【作者】 陈政强；

【导师】 战文斌； 陈昌生；

【作者基本信息】 中国海洋大学 ， 水产养殖学， 2009， 博士

【摘要】 本文对九孔鲍在副溶血弧菌感染过程中以及在低氧胁迫和饥饿胁迫条件下机体细胞和体液免疫防御因子的变化,九孔鲍野生与养殖群体免疫防御因子及生化遗传差异性等内容进行研究。结果表明,在副溶血弧菌感染过程中九孔鲍血淋巴细胞当中的吞噬细胞和体液中的一些免疫因子发挥重要作用,吞噬细胞存在呼吸爆发现象,呼吸爆发产生的活性氧和活性氮是杀灭被吞噬的异源生物的主要因素；胞内MPO将H2O2转化为次氯酸(hypochlorous acid, HOCl)和氯胺(chloramines, RNCl)等次生氧代谢产物,强化杀菌效果；胞内ACP对异物发挥消化、降解作用；胞内CAT则积极参与胞内氧自由基的淬灭和清除作用；九孔鲍血淋巴液中的凝集素、溶菌酶、酚氧化酶等参与抗感染免疫过程,凝集素发挥免疫识别、免疫调理、凝集限制、免疫溶解等作用,溶菌酶发挥水解革兰氏阳性菌的作用,酚氧化酶则可能参与病原微生物的抑制和免疫识别,九孔鲍存在通过细胞增殖强化免疫效应的机制；此外,九孔鲍血淋巴液中还发现免疫球蛋白样物质IgG-like, IgA-like, IgM-like,补体样物质C3-like,C4-like,C反应蛋白样物质CRP-like等,它们与机体抗感染免疫应答具有一定的相关性。平均体重为14.25g±2.21g的九孔鲍在水温为22.1℃±1.3℃,盐度为30.72±0.54,pH8.20±0.14的海水环境中,溶解氧含量由7.49±0.14 mg·L-1下调至2.53±0.16 mg·L-1后120内并未见到个体死亡,而注射感染5.0x105 cells-abalone-1副溶血弧菌后120h内的累计死亡率高达91.11±7.70%,比对照组11.11±3.83%的累计死亡率高出80%,血淋巴的抑菌清除率下降至-(3340.47±298.57)%,显而易见,低氧胁迫使得九孔鲍拮抗副溶血弧菌感染的抵抗力显著下降。低氧(2.53±0.16 mg·L-1)胁迫下,九孔鲍血淋巴细胞数量(THC)下降30.62±4.87%、血淋巴细胞吞噬率下降62.77±5.79%、呼吸爆发产生的超氧阴离子数量下降22.21±5.89%。低氧胁迫下九孔鲍血淋巴细胞内MPO活性上升9.63±7.59%～22.90±13.73%、CAT活性提高7.68±6.83%～56.28±13.96%,反映出低氧胁迫下九孔鲍体内应激反应产生的物质氧化加剧,血淋巴细胞内产生大量的活性氧自由基。溶解氧含量由7.49±0.14 mg·L-1下调至4.51±0.12 mg·L-1后九孔鲍120h内的累计死亡率达到37.78±3.85%,血淋巴抑菌率下降至-(883.56±123.22)%;THC、血淋巴细胞吞噬率、呼吸爆发产生的超氧阴离子O2-数量等最大降幅分别达到12.51±6.59%、21.90±15.84%、12.93±5.74%;MPO活性在原有水平的-(10.61±4.20)%-7.13±6.45%之间波动,CAT活性在-(5.17±18.08)%～16.26±10.85%之间变化。在水温24.3℃±1.7℃、盐度为30.42±1.63、pH值为8.15±0.11环境条件下,饥饿胁迫20d九孔鲍尚能存活。在停食5d时,九孔鲍血淋巴液蛋白含量不仅没有下降,甚至略有升高,而7d后,九孔鲍血淋巴液蛋白质含量逐渐大幅度下降,由此推测,九孔鲍在饥饿胁迫早期并没有动用体蛋白作为能源物质,而饥饿7d后,体蛋白被大量消耗于机体的生命维持。饥饿胁迫状态下九孔鲍血淋巴细胞吞噬活性下降、血淋巴细胞呼吸爆发水平下降、体液溶菌酶活性、凝集活性下降、抗感染免疫机能下降。但是,九孔鲍经历5d时间的饥饿,血淋巴抗菌清除效率、血淋巴细胞吞噬活性、血淋巴细胞呼吸爆发产生的超氧阴离子水平、体液溶菌酶活性等免疫指标都不仅变化不大,有时甚至略有升高,说明九孔鲍具有面对饥饿胁迫的应激适应机制,饥饿之初,由于营养不足,机体通过提高免疫机能加强来适应环境、保护自我。饥饿胁迫持续时间超过7d,这种本能的逆境适应方式和机能维持终因体内的营养状况严重不足和体内资源的过度消耗而改变和衰退。饥饿胁迫下的九孔鲍血淋巴液中的溶菌酶活性、凝集活性下降可能由体液蛋白含量下降引起,胞内颗粒性物质减少则可能是血淋巴液中溶菌酶及凝集素活性减弱的直接原因。对九孔鲍野生与养殖群体的细胞与体液免疫因子进行比较,结果表明：九孔鲍野生群体与养殖群体之间、养殖鲍的提纯复壮群体与养殖性状退化群体之间的免疫因子及机体免疫防御机能均有显著的差异性。养殖鲍的提纯复壮群体(养殖10个月,个体大小为51.8mm～59.2mm)细胞与体液免疫因子活性都明显高于养殖性状退化的养殖群体(养殖20个月,个体大小为37.4mm～43.1mm)p<0.01),与野生群体(个体大小为67.7mm～76.7mm)相比,九孔鲍提纯复壮群体的细胞免疫因子活性略高于野生群体,体液免疫因子活性则明显低于野生群体。九孔鲍血淋巴细胞在吞噬活动中引发呼吸爆发产生活性氧的水平与九孔鲍自身生理、环境温度以及异物数量等因素有显著的相关性p<0.01)。环境温度由25℃下降至18℃,九孔鲍的细胞与体液免疫因子活性都有明显的下降。养殖九孔鲍退化群体在25℃和18℃温度条件下血淋巴液的抗菌活力水平差异性不明显。酚氧化酶存在于九孔鲍血淋巴液中,但,活性水平不高,在各类九孔鲍群体之间的差异性也不明显。对深圳野生与养殖、广东碣石养殖及南澳野生4个九孔鲍群体8种酶共16个位点进行了等位酶检测分析。研究表明,以P095为标准,上述4个九孔鲍群体的位点Sod-1、Aat、Me、Mdh-1、Est-1、Est-2为多态,多态位点比例为37.5%；野生群体的观察杂合度分别为：0.1604(深圳野生)、0.1604(南澳野生),养殖群体的观察杂合度分别为：0.1521(广东碣石养殖)、0.1562(深圳养殖)；野生群体的期望杂合度分别为：0.1697(深圳野生)、0.1766(南澳野生),养殖群体的期望杂合度分别为：0.1534(广东碣石养殖)、0.1557(深圳养殖)；野生群体的平均有效等位基因数分别为：1.3633(深圳野生)、1.3943(南澳野生),养殖群体的平均有效等位基因数分别为：1.3072(广东碣石养殖)、1.3120(深圳养殖)。九孔鲍野生群体间遗传距离为：0.0052,养殖群体间遗传距离为：0.0131,4个群体平均遗传距离为：0.011。综合分析后认为,九孔鲍养殖群体遗传多样性水平低于野生群体,群体间分化较低(FST=0.0411,Nm=5.8340)。更多还原

