It is important to assess the chronic effects of chemical, physical, and biological stressors on organisms in the environment. Appropriate methods must enable rapid, inexpensive, and multibiomarker analyses of organism health. Here we investigate withering syndrome in red abalone (Haliotis rufescens), an important wild and farmed shellfish species along the Pacific coast, using a metabolomic approach that combines the metabolic profiling capabilities of nuclear magnetic resonance spectroscopy (NMR) with pattern recognition methods. Foot muscle, digestive gland, and hemolymph samples were collected from healthy, stunted, and diseased abalone, and the extracts were analyzed by NMR. Following spectral preprocessing, principal components analyses of the metabolite profiles were conducted. Our results confirm that NMR-based metabolomics can successfully distinguish the biochemical profiles of the three groups of animals, in every type of tissue or biofluid studied. Furthermore, this discovery-based approach successfully identified novel metabolic biomarker profiles associated with withering syndrome. The application of these methods for investigating other environmental stressors is discussed, as are the advantages of NMR-based metabolomics for biomonitoring, particularly in conjunction with gene and protein expression profiling.

No abstract

Withering syndrome in California red abalone (Haliotis rufescens) is caused by the Rickettsiales-like prokaryote (WS-RLP) Candidatus Xenohaliotis californiensis. WS-RLP infection is not sufficient to cause withering syndrome, and for reasons not yet well understood additional stressors such as elevated water temperature appear to influence disease development. Using nuclear magnetic resonance (NMR) based metabolomics, we have investigated the influence of food availability, temperature, and bacterial infection, both individually and in combination, on the metabolic status of the red abalone. Food limitation caused dramatic reductions in all observed classes of foot muscle metabolites, while at the same time metabolite levels within the digestive gland were preserved or increased. We also found that food limitation in combination with elevated temperature led to greater metabolic perturbations in both tissue types than those observed under food limitation alone. WS-RLP infection and food-limitation resulted in many of the same metabolic changes within the tissues studied, although the effects of infection were less severe. We observed increased levels of homarine in the digestive gland of both food-limited and WS-RLP-infected animals, yet only observed increased homarine levels in the foot muscle of infected abalone. These results further support the recently established glucose-to-homarine ratio in foot muscle as a potential marker for differentiating WS-RLP-infected animals from those of both healthy and food limited abalone. Furthermore, we found that the NMR metabolic data correlates well with histological measurements supporting the use of the metabolomics approach for characterizing both normal and pathological events in marine species, particularly during periods of environmentally relevant stress.

The antibiotic oxytetracycline (OTC) has shown immense promise for combating the causative agent of Withering syndrome (WS), a Rickettsia-like procaryote (WS-RLP) that has severely impacted California abalone (Haliotis spp.) populations. Using histology and nuclear magnetic resonance (NMR) spectroscopy based metabolomics, the effects of OTC treatments (10, 20, or 30 days) on WS-RLP infected abalone in seawater temperatures of 13.4 +/- 1.2 and 17.3 +/- 1.3 degrees C were investigated over 160 days. The highly efficacious nature of OTC in combating WS-RLP at both temperatures was demonstrated by histology. Metabolomics revealed, however, that the most significant metabolic changes in foot muscle depended upon posttreatment duration, irrespective of treatment and temperature. This was quite unexpected and would have been overlooked using histology alone. Metabolic changes in all animals at both temperatures included decreased levels of amino acids and carbohydrates and elevated taurine, glycine-betaine, and homarine. Subtle metabolic differences between OTC-treated and untreated abalone were observed at 17.3 degrees C only. These findings provide clear evidence that OTC eradicates WS-RLP which in turn reduces the metabolic decay associated with WS at elevated seawater temperature. Furthermore, this study documents the sequential metabolic changes that occur during pre-clinical WS, and demonstrates the application of metabolic phenotyping for understanding environmental effects on host-pathogen-drug interactions.