 310.750.310.730.690.490.410.360.500.570.86

1.020.740.330.710.690.490.440.380.480.560.85

1.100.750.390.700.760.550.400.350.550.580.84

1.340.740.420.690.700.500.390.300.510.610.79

1.310.730.300.730.680.520.390.290.510.610.88

1.520.710.330.780.730.530.400.290.550.550.97

1.320.740.360.730.720.530.410.340.520.590.87

0 .*Collected and experimental time of Artemia eggs: 1995~1996

(1) Establish normal criterion Hatching rate (%) and Dye cells/not Dye cells

SNY KSK SLP SYC SHY SYK RUS QGH J GD TXC GSL

0

20

40

60

80

100

Dry eggsWet eggsH %

0.2

0.4

0.60.8

1.0

1.2

1.4

Strains

Dye

cel

ls /

not

dye

cel

ls

Hat

chin

g ra

te (

%)

(Collected and experimental time of Artemia eggs: 1995~1996)

(1) Establish normal criterion Quality grading criterion

Hatching rate 100%~80% 79%~60% 59%~40% 39%~20% 19%~0

Dye/not dye cells 1.3785-1.1534 1.1421-0.9283 0.9171-0.7032 0.6920-0.4781 0.4669-0.253

Quality grading I II III IV V

0

20

40

60

80

100

0 0. 5 1 1. 5

Hat

chin

g ra

te

The rate of dye cells / not dye cells

3. Results (2) Hormone treatment SHY strain (from Haiyang of Shangdong, China)

0. 2

0. 4

0. 6

0. 8

1

1. 2

1. 45ppb15ppb25ppb35ppb45ppbBl ank

30 60 90 120 150 minTreatment time

The

rat

e of

dye

cel

ls /

not d

ye c

ells

(Collected and experimental time of Artemia eggs: 1999~2000)

(2) Hormone treatment SHY strain(Haiyang of Shangdong, China)

Treatment dosage

0. 2

0. 4

0. 6

0. 8

1

1. 2

1. 4 30mi n60mi n90mi n120mi n150mi nBl ank

5 15 25 35 45 ppb

The

rat

e of

dye

cel

ls /

not d

ye c

ells

(Collected and experimental time of Artemia eggs: 1999~2000)

(2) Hormone treatment RUS strain(Russia)

Treatment time

0. 2

0. 4

0. 6

0. 8

1

1. 2 5ppb15ppb25ppb35ppb45ppbBl ank

30 60 90 120 150 min

The

rat

e of

dye

cel

ls /

not d

ye c

ells

(Collected and experimental time of Artemia eggs: 2000~2001)

(2) Hormone treatment RUS strain (Russia)

0. 2

0. 4

0. 6

0. 8

1

1. 2 30mi n60mi n90mi n120mi n150mi nBl ank

5 15 25 35 45 ppb

Treatment dosage

The

rat

e of

dye

cel

ls /

not d

ye c

ells

(Collected and experimental time of Artemia eggs: 2000~2001)

(2) Hormone treatment QGH strain(fromGahai of Qinghai,China )

Treatment time

0. 2

0. 4

0. 6

0. 8

1

1. 2 5ppb15ppb25ppb35ppb45ppbBl ank

30 60 90 120 150 min

The

rat

e of

dye

cel

ls /

not d

ye c

ells

(Collected and experimental time of Artemia eggs: 1999~2001)

(2) Hormone treatment QGH strain(fromGahai of Qinghai,China )

Treatment dosage

0. 2

0. 4

0. 6

0. 8

1

1. 2

30mi n

60mi n

90mi n

120mi n

150mi n

Bl ank

5 15 25 35 45 ppb

The

rat

e of

dye

cel

ls /

not d

ye c

ells

(Collected and experimental time of Artemia eggs: 1999~2001)

(2) Hormone treatment KSK strain(from Kazakhstan)

Treatment time

0. 2

0. 4

0. 6

0. 8

1

1. 2 5ppb15ppb25ppb35ppb45ppbBl ank

30 60 90 120 150 min

The

rat

e of

dye

cel

ls /

not d

ye c

ells

(Collected and experimental time of Artemia eggs: 2000~2001)

(2) Hormone treatment KSK strain (from Kazakhstan)

Treatment dosage

0. 2

0. 4

0. 6

0. 8

1

1. 2 30mi n60mi n90mi n120mi n150mi nBl ank

45 ppb3525155

The

rat

e of

dye

cel

ls /

not d

ye c

ells

(Collected and experimental time of Artemia eggs: 2000~2001)

Efficiency compare of different treatment method

0

10

20

30

40

50

60

70

80

90

100

Not treatmentDecapsul ati onNaOHHClCH3OHFreezeHormone

Not treatment 20 45 12

Decapsul ati on 71 74 35

NaOH 56 50 46

HCl 38 42 35

CH3OH 59 44 25

Freeze 74 80 64

Hormone 85 90 72

SHY QGH KSK

0

20

40

60

80

100

SHY QGH KSK RUS

Not treatment

Decapsulation

NaOH

HCl

H2O2

CH3OH

Freeze

Hormone

Hat

chin

g ra

te

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

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.

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.

Thank you very much！

END