栉孔扇贝免疫致敏（immune priming)现象及其机制的初步研究

栉孔扇贝是我国重要的海水养殖品种，自1997年以来陆续爆发的大规模死亡，不仅造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。本研究通过比较扇贝在连续刺激条件下的不同免疫反应，推测栉孔扇贝中有无免疫致敏和记忆现象，观察短期免疫刺激对扇贝再次遇到相同病原物刺激时死亡率的影响，探讨栉孔扇贝的免疫致敏作用，并对其机制进行初步探讨，以期更好地了解扇贝的免疫防御机制，为扇贝病害防治提供参考。 利用荧光实时定量PCR技术检测了扇贝受到鳗弧菌连续等量刺激后两种重要病原识别受体（PRR）基因的表达。结果显示，在受到连续两次刺激后CfPGRP-S1、LGBP基因的表达量均有不同程度的增加。其中CfPGRP-S1基因在受到第二次刺激后表达量增加的幅度高于第一次，且基因表达显著性增加的时间比第一次提前；LGBP基因在第二次刺激后，表达量显著性增加的时间也比第一次提前。同时，对同批处理的扇贝进行重要免疫指标的检测分析发现两次刺激后吞噬率变化不显著，而第二次刺激后吞噬指数的最大峰值高于第一次，并且最大峰值出现的时间比第一次提前；血淋巴中的抗菌活力和肝胰腺中SOD、MDA含量在两次刺激前后的变化不显著。在脊椎动物中，识别能力和抗菌能力的提高是免疫记忆的两个重要特点，栉孔扇贝在第二次免疫刺激后PRR识别能力的提高和吞噬作用的增强，说明扇贝中可能存在类似脊椎动物免疫记忆的免疫致敏作用。对扇贝进行两次鳗弧菌浸泡刺激，第一次为短期浸泡刺激，第二次为长期浸泡刺激。结果发现，第一次浸泡过的扇贝在遭受相同病原第二次刺激时，在一定时间段内其死亡率比未受第一次刺激的扇贝极显著降低，表明第一次的短期病原刺激增强了扇贝对第二次刺激的抵抗能力。同时比较第二次刺激过程中扇贝免疫指标的变化发现经过短期刺激扇贝的吞噬作用、ACP和AKP活性比未受过刺激的扇贝显著增强，而其他指标包括POL、SOD则差异不显著，说明增强的吞噬作用、ACP和AKP活性与扇贝的死亡率下降有关。研究结果表明栉孔扇贝中存在免疫致敏（immune priming）现象，短期刺激可以提高扇贝的免疫反应能力，增强扇贝机体对相同病原物的抵抗能力。

Growth, survival and immune activity of scallops, Chlamys farreri Jones et Preston, compared between suspended and bottom culture in Haizhou Bay, China

We examined the growth, survival and immune response of the scallop, Chlamys farreri, during a 1-year period in deep water of Haizhou Bay. Scallops were cultured using two methods: (1) in lantern nets at a 5 m depth and (2) in a bottom culture system (sleeves) on the seabed at about a 25 m depth. Shell heights, meat dry weight and immune activities in the haemolymph (superoxide dismutase and myeloperoxidase) were measured bimonthly or quarterly from July 2007 to June 2008. Survival was measured at the end of the study and environmental parameters in the experimental layers were monitored during the experiment. The growth and immune activities of scallops were lower when the water temperature was high, which was consistent with the main mortality occurring in summer. The growth and immunity of scallops were higher in the suspended culture than in the bottom culture during the experiment, with the exception of shell growth during the last study period. Survival of scallops in the suspended culture (54.6 +/- 12.3%) was significantly lower than that in the bottom culture (86.8 +/- 3.5%) at the end of this study. We conclude from our results that the high mortality of C. farreri can be prevented by culturing them in a bottom culture system before November of the first year, and then transferring them to a suspended culture to improve scallop production.

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l(-1) ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W > NW > N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l(-1) ammonia-N reduced the immunocompetence of P japonicus and may have decreased the virulence of WSSV (C) 2004 Elsevier B.V. All rights reserved.

Immune condition of Chlamys farreri in response to acute temperature challenge

The effects of acute temperature challenge on some immune parameters of haemocyte in Zhikong scallop, Chlamys farreri, recognised as a temperature sensitive bivalve species, were evaluated over a short period of time. Scallops were suddenly transferred from 17 degrees C to 11 degrees C, 23 degrees C and 28 degrees C for a period of 72 h. Total haemocyte count (THC), percentage of phagocytic haemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of temperature stress. Results demonstrated that the percentage of phagocytic haemocytes and ACP activity in cell-free haemolymph of scallops challenged at 28 degrees C for 72 h significantly decreased. By contrast, reactive oxygen species production by haemocytes increased when compared to the initial values. It is concluded that haemocyte activities of C. farreri appear to be compromised when scallops were transferred from 17 degrees C to 28 degrees C. Meanwhile, no obvious negative effect of acute temperature stress was detected on haemocyte activities of C. farreri challenged at 11 degrees C, which highlighted the high tolerance of scallops to acute decrease of seawater temperatures. (C) 2007 Elsevier B.V. All rights reserved.