【Abstract】 The roles and variation of cellular and humoral effectors involved in immune defense system of the abalone, Haliotis diversicolor supertexta, in duration of anti-infectious immune response against to Vibrio parahaemolyticus or exposed to hypoxic and starvation stress were studied in this paper, in addition, the variation in immune defense effecors and biochemical genetics among wild and cultivated populations were investigated either. The results showed that phagocytes involved in haemocytes and some humoral immune effectors of the analone acted the most important roles in immune response against toⅤ. parahaemolyticus infection, and reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) produced by respiratory burst which occurred concurrently with phagocytosis employed the major effectors among bactericidal agents in phagocytes, and the bactericidal effects of ROIs highly enhanced by the way of intracellular myeloperoxidase (MPO) catalyzed the transformation of hydrogen peroxide (H2O2) to some subordinated reactive oxygen metabolites such as hypochlorous acid (HOCl) and chloramines (RNCl), while the intracellular acid phosphatase (ACP) acted in digestion and biodegradation for engulfed foreign microorganisms, and catalase (CAT) served as antioxidant in quenching and clearance to oxyradicals. All are they the main anti-infectious immune effectors in immunocytes. Furthermore, some effectors in haemolymph of the abaone such as agglutinin, lysozyme, and phenol oxidase were all took parts in the immune response against toⅤ. parahaemolyticus infection either. Agglutinin in haemolymph performed functions in recognition for foreign material, opsonization, inhibition and haemolysis. Lysozyme took effect in bacteriolysis for gram-positive bacteria, and phenol oxidase may contribute to bacteriostasis and foreign material recognition. There was a pathway of potentiation immune effection through haemocytes proliferation which had been found in H. diveersicolor supertexta in duration of infection with V. parahaemolyticus. In haemolymph of the abalones some kinds of substance similar to immunoglobulin, complement and C reaction protein had been found and been named IgG-like, IgA-like, IgM-like, C3-like, C4-like and CRP-like respectively. All of them had some dependencies in anti-infectious immune response against to V. parahaemolyticus certainly.The abalones of H.diversicolor supertexta with an average body weight 14.25g±2.21g can tolerate 120 hours exposure to a hypoxic condition while dissolved oxygen reduction from 7.49±0.14 mg·L-1 to 2.53±0.16 mg·L-1 without onset mortality occured at a water temperature 22.1℃±1.3℃, and a salinity level of 30.72±0.54%o, and pH 8.20±0.14, but they suffered 91.11±7.70% cumulative mortality rate which was 80% more than of the control animals duo to challenged withⅤ. parahaemolyticus at a dose of 5.0x105 cells-abalone-1, and a clearance efficiency in the haemolymph of H.diversicolor supertexta decreased to-(3340.47±298.57)% inhibition rate. Obviously, the resistance of the abalones against toⅤ. parahaemolyticus infection decreaced and the susceptivity inceassed correspondingly at the hypoxic stress above, in which the total haemocytes counts (THC) and percentage of phagocytosis and the amount of superoxide anion (O2-) produced by respiratory