Evaluation of fluorogenic substrates in the assessment of digestive enzymes in a decapod crustacean Maja brachydactyla larvae

Abstract

The potential use of fluorescent substrates for measuring digestive enzyme activities has been adapted to decapod crustacean larvae and compared to traditional analytical techniques based on spectrophotometry. The ontogeny of three digestive enzymes (trypsin, amylase, and esterase) from the hepatopancreas of Maja brachydactyla has been selected to illustrate the advantages and disadvantages of both spectrophotometric (SP) and fluorometric (FL) methods. The three enzyme activities showed the same pattern with either of the techniques used. Standardised discriminant function coefficients indicated that the stage of development in spider crab larvae was most strongly discriminated by the activity of amylase, whichever method of analysis was considered. The FL method allows the detection of up to three enzyme activities in a single larva (< 100 μg dry weight), whereas SP needs more than 20 times this weight. The FL method allows studying the inter-individual variability, while the SP approach only permits one to evaluate the inter-population variability (pool of individuals). Nevertheless, the higher sensitivity of the FL method implies a larger variability in the results compared to SP. There are no differences in feasibility between the two methods when using kinetic analyses. Although data from different experiments assayed by SP methods are easily standardised in units of dry weight or soluble protein concentration, this standardisation is difficult when using FL substrates, since differences between the sensitivity of the equipment used for the analysis might be higher than one order of magnitude. The availability of FL substrates in the market is constantly increasing, although it is still scarce in relation to the high number and types of substrates normally used in SP methods. This may be a limitation to the study of certain enzyme activities. The cost of the analysis is based on the price of the specific substrates, FL substrates being more expensive than SP ones. Both SP and FL methods are useful to measure digestive enzymes in decapod crustacean larvae. Selection of the method used will depend on the objectives and interests of the researcher. For studies based on the characterization of enzyme activities in crustacean populations without any limitation on the number and amount of samples, the SP method is recommended. When several experimental treatments, dietary regimes, and/or moulting stages are considered, the FL analysis might be more appropriate due to the low number of individuals needed for sampling. If the study is conducted to assess the individual variability of the population, then, the FL method needs to be applied.