| At present,the artificial farming area of H.discus hannai has been extended to the southern part of China,such as Fujian and Guangdong provinces.However,sick prevention and management of aquaculture environment are still important,because of the widespread and high-density.Therefore,it is expedite priority to obtain high-quality abalone variety.Hybridization has been demonstrated to be an useful and effective method in abalone breeding and Hybrid Haliotis fulgens(?)×H.discus hannai?(DF)has shown great heterosis for growth than Haliotis discus hannai(DD).In recent years,DF showed good heterosis and suer-parent advantage in survival rate,but we found mass death of small size stage(1-2cm)DF in June and July.However,there was no similar phenomenon with the same size DD.At the same time,the observation showed that overall survival rate of large size stage DF(6-7cm)was higher than the same size DD.Based on above phenomenons,this study investigated the differences between sick DF and healthy DF and DD.We researched the differences in survival rate and immune-related indicators and genes of DD and DF under Vibrio Harveyi stress,the main results were summarized as follows:(1)Analysis the differences between sick DF and healthy DF and DDComparing the number of culturable bacterias in sickd DF and healthy DF and DD,we found the number of sickd and healthy DF's internal was significantly higher than foot muscle.The number of sickd DF's culturable bacteria was still larger than healthy DF and DD.The 16S rRNA sequence analysis of isolated culturable bacterias found that 45 strains were belonged to vibrio genus,another 13 strains belonged to Shewanella genus.Histological analysis of sickd and healthy DF showed that there were multiple vacuoles in the foot muscles and internal of sickd DF without obvious lesions in the sputum and hepatopancreas.Healthy DF possessed tight organizations.(2)Evaluation of vibrio harveyi resistance ability of DF and DDStrain vibrio harveyi was diluted into the suspension liquid of different concentrations:5×106cfu·-mL-1?5×107cfu·ML-1?5×107cfu·mL-1?1×108 cfu·mL-1.Small size stage DD and DF(1.73±0.55 and 1.53±0.46cm in shell length respectively)were soaked with harveyi suspension.Large size stage DD and DF(6.23± 1.10 and 6.15±1.35cm in shell length respectively)were injected with harveyi suspension in the treatment group while with sterile seawater in the control group.Each abalone was foot-injected with 50?L harveyi suspension or sterile seawater.Then the tested abalones were cultured at 20? and 28?,and the death of abalones was recorded regularly for 3 days.The results showed that:(1)The average cumulative mortality rate of small-sized DD was 0%,11.11%,20%,33.33%,50%,70%after soaking at 20?for 3 days and 0%,14.44%,26.67%,37.78%,60%,74.44%for DF.The average cumulative mortality rate of DD was 0%,15.56%,23.33%,36.67%,61.11%at 28?for 3 days and 0%,17.78%,26.67%,46.67%,71.11%,86.67%for DF.(2)The average of cumulative mortality rate of large-sized DD was 1.11%,24.44%,40%,53.33%,70%,73.33%after injected at 200? for 3 days and 1.11%,16.67%,26.67%,46.67%,53.33%,63.33%for DF.The average cumulative mortality rate of DD was 2.22%,26.67%,50%,60%,71.2%,76.67%at 28? for 3 days and 2.22%,20%,30%,50%,53.33%,66.67%for DF.(3)The lethal doses LD50 of small-sized DD and DF at 20? were 4.8x 107?3×107 cfu·mL-1.The LD50 at 28? were 2.6x107 cfu·mL-1?1.7×107 cfu·mL-1 respectively.The lethal doses LD50 of large-sized DD and DF at 20? were 1.0×107 cfu·mL-1?2×107 cfu·mL-1.The LD50 at 28? were 1.2×106cfu·mL-1?2.4×l06cfi·mL-1.The above results showed that the mortality of the tested abalones is proportional to the concentration of the treatment solution.The vibrio harveyi resistance ability of small-sized DD was stronger than the same size DF,and the large size stage was the opposite.At the same time,the conditions of pathogenic stress for different size abalones mainly included normal treatment at 20? and 28?,normal treatment after 3 hours of dry dew,normal treatment after 6 hours of dry dew,and low salt treatment of no-dryness.The results showed that there was no significant difference in the survival rate of small-sized DD and DF at normal processing of 20? and dry dew 3 hours of 28?.The other 7 conditions showed the survival rate of DD was significantly higher than that of DF.The large size stage was the opposite.The conclusion was consistent with the previous results.