Utilization of dried blood spots for assessing dusky kob (Argyrosomus japonicus) glycaemia and metabolome in South African aquaculture.
Mdlalose, Thabani Irvin.
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Dusky kob (Argyrosomus japonicus) is an important marine fish in South African aquaculture, and has the potential to become commercially significant. The continuous growth of Dusky kob culture faces challenges with regards to fish health. There is currently a lack of on-farm tools for fish health assessment and Dusky kob are known to be prone to parasitic infections. Fish blood glucose is a reliable indicator of biological and environmental stress; however, methods of glycaemia diagnosis are often lab based and cannot be conducted rapidly. This limits the use of glucose to assess the condition of fish on-site. Furthermore, the gill parasite Diplectanum oliveri continuously affects Dusky kob and the only know method of detection is microscopic analysis which requires destructive sampling. The drawbacks of fish health assessment are further amplified by the lack of reliable methods of sample collection, storage and transportation. Conventional methods of blood sample collection have limitations, including complex protocols (collection and storage), sample degradation during storage and large volumes required for analysis. Additionally, blood samples (plasma, serum and whole blood) are often restricted to single use due to sample degradation, thus do not allow for repeated analysis. Dried blood spots (DBS) offer an alternative method of sample collection, with benefits including ease of use (collection and handling), storage and transportation. Furthermore, DBS require relatively small volumes and ensure sample integrity is maintained due to the elimination of a liquid medium thus inhibiting metabolic reactions. DBS further allow for repeated analyses across a wide range of blood parameters and numerous assays. The use of DBS is currently limited to human health research, however, can be beneficial in fish health assessment and biomarker discovery in aquaculture. In the present study, the utility of DBS as (1) a reliable method of blood collection and (2) an effective medium for biomarker discovery in fish was investigated. Dusky kob DBS were collected from four farms namely; Mtunzini Fish Farm (currently Zini Fish Farms, KwaZulu-Natal), Oceanwise (currently Ocean Choice), Pure Ocean (both Eastern Cape) and Blue Cap (Western Cape) fish farms between February – May 2015. The use of a hand-held diabetic glucometer as a reliable tool for on farm glucose measurement tested against laboratory based enzymatic analyses of DBS, plasma and whole blood. Lastly, the application of metabolomics analyses of Dusky kob DBS was examined as a potential for a non-destructive alternative method for the detection of parasitic infections. A consistent over-estimation of glucose by DBS was observed, while the use of a diabetic glucometer was shown to be a reliable tool for on-farm glucose measurements. The accuracy of the glucometer was evident by the correlation with plasma glucose (R2 = 0.973). Plasma is the recommended medium for glucose analysis since the removal of red blood cells inhibits glycolysis. Additionally, targeted metabolomic analysis of DBS by LC-MS and GC-MS identified 53 metabolites. Six amino acids (citrulline, glutamine, lysine, methionine, phenylalanine and proline) were significantly altered in accordance to parasite intensities and/or geographical distributions. The results indicate that hand-held glucometers can be used on-farm for accurate preliminary measures of glucose, which is further beneficial for rapidity and routine analysis. Continuous measurement of glucose can aid in detecting hyper- or hypoglycaemia. The ease of use provided by DBS is essential for biomarker discovery and fish health assessment. In addition to simple storage and transportation, DBS also ensure the stability of blood parameters including metabolites. Metabolomics analysis offer an essential platform for the early detection of parasites infecting farmed fish and eliminates the need for destructive sampling. The examination of metabolites reveals compounds that are essential in the innate and adaptive immune response of fish during infection. Understanding the roles of these compounds can be used to develop and implement corrective measures.