( White Spot Syndrome,WSS) 1990,

Transcription

1 PERIODICAL OF OCEAN UNIVERSITY OF CHINA 37 (3) : May, 2007 Ξ, ΞΞ,,,, (, ) : (White Spot Syndrome WSS), , 128, 20, 5 PCR PCR ( White Spot Syn2 drome Virus WSSV),5 10,56. 3 % ;, %,6,8,9,10 1, 60 % 20 1, 5 % ;, 40 %, WSSV, WSSV : ; ; ; PCR : S : A : (2007) ( White Spot Syndrome,WSS) 1990, [127 ], [8213 ],,, 1 2,, [9,11,13214 ],WSSV [14215 ], WSSV, 1 ( B ra2 chionus urceus) ; WSSV, WSSV,,, WSSV [9,11,13214 ], WSSV, ,, WSSV, WSSV , , ;5 20 ( Fenneropenaeus chi nesis),6, , ,, 2 h ( Calanoida),, ml, SEMP2Tris ( ED2 TA 70 mm, 0. 5 %,Tris HCl 10 mmol/ L, ) [16 ], cm, 30 s, (1 1), r/ min 5 min,300,, ; Ξ ΞΞ : ( ) ; ( G ) ; (2001BA505B) : ; : : (19712),, E2mail com ; Tel : edu. cn

2 ml, SEMP2Tris, DNA SEMP2Tris, - DNA [17218 ] : SEMP2Tris, 10 min, r/ min 8 10 min ; 180 L, 2 mol/ L NaCl 20 L 400 L, h ; r/ min 8 10 min,, 800 L 70 % 1 ; r/ min 8 min,, 36 L TE ( 10 mmol/ L Tris Hcl,1 mmol/ L ED TA p H8. 0) PCR PCR 25 L : L,10 Buffer2. 5 L,25 mmol/ L MgCl L,dN TP 2. 5 L ( da TP dctp d GTP 2 mmol/ L,dU TP4 mmol/ L),10 mol/ L 2. 5 L,5 U/ L Taq L, 1 L DNA 5 - C C A A G A C A T A C T A G C G G A T A - 3 (O 1 ) 5 - G A C G A C C C T G A C A G A A T T A C - 3 (O 2 ) 235 bp ; 5 - G G G A A G T G A A T A C G C A G T G A - 3 ( I 1 ) 5 - G T T C T A G G G C A A A C A A T G G C - 3 ( I 2 ), 101 bp [16 ] PCR 95 2 min 1 ;95 30 s,54 30 s,72 1 min,30 ;72 5 min, PCR PCR min, 1 L,80 2 h, h, DIG, h ; 2 SSC/ 0. 1 %SDS,0. 1 SSC/ 0. 1 %SDS ; DIG 30 min, NB T/ BCIP [18 ] DNA ; PCR PCR [19 ] PCR , 72, % 5 48, 18, %( 1),, WSSV ,42, 70 %( 2), , 60 %( 3) 3, 5, 90 %, 8, 80 % ( 1) WSSV Fig. 1 The WSSV carrying status in the copepoda, which were sampled from the pond in May 2003, detected by PCR2dot2 bolt hybridization (A12E8 48 ; F12F2 ; F42F5 A12E8 : copepoda samples ; F12F2 : negative controls ; F42F5 : positive controls) WSSV Fig. 2 The WSSV carrying status in the copepoda, which were sampled from the pond in J uly, August and September 2003, detected by PC R2dot2bolt hybridization (A12F10 60 ; G12G4 ; G52G8 A12F10 : copepoda samples ; G12G4 : negative controls ; G52G9 : positive controls)

3 3, : WSSV Fig. 3 The WSSV carrying status in the copepoda, which were sampled from the pond in October 2003, detected by PCR2 dot2bolt hybridization (A12C4 20 ; C52C6 C72C8 A12C4 : copepoda samples ; C52C6 : positive controls ; C72C8 : negative controls) 1 Table 1 Statistical results of different positive intensities in the Detection results Positive cases positive copepoda samples / % May J une August 9 10 September October Detection cases Detection rate : + ; + + ; Notes : + means weak positive ; + + means secondary positive ; means strong positive WSSV Fig. 4 The WSSV carrying status in the resting eggs of copepoda, which were sampled from the pond mud in May 2003, detected by PCR2dot2bolt hybridization (A12C4 20 ;C52C6 ; C72C8 A12C4 copepoda samples ;C52C6 : positive controls ;C72C8 : negative controls) 2. 2 WSSV 10,, 12 20, PCR2 1,5 %( 4),25,36 h,, N I N II 300,20 30 PCR,5 2, 40 %( 5) 5 WSSV PCR Fig. 5 The WSSV carrying status in the nauplins, which were hatched from the resting eggs carrying WSSV, detected by nest PCR (1 DNA ;2 ;3 ; 428 ; 1 : DNA Markers ; 2 : positive controls ; 3 : negative controls ; 428 : 3 nauplins samples) 3. 1 WSSV, ( EL ISA PCR ) WSSV [9,11213,20 ] WSSV, WSSV, WSSV,,,,, %, 6 40 %,8 60 %, PCR2, PCR2, PCR2 PCR PCR2 [14216,21 ],, WSSV 3. 2 WSSV, WSSV

