RUI: Hypercapnic Hypoxia Impacts Shrimp Immune Defenses Against Bacterial Pathogens

和陆地生物一样，大多数海洋生物也需要氧气才能生存。近年来，世界沿岸水域的溶解氧含量一直在下降。由于沿海地区的集约化农业和城市化，导致了水中氧气（缺氧）的减少。在水生环境中，缺氧通常伴随着水质其他方面的变化，包括溶解的二氧化碳和酸度或ph值。实验室和实地调查支持这样一种观点，即水质的这一系列变化，称为高碳酸血症性缺氧，可以增加驻留生物在接触致病微生物或病原体后感染和患病的风险。沿海地区是重要的商业鱼鳍和贝类主要群体的重要繁殖地。对虾，包括白虾、褐虾、太平洋虾和虎虾，是在沿海河口生活和繁殖的重要甲壳类动物。初步研究表明，在接触副溶血性弧菌细菌病原体后，高碳酸血症性缺氧会增加太平洋对虾（凡纳滨对虾）的感染率和死亡率。高碳酸缺氧可直接增强细菌本身的分布、存活或生长，或抑制虾宿主的免疫防御。本研究的目的是研究宿主凡纳美弧菌与其病原体副溶血性弧菌之间相互作用的三个因素，这些因素可能对高碳酸血症缺氧敏感。血细胞是甲壳类动物中主要负责免疫防御的细胞，可能对溶解氧和二氧化碳的水平或ph值敏感。为了检验这种可能性，本研究的第一个目标是确定伴随亚致死高碳酸缺氧的溶解气体和酸度的组织水平变化是否会抑制分离的血细胞杀死或抑制细菌生长的能力。一些证据也表明，对高碳酸血症缺氧的生理反应，如通气率、血淋巴循环和细胞-细胞聚集的增加，可能会改变虾体内血细胞的正常分布，降低清除感染性病原体的效率。为了解决这种可能性，本研究的第二个目的是检查注射活细菌后，高碳酸血症缺氧是否会改变血细胞向鳃或其他主要组织区室的正常运动模式。这些运动将使用分子和免疫探针对参与免疫防御的血细胞相关分子进行监测。第三组实验将检验氧气、二氧化碳和pH值的变化是否会改变虾清除细菌感染的效率。在这些研究中，将给南美嗜血杆菌注射一种能产生绿色荧光蛋白的转基因副溶血性弧菌。基因改造将使研究人员能够追踪细菌的去向，在哪里被杀死，以及它是如何最终从虾中消灭的。这些研究结果有望为保护对虾这一重要的自然资源提供基础信息，同时增加我们对甲壳类动物呼吸功能、离子调节和抗病性之间相互作用的理解。

Like their terrestrial counterparts, most marine organisms require oxygen to live. In recent years the levels of dissolved oxygen in waters along the world's coastlines has been decreasing. This decline in water oxygen (hypoxia) has been attributed to many factors stemming from intensive agriculture and urbanization in the coastal zone. In aquatic environments, hypoxia is often accompanied by changes in other aspects of water quality, including dissolved carbon dioxide and acidity or pH. Laboratory and field investigations support the idea that this suite of changes in water quality, referred to as hypercapnic hypoxia, can increase the risk of resident organisms to infection and disease following exposure to a disease-causing microbe, or pathogen. Coastal zones are critical breeding grounds for major groups of commercially important fin and shellfish. Penaeids, including the white, brown, Pacific and tiger shrimps, are such an important group of crustaceans that live and breed in coastal estuaries. Preliminary studies have shown that hypercapnic hypoxia increases the rate of infection and death in the Pacific shrimp, Litopenaeus vannamei, following exposure to the bacterial pathogen, Vibrio parahaemolyticus. Hypercapnic hypoxia may directly enhance the distribution, survival, or growth of the bacterium itself, or suppress immune defenses of the shrimp host. The objectives of the proposed studies are to examine three factors of the interaction between the host L. vannamei and its pathogen, V. parahaemolyticus, that might be sensitive to hypercapnic hypoxia. Hemocytes, those cells principally responsible for immune defense in crustaceans, may be sensitive to levels of dissolved oxygen and carbon dioxide, or to pH. To examine this possibility, the first objective of this study is determine whether tissue-level changes in dissolved gasses and acidity that accompany sublethal hypercapnic hypoxia can suppress the ability of isolated hemocytes to kill or inhibit the growth of bacteria. Several lines of evidence also suggest that physiological responses to hypercapnic hypoxia, such as increases in ventilation rate, hemolymph circulation and cell-cell aggregation, might alter the normal distribution of hemocytes in the shrimp and reduce the efficiency of clearing an infectious pathogen. To address this possibility, the second objective of this study is to examine whether hypercapnic hypoxia alters the normal pattern of hemocyte movement to the gill or to other major tissue compartments following an injection of live bacteria. These movements will be monitored using both molecular and immunological probes specific for hemocyte-associated molecules that are involved in immune defense. A third set of experiments will examine whether changes in oxygen, carbon dioxide and pH can alter the efficiency with which the shrimp clears a bacterial infection. In these studies, L. vannamei will be injected with a genetically-modified V. parahaemolyticus that produces green fluorescent protein. The genetic modification will allow investigators to track where the bacteria goes, where it is killed, and how it is ultimately eliminated from the shrimp. The results of these studies are expected to provide basic information that can be used toward protecting a key natural resource, the Penaeid shrimp, while increasing our understanding of interactions between the functions of respiration, ion regulation and disease resistance in crustaceans.