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 degrees C, 17 degrees C and 25 degrees C respectively. Thereafter, a recovery period of 24 h at 17 degrees C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immumomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 degrees C and 24 h of air exposure at 17 degrees C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 degrees C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 degrees C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 degrees C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops. (c) 2007 Elsevier B.V. All rights reserved.

Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus

Invertebrates are increasingly raised in mariculture, where it is important to monitor immune function and to minimize stresses that could suppress immunity. The activities of phagocytosis, superoxide dismutase (SOD), catalase (CAT), myeloperoxiclase (MPO), and lysozyme (LSZ) were measured to evaluate the immune capacities of the sea cucumber, Apostichopus japonicus, to acute temperature changes (from 12 degrees C to 0 degrees C, 8 degrees C, 16 degrees C, 24 degrees C, and 32 degrees C for 72 h) and salinity changes (from 30 parts per thousand to 20 parts per thousand, 25 parts per thousand, and 35 parts per thousand for 72 h) in the laboratory. Phagocytosis was significantly affected by temperature increases in 3 h, and by salinity (25 parts per thousand and 35 parts per thousand) changes in 1 h. SOD activities decreased significantly in 0.5 h to 6 h samples at 24 degrees C. At 32 degrees C, SOD activities decreased significantly in 0.5 h and 1 h exposures, and obviously increased for 12 h exposure. CAT activities decreased significantly at 24 degrees C for 0.5 h exposure, and increased significantly at 32 degrees C in 3 h to 12 h exposures. Activities of MPO increased significantly at 0 degrees C in 0.5 h to 6 In exposures and at 8 degrees C for 1 h. By contrast, activities of MPO decreased significantly in 24 degrees C and 32 degrees C treatments. In elevated-temperature treatments, activities of LSZ increased significantly except at 32 degrees C for 6 h to 12 h exposures. SOD activity was significantly affected by salinity change. CAT activity decreased significantly after only 1 h exposure to salinity of 20 parts per thousand.. Activities of MPO and LSZ showed that A. japonicus tolerates limited salinity stress. High-temperature stress had a much greater effect on the immune capacities of A. japonicus than did low-temperature and salinity stresses. Crown Copyright (C) 2008 Published by Elsevier Inc. All rights reserved.

Immune condition of Apostichopus japonicus during aestivation

Aestivation is an indispensable state in the life history of sea cucumbers, Apostichopus japonicus. The immune characteristics of the coelomic fluid of A. japonicus, were investigated during aestivation. Samples were collected between July and November 2006 from a coastal pond located off the Yellow Sea in Jiaonan, Shandong Province, China. The total coelomocytes counts (TCC), total superoxide dismutase (T-SOD), catalase (CAT), myeloperoxidase (MPO), and lysozyme (IZM) in the coelomic fluid were measured. The activities of catecholamines, [adrenaline (AD), noradrenaline (NA), and dopamine (DOP)] were estimated. TCC decreased from July to September, indicating weakness of the cellular immune activity at that time. Activities of SOD, CAT, MPO, and LZM changed significantly from July to October. Catecholamines AD and NA in coelomic fluid were significantly higher on August 21 and November 27. There was no significant variation in DOP during the sampling period. Thus, immune characters in coelomic fluid of A. japonicus changed significantly during aestivation. Water temperature was significantly and negatively correlated with TCC, and salinity was significantly and positively correlated with AD and NA. The mechanism of aestivation in A. japonicus is complex and might not be attributed only to environmental changes, such as temperature and salinity, as shown in previous studies. (C) 2008 Elsevier B.V. All rights reserved.

Survival, growth and immune activity of scallop Chlamys farreri cultured at different depths in Haizhou Bay (Yellow Sea, China) during hot season

Survival, growth and immune response of the scallop, Chlamys farreri, cultured in lantern nets at five different depths (2, 5, 10, 15, and 20 m below the sea surface) were studied in Haizhou Bay during the hot season (summer and autumn) of 2007. Survival and growth rates were quantified bimonthly. Immune activities in hemolymph (superoxide dismutase (SOD) and acid phosphatase (ACP)) were measured to evaluate the health of scallops at the end of the study. Environmental parameters at the five depths were also monitored during the experiment. Mortalities mainly occurred during summer. Survival of scallops suspended at 15 m (78.0%) and 20 m (86.7%) was significantly higher than at 2 m (62.9%), 5 m (60.8%) or 10 m (66.8%) at the end of the study. Mean shell height grew significantly faster at 10 m (205.0 mu m/d) and 20 m (236.9 mu m/d) than at 2, 5 or 15 m in summer (July 9 to September 1); however, shell growth rate at 20 m was significantly lower than at the other four depths in autumn (September 2 to November 6). In contrast to summer, scallops at 5 m grew faster (262.9 mu m/d) during autumn. The growth of soft tissue at different depths showed a similar trend to the shell. Growth rates of shell height and soft tissue were faster in autumn than in summer, with the exception of shell height at 20 m. SOD activity of scallops increased with depth, and ACP activity was significantly higher at 15 and 20 m than at other depths, which suggests that scallops were healthier near the bottom. Factors explaining the depth-related mortality and growth of scallops are also discussed. We conclude that the mass mortality of scallop, C. farreri, during summer can be prevented by moving the culture area to deeper water and yield can be maximized by suspending the scallops in deep water during summer and then transferring them to shallow water in autumn.