burst decreased at 30.62±4.87%,62.77±5.79%,22.21±5.89% respectively. What intracellular MPO activity and CAT activity went up from 9.63±7.59% to 22.90±13.73% and 7.68±6.83% to 56.28±13.96% respectively while dissolved oxygen reduction from 7.49±0.14 mg·L-1 to 2.53±0.16 mg-L"1 suggests that the intracellular oxidation introduced by hypoxic stress response intensified and there must be much more oxyradical. The trial abalones of H. diversicolor supertexta possessed a cumulative mortality rate of 37.78±3.85% and a haemolymph inhibition rate of -(883.56±123.22)%, and the maximal downtrends of 12.51±6.59%, 21.90±15.84%,12.93±5.74% in THC and percentage of phagocytosis and the amount of O2-production respectively, and a variation of MPO activity range from -(10.61±4.20)% to 7.13±6.45% and CAT activity from -(5.17±18.08)% to 16.26±10.85% while dissolved oxygen went down from 7.49±0.14 mg·L-1 to 4.51±0.12 mg·L-1 due to infection with V. parahaemolyticus.The abalones of H.diversicolor supertexta with an average body weight 14.25g±2.21g can survive in a period of 20 days starvation at 24.3℃±1.7℃water temperature, 30.42±1.63%o salinity level and pH 8.15±0.11. The concentration of haemolymph protein in the abalones increased slightly in 5 days exposure time to starvation and dropped progressively with a wide range from 7d. Based on this, it will be surmised that there was hardly any humoral protein consumed for living energy in early starvation and mass humoral protein consumed for life support from 7d to starvation. The abalones survived with decreases in phagocytosis activity of haemocytes and respiratory burst and haemolymph lysozyme activity and haemolymph agglutination titers and anti-infectious immunity in duration of starvation, however, rather than did not greatly alter, some immune parameters such as the haemolymph clearance efficiency and phagocytosis activity of haemocytes and respiratory burst and haemolymph lysozyme activity sometimes went up somewhat in 5 days early starvation. Consequently, it would be suggested that the abalones were born with functions of starvation stress adaptation. In early duration of starvation the abalones did protect themselves and adaptate to surroundings by improving immune effection owing to subnutrition, and this biophilia styles of stress adaptation and natural ability maintaining changed or degenerated from 7d to starvation resulted from serious poor nutritional state and overmuch intravital resources degradation. Perhaps the haemolymph lysozyme activity and agglutinating titers decreased resulting from reduction of haemolymph protein, while the intracellular granules reduction may result in the haemolymph lysozyme activity and agglutinating titers dropped down directly.Comparion of wild population with a body length ranged from 67.7mm to 76.7mm and cultivated population of the abalone, Haliotis diversicolor supertexta, in cellular and humoral immune effectors showed that there were clear differences both in activities of immune defense effectors and immune defense function either