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Title: Biological responses to hypoxic and low salinity stresses in the green-lipped mussel Perna viridis
Other Titles: Fei cui yi bei zai que yang ji di yan du xie po xia zhi sheng wu fan ying
Authors: Wang, Youji ( 王有基)
Department: Department of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2011
Publisher: City University of Hong Kong
Subjects: Perna -- Effect of oxygen on.
Perna -- Effect of salt on.
Hypoxia (Water)
Notes: CityU Call Number: QL430.7.M95 W36 2011
xxi, 257 leaves : ill. (some col.) 30 cm.
Thesis (Ph.D.)--City University of Hong Kong, 2011.
Includes bibliographical references (leaves 191-257)
Type: thesis
Abstract: Hypoxia has been recognized as a major problem on a global scale in many estuarine and coastal regions influencing marine animals over the last several decades. Environmental stresses rarely occur in isolation. Their effects on marine invertebrates, however, are frequently studied in isolation, regardless of potential interaction among the stresses. Estuaries and coastal areas with intensive aquaculture activities are highly stressful environments, with fluctuating salinity and common occurrence of hypoxia. The green-lipped mussel Perna viridis is widely distributed in the Indo-Pacific region and extensively cultured in Asia as a cheap protein source owing to its fast growth and natural abundance. The present study investigated the biological responses to hypoxia and hyposalinity. Parameters studied included hemocyte immunology, physiology, biochemistry and anti-predatory responses. In bivalves, physiological responses to environmental stresses are mediated, at least partially, by hemocytes. Therefore, to provide a better understanding of the role of hemocytes in internal defence and to expand the knowledge of environmental immunotoxicology in Perna viridis, it is essential to explore the fundamental knowledge of hemocytes. Two major types of hemocytes were identified in the hemolymph, including granulocyte and hyalinocyte. Flow cytometry has revealed that the granulocytes are more active than the hyalinocytes in terms of cell phagocytosis, esterase activity and reactive oxygen species (ROS) generation and have a higher lysosomal content. The immune functions assessed by the flow cytometry indicated that the granulocytes are the main hemocytes involved in the cellular defence in the green-lipped mussel. The impact of hypoxia on mussels may aggravate hemocytes in Perna viridis at low salinities. An experiment was conducted to examine the combined effects of hypoxia and salinity on immune responses in hemocytes of P. viridis. Using flow cytometry, P. viridis were exposed to six combinations of oxygen level (1.5 and 6.0 mg O2 l-1) and salinity (15, 22 and 30), and immune responses were examined at 24, 48, 96 and 168 hours. The mussels were then transferred to a normoxic condition (6.0 mg O2 l-1) at salinity of 30 for further 24 hours to study their recovery from the combined hypoxic and salinity stress. Results showed that hypoxia and low salinity affected the immune functions in P. viridis; interactive effects of dissolved oxygen (DO) and salinity were also observed. Hypoxia and hyposalinity resulted in higher hemocyte mortality. Phagocytosis at low salinities and hypoxia was lower compared to the normal DO and salinity treatment. The activity of esterase and ROS production at low salinity and hypoxia were also reduced. Lysosomal content and total hemocyte counts were lower at low salinity and hypoxia compared to other treatments. After 24 hours of recovery, hypoxia and salinity effects were still significant; this indicated that the immune functions of P. viridis could not recover from combined stresses in a short time. The effect of hypoxia and low salinity on the energy budget of Perna viridis was also studied. Juvenile mussels were maintained for four weeks under different combinations of dissolved oxygen concentration (1.5, 3.0 and 6.0 mg O2 l-1) and salinity (15, 20, 25 and 30) in a 3 × 4 factorial design experiment. Clearance rate (CR), absorption efficiency (AE), respiration rate (RR) and scope for growth (SFG) decreased with decreasing salinity and DO, while excretion rate (ER) increased with decreasing salinity and increasing DO. The O:N ratio was < 10 at salinities of 15 and 20, irrespective of DO levels. SFG was negative in most of the treatments, except for those under 6.0 mg O2 l-1 or at a salinity of 30 when DO was lower. The combined effect of hypoxia and salinity on body composition and growth was studied. Mussels were cultured for six weeks under four different salinities (15, 20, 25 and 30) and three dissolved oxygen concentrations (1.5, 3.0 and 6.0 mg O2 l-1) in a 4 × 3 factorial design. All growth parameters (shell length (SL), tissue dry weight (TDW), condition index (CI), specific growth rate (length: SGRL weight: SGRW)) were reduced under reduced DO and salinities, but interactive effects between these two factors were statistically indistinguishable, except for SGRW. Higher percentages of crude fat (CF) and crude protein (CP) and lower percentages of crude carbohydrate (CC) were obtained at reduced salinities and DO. When changes in biochemical content (weight individual-1) were compared, both CP and CC content decreased significantly as salinity or DO decreased, whereas no pattern was observed for CF. Energy content (EC, calories g-1) was not significantly different among DO treatments, but significantly varied with salinity. Total energy content (calories individual-1), however, increased significantly with both DO and salinity, but the interaction between salinity and DO was statistically indistinguishable. In marine mussels, anti-predator responses, which include thicker shell and increased byssus production, occur upon exposure to predators. However, energy supply is limited by aerobic metabolism and osmoregulation. If energy allocation to the induction of anti-predator responses remains unchanged under hypoxia and low salinity, there is a disproportionate decrease in energy supply for other physiological processes, which may reduce the tolerance of the mussels to hypoxic or low salinity stress and lessen the chance of survival. In contrast, a larger reduction in the anti-predator response, as compared with other physiological processes under hypoxia, may elevate predation risk. To understand how the mussels responded to this dilemma, Perna viridis were exposed to combined stressors of hypoxia and low salinity, with two dissolved oxygen concentrations (1.5 and 6.0 mg l-1) and two salinities (15 and 30) in the presence of the predator, the swimming crab Thalamita danae, and byssus production was monitored for 48 hours. Fewer byssus threads, which were also shorter and thinner, were produced at reduced oxygen and salinity levels, no matter if the predator was present or not; the frequency the mussels shedding their stalks was also lower. Mussels exposed to the predator, however, have enhanced byssus thread production at all oxygen and salinity levels as compared with the control. This has highlighted the significance of anti-predator responses for the survival of individuals, even under a stressful environment in which energy supply is limited by aerobic metabolism. Interactive effects between oxygen level, salinity and predator exposure were observed for the byssus thread production. The present study has demonstrated the wide-ranging effects of hypoxia and low salinity on the immune response, physiology, biochemical composition and anti-predator responses in Perna viridis. As P. viridis is widely cultured in sheltered harbours and bays in Southeast Asia, which may suffer from eutrophication, the results in this study may provide useful guidelines for culture practices and site selection for this species.
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