(3)Immune-physiological responses of DD and DF under pathogenic stressIn order to further determine the immune differences of different stage DD and DF.We studied the effect of vibrio treatment on the non-specific immune indexes.PO(phenol oxidase)and SOD(superoxide dismutase)of small-scale DD and DF immersed at 20? and 28 ? were determined.The results showed that:(1)There was no significant difference in PO activity between DD and DF at 20?,the PO activity only decreased after 12 hours treatment.The PO activity of DD was significantly higher than that of DF at 28?,and it declined during the first 24 hours then reducing.(2)The SOD activity increased on 6 hours then declining rapidly at 20?.However,the SOD activity increased on 12 hours then reducing at 28?.That meaned there was a certain delay in the high temperature.Overall,the SOD activity of DD was significantly higher than thar of DF.The results meaned that small size stage DD had a stronger immune response than DF under vibrio stress.We determined the total number of blood cells(THC),phagocytic activity of blood cells,total bacterial count of blood cells,PO and SOD activity in large-scale DD and DF which were injected with vibrio at 20? and 28?.The results showed that:(1)The relative value of THC increased significant at the 6th hour after treatment at 20?.When the temperature was 28? it increased at the 12th hour with a certain reaction delay.The relative THC values of DF was significantly higher than DD at both temperature.(2)The phagocytic activity of blood cells increased first then decreased during the treatment at 20? and 28?.What's more,the phagocytic activity was significantly higher than that of DD.(3)The total bacteria number of blood cells in treated group was significantly higher than that in the control group at 20?.After treatment for 48 hours,the total bacteria number of DD blood was significantly higher than that in the DF,the same as 28?.At the same time,the total bacteria number of blood cells in treatment group was also significantly higher than the control group.(4)The PO activity increased first then decreased in treatment group at 20?,showing a gradual decline at 28?,which was consistent with the control group.There was no significant difference in PO activity between DD and DF in the whole process.(5)The SOD activity of DD decreased at first then increased at 20?.But the SOD activity of DF always increased.At 28?,DD and DF showed a trend of increasing first and then decreasing.The overall performance of DD was significantly higher than that of DD.The above results showed that the selected immune parameters could be affected by pathogenic bacteria.The whole fluctuation of the large size DD against the pathogenic stress was obvious,indicating that the tolerance of the large size DD to pathogenic stress was weaker than that of the same size DF.(4)Effects of pathogenic stress on expression of immune related genes in DD and DFIn the study,the expression of 8 immune related genes was analyzed.Among them,TRAF,TLR and TNF genes were associated with the tolling signaling pathway,which was mainly involved in the immune response against gram-negative bacteria.TRAF was significantly up-regulated in different stages and temperature treatments.The gene expression level of DF was significantly higher than DD in large size stage.The TLR gene could be used for immune defense by cell adhesion,and its expression was significantly reduced in small scale.The TNF gene of small size DD and DF showed.a decreasing trend at different temperatures,but the TNF genes of large stage DD and DF was steadily increasing.The MIF gene allowed macrophages to function at specific sites.In the study,the MIF gene has been significantly reduced.The Lys gene could synthesize proteins resistanted to gram-negative bacteria,and the expression levels was signigicantly decreased and then increased in the different size and treatment temperature.The Spi gene could control the activity of serine proteases at appropritate time and space and it was significantly up-regulated in the treatment group.The Cat gene could balance excess hydrogen peroxide to ensure the health.The expression of Cat gene significant dropped might be due to the invasion of pathogenic bacteria.The SOD gene could remove excessive superoxide in the body,and the expression level of SOD gene was significantly decreased in the treated group.Altogether,the Lys gene was the only one which was similar in different sizes.The remaning 7 genes had opposite expresson in different sizes among the immune relative gene of DD and DF. |
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