LASER CAPTURE MICRODISSECTION OF INTESTINAL TISSUE FROM EUROPEAN SEA BASS LARVAE  

The increasing demand for a sustainable larviculture industry has promoted research regarding optimal environmental and nutritional parameters so as to avoid diseases. The mucosal surface of the intestinal tract is known to constitute the first line of defence against pathogen invasion. Therefore, many areas of intense research have hinged upon a better understanding of intestinal mucosal health, with a focus on the impact of nutrition, immunostimulants, pre- and probiotics, and exposure to pathogens. Transcriptomic methods offer a rapid and valuable approach to characterize these effects. However, studies resorting to gene expression analyses in fish larvae remain scarce and resort to using homogenized whole larval bodies for RNA extraction. This is rooted in the small size of fish larvae in the first weeks following hatching. Laser capture microdissection (LCM) circumvents the difficult sampling of the tiny fish larvae by allowing researchers to isolate specific cell populations or individual cells from sections of complex tissues. However, RNA degradation constitutes a major drawback of this technique, considering that RNA quality has a major impact on the following gene expression profiling results. In addition to this, the choice of a proper normalization method to correct for sample input is crucial.

In the current study, we optimized an LCM protocol to procure intestinal tissue from fish larvae whilst maintaining a high RNA quality for downstream gene expression analysis by RT-qPCR. European sea bass (Dicentrarchus labrax, Linnaeus, 1758) was employed as a model species, as it is the most extensively cultured seawater fish species from the Mediterranean. For that purpose, fixation protocols were compared focusing on preserving tissue morphology and RNA integrity, two critical success factors for LCM and consequent RT-qPCR. Furthermore, a 3'/5' integrity assay was optimized for LCM samples of fish tissue, comprising low RNA concentrations. In addition, a set of reliable reference genes were identified out of nine candidate reference genes to perform gene expression analysis in several developmental stages of sea bass larvae  under various experimental conditions.

We demonstrated that a meticulous optimization of the LCM procedure allows recovery of high quality mRNA from intestinal tissue. According to the geNorm and Normfinder algorithms, translation elongation factor (ef1a) and 40S ribosomal protein S30 (faua) were the most stable genes to be implemented as reference genes for an appropriate normalization of intestinal tissue from sea bass larvae across a range of experimental settings. The developed methodology offers a rapid and valuable approach to characterize actors in the intestinal tissue of fish larvae and their changes following pathogen exposure, nutritional/environmental changes, probiotic supplementation or a combination thereof.