4 408 WSSV, [22224 ], [25 ], WSSV,, [14 ] WSSV,, WSSV,,, 5 WSSV, WSSV 12 5 % WSSV, WSSV 40 %, WSSV, WSSV WSSV,,,, 20, 80 % [26 ] WSSV ( ),, WSSV,,,, : [ 1 ],,,. [J ]., 1995, 16 (1) : [ 2 ],,,. [J ]., 1995, 16 (1) : [ 3 ],,. ( Penaeus chinensis) [J ]., 1995, 2 (3) : [ 4 ],,,. [J ]. (C ), 1997, 27 (5) : [5 ] Wongteerasupaya C, Vickers J E, Sriurairatana S, et al. Anon2oc2 cluded baculovirus that occurs in cells of ectoder and mesodermal origin and causes high mortalities in the black tiger prawn Penaeus [ J ]. Monodon. D/ S Aquat Org, 1995, 21 : [ 6 ] Lightner D V. A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp [ M ]. Louisiana, USA : Baton Rouge, [ 7 ],,,. [J ]. :, 2005, 35 : [ 8 ],,, [J ]., 1994, 19 (2) : [ 9 ],,, [J ]., 1995, 16 (1) : [ 10 ] Wang C H, Lo C F, Leu J H, et al. Purification and genomic analy2 sis of baculovirus associated with white spote syndrome ( WSBV) of Penaeus monodon [J ]. Dis Aquat Org, 1995, 23 : [ 11 ] LO C F, HO C H, PEN G S E, et al. White spot syndrome bac2 ulovirus ( WSBV) detected in cultured and captured shrimp, crabs and arthropods [J ]. Dis Aquat Org, 1996, 27 : [ 12 ],,. [J ]., 1999, 18 (1) : [13 ],,,. ( WSSV) [J ].,2000, 21 (3) : [14 ] Yan D C, Dong S L, Huang J, et al. White spot syndrome virus (WSSV) detected by PCR in rotifers and rotifer resting eggs from shrimp pond sediments[j ]. Dis Aquatic Org, 2004, 59 (1) : [ 15 ],,,. PCR2 ( ( WSSV) [J ]., 2004, 14 (3) : [16 ],,,. PCR ( WSSV) 2DNA ( UN G) [J ]., 2003, 24 (3) : [ 17 ],,. PCR ( HHNBV) DNA [J ]., 2000, 21 (1) : [18 ],,,. ( Penaeusi chinensis) ( WSSV) [J ]., 1999,29 : [ 19 ],,,. DNA [J ]., 2003, 25 (supp2) : [ 20 ],,,. DNA [J ]., 2001, 8 (4) : [ 21 ],. [J ]., 2003, 27 (8) : [ 22 ]. [J ]., 1995, 6 : 628. [ 23 ],. [J ]., 2002, 26 (4) : [ 24 ],. [J ]., 2003, 22 (1) : [ 25 ],,,. [J ]., 1995, 3 : [26 ] Nancy H M, Margaret M. Copepod diapause eggs : a potential source of nauplii for aquaculture [J ]. Aquaculture, 2001, 201 : ( 412 )

5 412 Preliminary Observations on the Develop ment Stage s and Nucle ar Numbers of Encyste d Embryo s of Four Artemia Populations WAN G Su2Feng 1,2, SUN Shi2Chun 1 (1. College of Fisheris, Ocean University of China, Qingdao , China ; 2. Environmental Management College of China, Qinhuangdao , China) Abstract : The embryonic development stage of encysted embryos of four A rtem ia populations [ Ga Hai, Qinghai ; Aqqikkol Hu, Xinjiang ; Xizang (exact locality not known) ; Jingyu Hu, Xinjiang ] from the Qinghai - Tibet Plateau was studied with the paraffin section method, and the nuclei numbers of encysted embryos were estimated by counting nuclei on squashed preparations. The result s showed t hat t he encysted A rtem ia embryos of all four A rtem ia populations were at the stage of gastrula, and consisted of , , and nuclei, respectively. Key words : A rtem ia ; encysted embryos ; cell number ; embryonic stage ( 408 ) Primary Inve stigation on the WSSV Carrying Status in Copepoda and Their Re sting Eggs in Shrimp Ponds ZHAN G Jia2Song, DON G Shuang2Lin, DON G Yun2Wei, YAN Dong2Chun, TIAN Xiang2Li, L IU Xiang2Yi ( Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao , China) Abstract : The paper describes the research on the WSSV carrying status in copepoda and their resting eggs. During May to October, 2003, in Rushan shrimp farm, 128 samples of copepoda were collected f rom t he shrimp ( Fenneropenaeus chi nensis) cult ural ponds, where White Spot Syndrome ( WSS) had taken place in In addition, 20 samples of copepoda resting eggs were separated f rom t he pond mud in Decemeber, 2003, and 5 samples of nauplins hatched from that kind of resting eggs were detected. These samples were de2 tected for WSSV by the method of PCR2dot2blot hybridization or nest2pcr, both of which could be used in the detection of trace WSSV. The results showed that the WSSV could infect the copepoda and the average positive rate of the copepoda samples reached %. The positive rate in May was % and in J uly, August, September and October it was more than 60 %. Furthermore, one positive case was detected from the copeopda resting eggs samples and two positive cases in the nauplins hatched from this kind of resting eggs. The results indicated that copepoda was one of the vectors of WSSV, and it was extremely possible that copepoda resting eggs carrying WSSV hatch new WSSV carrying copepoda t he next year. Key words : white spot syndrome virus ; copepoda ; resting eggs ; PCR2dot2blot hybridization