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1 South China Fisheries Science Vol. 12, No. 6 Dec., 2016 doi: / j. issn , 1, 1, 1, 2, 3 ( 1.,, ; 2., ; 3.,, ) : ( ARS) ( CAL) 2 ( Acanthopagrus schlegelii), 2 2 ;, 2 ARS CAL, ARS CAL 2, ARS, CAL, ARS,, ; CAL, ARS : ; ; ; ; : S : A : ( 2016) Comparative study on immersion marking with alizarin red S and calcein for black porgy Acanthopagrus schlegelii of different sizes LIU Yan 1, SUN Dianrong 1, GENG Qian 1, YANG Changping 1, ZHAO Jing 2, DUAN Yan 3 ( 1. Key Lab. of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture; South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou , China; 2. Dalian Fisheries Research Institute, Dalian , China; 3. Key Lab. of Marine Biological Resources and Ecology, Liaoning Ocean Fishery Science Research Institute, Dalian , China) Abstract: We used alizarin red S ( ARS) and calcein ( CAL) to dip black porgy ( Acanthopagrus schlegelii) of two sizes by fluorescent markers technique. Acute staining results show that ARS and CAL had no significant effects on the survival rate of the juveniles of both sizes. Subsequent breeding test shows no significant effects on juveniles of both sizes. The markers effects of ARS and CAL on hard tissues of the juveniles increased with increasing dye concentration, but the markers effects of ARS were better than those of CAL. No significant difference was observed for ARS, but the marker effect of CAL of small size was better than that of big size. The marker effect of ARS of fin spines was the best, followed by scales and fins, and the worst was that of otolith. CAL marker effects were at the same level for spines, fins and scales, followed by otolith. ARS marking technology has more application and research value. Key words: Acanthopagrus schlegelii; alizarin red S; calcein; fluoresent markers technique; hard tissues : ; : : ( ) ( ) ; ( ) ( 2015 TS07 ) ; ( ) : ( ),,,, @ qq. com : ( ),,,, drsun73@ 163. com

18 12 ( Acanthopagrus schlegelii), 1. 2 [ 1 ],, ARS CAL 2, 40 60 m, ARS 3 ( 100 mg L - 1, 300 mg L - 1 500 mg L - 1 ), CAL, ;, 3 ( 50 mg L - 1 150 mg L - 1 250 mg ;, L - 1 ) 105 ( 3.

2 18 12 ( Acanthopagrus schlegelii), 1. 2 [ 1 ],, ARS CAL 2, m, ARS 3 ( 100 mg L - 1, 300 mg L mg L - 1 ), CAL, ;, 3 ( 50 mg L mg L mg ;, L - 1 ) 105 ( 3. 5 L - 1 ), [ 2-3 ] 24 h, ( 140 cm 70 cm 120 [ 4-5 ], cm) 48 h, [ 6 ] ; [ 7 ] [ 8] ( n = 30), 3 ( 140 cm 70 : 1) cm 120 cm, 80 cm,, 2), 3) L - 1 ), [ 9], 4), 5) ; , ( ), mg L - 1, ph , [ 10-11] 14 10, [ 12 ],, 25%,,, 0. 1 cm 0. 1 g, 60 d [ 13] ( ARS) ( CAL) 2, ,, ) AMG,, 2 :, ( ) cm;, ) EVOS-xl( ) AMG ( ) cm, LIU [ 12 ] ( L - 1 ) 5 m 3 ( 250 cm 250 cm 150 cm, 80 cm), 10 d ( 1 # ), 1, , ( ), ( 105 ) ( 30 ),,, EVOS-fl( ( 6 ( 1) 2, [ 14 ], 2, ( 1 ) ( 10 ) ( 3 5 ) mg L - 1, ph , ( 3 5 ) [ 1 5] 14 10, 25%,

6 : 19 Tab. 1 marker definition level 1 Marker definition level and division standard division standard 0 level 0 1 level 1 2 level 2 3 level 3 4 level 4 5 level 5 1. 5 Excel 2010 SPSS 19. 0, > 0.

