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.
