THE JOURNAL OF EXPERIMENTAL ZOOLOGY 249~350-356 (1989) zy RAPID COMMUNICATION Monitoring of Metabolic Responses of Intact zy HuZiotis (Abalones) Under zyxw Salinity Stress by 31P Surface Probe Localized NMR RICHARD M. HIGASHI, TERESA W.-M. FAN, AND JEFFREY M. MACDONALD zyxwvu Department of Environmental Toxicology (R .M.H., J.M.M.) and NMR Facility /T.W.-M.F.). University zyxwvu of California Davis, California 95616 zy ABSTRACT Surface probe localized "P NMR spectroscopy was employed to record the meta- bolic responses of the foot of intact Haliotis cracherodii and H. rufescens (black and red abalones) under hyper- and hypoosmotic stresses. Use of the surface probe allowed spectral localization on the foot of intact abalones, facilitated monitoring of different sizes of animals, and minimized constraints on aquatic chamber design normally imposed by homogeneous-field probes. Generally, hyperosmotic stress (51%) elicited more rapid changes of phosphate metabolites than hypoosmotic stress zyxwvuts (17%0). As with the well-studied hypoxic stress in intact mammalian and excised molluscan tissue, both salinity treatments caused drops in the phosphagen and increases in inorganic phos- phate levels. However, osmotic stress was distinct from hypoxic stress in that intracellular pH did not change and nucleotide triphosphate (NTP) concentrations dropped immediately. Although these findings are preliminary, they demonstrate the utility of the surface probe approach for studies of environmental stress in intact marine invertebrates. The development of in vivo NMR methodologies over the past decade has offered a practical means for investigating difficult problems of compartmen- tation, transport, and coupled metabolic fluxes (Gad- ian, '82). The versatility of in vivo NMR has been developed for and demonstrated in a number of bi- ological systems from isolated cells (Shulman et al., zyxwvu '79) to excised organs of mammals (Cohen et al., '79) and plants (Loughman and Ratcliffe, '84). The tech- nique of organ-localizedNMR on intact animals closely followed these developments (see Gadian, '82). Typ- ical applications have included measurements of in- tracellular pH, ion transport kinetics, compartmen- tation dynamics, rates of phosphate metabolite turnover, intermediary metabolite fluxes, and me- tabolite composition, to list just a few. In the past few years, in vivo NMR has expanded to studies of aquatic animals, including excised muscles of aquatic molluscs (Burt et al., '76; Barrow et al., '80; Elling- ton, '83) and nonlocalized spectroscopy of whole or- ganisms (Thompson and Lee, '85; Drinkwater and Crowe, '87). However, application of organ-localized NMR to study intact animals in the aquatic envi- ronment is yet at an infant stage. Following a report on 31P NMR study of hypoxic brain metabolism in intact submerged turtles (Wemmer et al., '82), lo- calized 31P NMR of intact aquatic crustaceans was reported almost simultaneously for crayfish tail (Butler et al., '85) and crab leg muscles (Briggs et al., '85). More recently, studies of tail muscles of intact shrimp (Kamp and Juretschke, '87) and prawn (Thebault et al., '87) have been reported. The cray- fish study lacked experimental feasibility since some animals were permanently altered and all were an- aesthesized as well as placed in 50% 'H20 solvent to accommodate their instrument needs, but the oth- ers appeared to address the physiological require- ments of their organisms. Certainly, for investigating normal and environ- mental stress physiology of marine organisms such as Haliotis (abalone), the spectrometer must be ca- pable of accommodating the intact organism under controlled conditions. As a simple, effective way of achieving this, we have assembled a NMR spectrom- eter-compatible aquatic chamber to provide proper control of salinity, temperature, and oxygenation. In combination with this, we chose to implement sur- face probe-"P NMR on abalones because the surface Received October 14, 1988; revision accepted November 16, 1988. Address reprint requests to Richard M. Higashi, University of Cal- R.M. Higashi and J.M. Macdonald are now at University of Cali- T.W-M. Fan is now at Department of Environmental Toxicology, ifornia Bodega Marine Laboratory, Bodega Bay, CA 94923. fornia Bodega Marine Laboratory, Bodega Bay, CA 94923. University of California Davis, Davis, CA 95616. zyx 0 1989 ALAN It. LISS, INC.