3 6 : 19 Tab. 1 marker definition level 1 Marker definition level and division standard division standard 0 level 0 1 level 1 2 level 2 3 level 3 4 level 4 5 level Excel 2010 SPSS 19. 0, > 0. 05) ( 2 3) ARS 24 h ARS, 300 mg L mg L ARS 500 mg L - 1, ; CAL 48 h, d ARS, % ( 7 /630), CAL ; ARS CAL 2, 0( 0 /630), CAL, 2 ( P >, ARS CAL, 0. 05), ARS CAL d 20 d 40 d 60 d ( P > ) ( 1) 2. 2, ( 3 ( 0. 63% ), ), 2, [ 9 ] [ 17] MORAN [ 18 ] [ 1 9] ( 3), ARS,, CAL [ 2 0] , ARS ; CAL , CAL 50 mg L - 1 ( P <0. 01) ; CAL < 0. 05) ; ( P, ARS CAL,,, ARS 300 mg L - 1 [ 16 ] 2, CAL, 150 mg L - 1 ( P < 0. 01), 250 mg L - 1 ( P,

4 d 20 d 40 d 60 d Fig. 1 Mean body length and weight of black porgy of two sizes after ARS and CAL treatments Data were collected on the 0, 20 th, 40 th and 60 th day during the growth experiment.

5 6 : h, 2 60 d ( ) Fig. 2 Marker effects of black porgy of two sizes detected in scale, fin ray, fin spine and otolith by batches of ARS and CAL treatments No markers were detected in the control ( vertical bars show standard error). [ ],,, CAL( 250, mg L - 1 ), [ 23 ],, ARS,,, [ 24] [ 25 ] [ 26 ] ; CAL, [ 1 5] [ 29 ],,,, ARS [ 9 ] CAL,, :, BAER R ESCH [ 27 ] ARS, < ph, ARS 150 mg L mg L - 1 ARS CAL, ( 40 d ) [ 28 ],, < 2

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7 6 : 23 Fig. 3 3 Photomicrographs of black porgy of two sizes in scale, fin ray, fin spine and otolith by batches of ARS and CAL treatments, ECKMANN [ 2 1] BAER R ESCH [ 27 ] BASHEY [ 30 ] ( Salmo trutta) ( Coregonus albula) ( Poe-, cilia reticulata),, ARS 24 h [ 31 ] ARS CAL,, : ARS,, [ 1]. [ M]. :, 1962:

24 12 [ J]., 2013, 9 ( 5 ) : 100-108. [ 3]. [ J].,, 2011, 32( 6 ) : 135-140. 2005, 1 ( 1) : 11-20. [ 4],,,. [ J]., 2013, 31( 2 ) : 273-280. [ J]., 1998, 7 ( 2) : 167-171., 1991( 50) : 1-10.