between wild population and cultivated population or between purification and rejuvenation cultivated population which gained a body length ranged from 51.8mm to 59.2mm in 10 months of cultivation and degenerate cultivated population which only gained a body length ranged from 37.4mm to 43.1mm in 20 months of cultivations. Comparing with degenerate population, activities of both cellular and humoral immune effectors in purification and rejuvenation cultivated abalone population of H. diversicolor supertexta were obviously higher. While, comparing with wild population, only activities of cellular immune effectors in the purification and rejuvenation cultivated abalone population were somewhat higher, but the activities of humoral immune effectors were obviously lower. The amount of reactive oxygen intermediates (ROIs) produced by respiratory burst which occurred concurrently with phagocytosis were much in relation to the physical states of the abalones themselves and the environmental temperatures and the quantities of the stimulants. Activity of both cellular and humoral immune effectors had a significant decrease as the environmental temperature dropped down from 25℃to 18℃, while the antibacterial activities of the degenerative abalone population only had a little difference under two kinds of surrounding temperatures. There was some phenol oxidase in the hemolymph of the abalones, but their activities were low and had no marked activity difference within three above different populations.Conducted an investigation into genetic variation and diversity of wild and cultivated abalone populations of Haliotis diversicolor supertexta from Shenzhen and wild population from Nanao and cultivated population from Guangdong Jieshi altogether 4 populations in 8 enzyme systems presumably encoded by 17 allozyme loci using the assay of vertical slab polyacrylamide gel electrophoresis, it was found that six loci(Sod-1、Aat、Me、Mdh-1、Est-1、Est-2) presented polymorphic (P0.95) in the four populations above, and the percentage of polymorphic loci (P) was 37.5%; the observed heterozygosity (Ho) in both of wild populations were 0.1604, and the cultivated populations were 0.1521(GDSZ C.) and 0.15629(SZ C.) respectively; the expected heterozygosity (He) in wild populations were 0.1697 (SZ W.) and 0.1766 (NA W.) respectively, the cultivated populations were 0.1534 (GDSZ C.) and 0.1557(SZ C.) respectively; mean effective number of alleles per locus (Ae) in wild populations were 1.3633 (SZ W.) and 1.3943 (NA W.) respectively, while the cultivated populations were 1.3072 (GDSZ C.) and 1.3120 (SZ C.) respectively. The genetic distance between the two wild populations above was 0.0052, while the genetic distance between the cultivated populations was 0.0131, and the average of the four populations were 0.011. All of these results showed that there were a loss of genetic variation in cultivated populations of H. diversicolor supertexta, low level of differentiation was found between wild and cultivated populations (Fst=0.0411, Nw=5.8340).更多还原