Possible suppression of exogenous beta-1,3-glucanase gene gluc78 on rice transformation and growth

Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag7O and gluc78 from the biocontrol fungus Trichoderma atroviride were transformed into rice mediated by Agrobacterium tumefaciens singly and in all possible combinations. A total of more than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. Our data indicated that gluc78 gene had negative effects on transformation frequency and plant growth. Some regenerated plants with gluc78 gene were stunted; spontaneously produced brown specks; could not tassel. The combination with either one of the two other genes (ech42, nag70) present in the same T-DNA region reduced the negative effect of gluc78 on plant growth. These results indicated that expression of several genes in one T-DNA region interfered with each other and expression of exogenous gene in recipient plant was a complex behavior. (c) 2007 Published by Elsevier Ireland Ltd.

条件反射性免疫抑制及其脑机制的c-fos研究

To explore the neural mechanisms underlying conditioned immunomodulation, this study employed the classical taste aversion (CTA) behavioral paradigm to establish the conditioned humoral and cellular immunosuppression (CIS) in Wistar rats, by paring saccharin (CS) with intraperitoneal (i.p.) injection of an immunosuppressive drug cyclophophamide (UCS). C-fos immunohistochemistry method was used to observe the changes of the neuronal activities in the rat brain during the acquisition, expression and extinction of the conditioned immunosuppression (CIS). The followings are the main results: 1. Five days after one trial of CS-UCS paring, reexposure to CS alone significantly decreased the level of the anti-ovalbumin (OVA) IgG in the peripheral serum. Two trials of CS-UCS paring and three reexposures to CS not only resulted in further suppression of the primary immune response, but also reduced the numbers of peripheral lymphocytes and white blood cells. This finding indicates that CS can induce suppression of the immune function, and the magnitude of the effects is dependent on the intensity of training. 2. On day 5 following two trials of CS-UCS pairing, CS suppressed the spleen lymphocytes responsiveness to mitogens ConA, PHA and PWM, and decreased the numbers of peripheral lymphocytes and white blood cells. On day 15, only PHA induced lymphocyte proliferation was suppressed by CS. On day 30, presentation of CS did not have any effect on these immune parameters. These results suggest that the conditioned suppression of the cellular immune function can retain 5-15 days, and extinct after 30 days. 3. CTA was easily induced by one or two CS-UCS parings, and remained robust even after 30 days. These data demonstrate that CIS can be dissociated from CTA, and they may be mediated by different neural mechanisms. 4. Immunohistochemistry assays revealed a broad pattern of c-fos expression throughout the rat brain following the CS-UCS pairing and reexposure to CS, suggesting that many brain regions are involved in CIS. Some brain areas including the solitary tract nucleus (Sol), lateral parabrachial nucleus (LPB) and insular cortex (IC), showed high level c-fos expressions in response to both CS and UCS, suggesting that they may be involved in the transmission and integration of the CS and UCS signals in the brain. There were dense c-FOS positive neurons in the paraverntricular nucleus (PVN) and supraoptic nucleus (SO) of hypothalamus, subfornical organ (SFO) and area postrema (AP) etc. after two trials of CS-UCS paring and after the reexposure to CS 5 days later, but not in the first training and after the extinction of CIS (30 days later). The results reflect that these nuclei may have an important role in CIS expression, and may also response to the immunosuppression of UCS. The conditioned training and reexposure to CS 5 days later induced high level c-fos expression in the cingulate cortex (Cg), central amygdaloid nucleus (Ce), intermediate part of lateral septal nucleus (LSI) and ventrolateral parabrachial nucleus (VLPB) etc. But c-fos induction was not apparent when presenting CS 30 days later. These brain regions are mainly involved in CIS, and may be critical structures in the acquisition and expression of CIS. Some brain regions, including the frontal cortex (Fr), ventral orbital cortex (VO), IC, perirhinal cortex (PRh), LPB and the medial part of solitary nucleus (SolM), showed robust c-FOS expression following the conditioning training and reexposure to CS both on day 5 and day 30, suggesting that they are critically involved in CTA.