8 24 12 [ J]., 2013, 9 ( 5 ) : [ 3]. [ J].,, 2011, 32( 6 ) : , 1 ( 1) : [ 4],,,. [ J]., 2013, 31( 2 ) : [ J]., 1998, 7 ( 2) : , 1991( 50) : Archiv fur Hydrobi- [ 6],,,. ologie, Special Issues, Advance in Limnology, 1998, 50: [ J]. ( ), 2009, 28 ( 4 ) : , 2001, 25 ( 1) : [ 8] LORENZEN K, LEBER K M, BLANKENSHIP H L. Responsible approach to marine stock enhancement: an update [ J]. Rev Fish Sci, 2010, 18( 2 ) : [ 9 ]. [ 24],,,. [ D]. :, 2013: [ 10],,,. [ J]. [ 25],,,., 2010( 4) : [ 11],. [ J]., 2011, 7( 1 ) : [ 12] LIU Q, ZHANG X M, ZHANG P D, et al. The use of alizarin red S and a lizarin complexone for immersion marking Japanese flounder Paralichthys olivaceuse( T. ) [ J]. Fish Res, 2009, 98 ( 1 /2 / 3 ) : [ 13],,,. 2 Appl Ichthyol, 2008, 24( 1 ) : [ J]., 201 4, 10 ( 3 ) : [ 14 ] TAYLOR M D, FIELDER D S, SUTHERS I M. Batch marking of otoliths and fin spines to assess the stock enhancement of Argyrosomus japonicus[ J]. J Fish Biol, 2005, 66 ( 4) : [ 15 ] FRENKEL V, KINDSCHI G, ZOHAR Y. Noninvasive, mass marking of fish by immersion in calcein: evaluation of fish size and ultrasound exposure on mark endurance[ J]. Aquaculture, 2002, 214 ( 1 /2 /3 /4) : calcein for inducing a nonlethally detectable mark in juvenile gup- [ 16],,,. pies[ J]. Trans Am Fish Soc, 2004, 133( 6) : [ J]., 2014( 2) : [ 17]. [ D]. :, 2013: [ 2],,,. [ 18] MORAN A L. Calcein as a marker in experimental studies newlyhatched gastropods[ J]. Mar Biol, 2000, 137( 5 / 6) : [ 19],,. [ J]. [ 20],,,. [ 21] ECKMANN R, CZERKIES P, HELMS C, et al. Evaluating the [ 5]. [ R]. effectiveness of stocking vendace ( Coregonus albula( L. ) ) eleutheroembryos by alizarin marking of otoliths[ J] [ 22] SECOR D H, WHITE M G, DEAN J M. Immersion marking of [ 7],,,. [ J]. larval and juvenile hatchery-produced striped bass with oxytetracycline[ J]. Trans Am Fish Soc, 1991, 120 ( 2) : [ 23],,,. ( Sinocyclocheilus grahami) [ J]., 2013, 34 ( 5) : [ J]., 2015( 24) : [ J]., 2011, 41 ( 2 ) : [ 26],,,. [ J]., 2014, 21 ( 6 ) : [ 27] BAER J, R ESCH R. Mass-marking of brown trout ( Salmo trutta L. ) larvae by alizarin: method and evaluation of stocking[ J]. J [ 28] HOYLAND J. Fluorescent probes in practice: potential artifacts. Fluorescent and luminescent probes for biological activity[ M]. San Diego: Academic Press, 1999 : [ 29] DRIESSEN M, KIENHUIS A S, PENNINGS J L, et al. Exploring the zebrafish embryo as an alternative model for the evaluation of liver toxicity by histopathology and expression profiling [ J]. Arch Toxicol, 2013, 87( 5 ) : [ 30] BASHEY F. A comparison of the suitability of alizarin red S and [ 31] LEIPS J, BARIL C T, RODD F H, et al. The suitability of calcein to mark poeciliid fish and a new method of detection[ J]. Am Fish Soc, 2001, 130( 3 ) : Trans

2 : 111 ( Pinctada maxima), 13, 11,,,,,, 15, 25 cm, cm, 4 5 kg 1-2],,, 3-7] 24] 25-26],,, ] min, 1, 5 m 4 m, 20 m , 9-22],,, (

2 : 111 ( Pinctada maxima), 13, 11,,,,,, 15, 25 cm, cm, 4 5 kg 1-2],,, 3-7] 24] 25-26],,, ] min, 1, 5 m 4 m, 20 m , 9-22],,, ( 12 2 2 0 1 6 4 South China Fisheries Science Vol. 12, No. 2 Apr., 2016 doi: 10. 3969/ j. issn. 2095-0780. 2016. 02. 016,,,, (,,, 510300) : ( Pinctada maxima),,,, 54. 55%, 0 0. 25 m - 2, 0 70. 31 gm - 2,