Fish Skin. Application of imaging techniques

Abstract

Aquaculture is probably the fastest growing sector in the food industry, producing almost 50 percent of the fish destined for human food worldwide. However, to supply this high demand, fish are farmed in intensive systems, where they are exposed to stress conditions. These factors promote the appearance of diseases, which are manifested mainly on the skin. Thus, this Doctoral Thesis, divided in two parts, aimed to study the skin from fish of interest in aquaculture (Part 1, with four Chapters) and apply imaging techniques to fish study (Part 2, with three Chapters). In Part 1, the effect of different external factors on the skin and its mucus in some fish species was studied. First, different immune parameters were analysed in the skin mucus of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) exposed to a constant light-dark (L:D) photoperiod. Results showed that the IgM levels and the activity of different enzymes (protease, antiprotease, peroxidase and lysozyme) as well as the bactericidal activity remarkably varied during the daily L:D cycle in those fish. Since fish skin is a primary target organ for entry and colonization of several common pathogens, normally found in the aquatic environment, skin integrity is necessary for fish health. Therefore, we studied the effect of dietary supplementation of guava leaf (Psidium guajava L.) at different percentages (1.5% and 3%) on skin mucus of hybrid tilapia (Oreochromis niloticus ¿ O. mossambicus). Immune parameters analysed increased significantly in the skin mucus of fish supplemented with the lowest percentage of guava leaf compared to no supplemented fish (control). In addition, guava leaf supplementation in experimental diets significantly reduced the bacterial load on fish skin after Vibrio harveyi infection, possibly due to the bactericidal properties of this plant. Because skin damage facilitates pathogen entry, we considered analysing the physical and the immune response in the skin of gilthead seabream after experimental wounds (above or below the lateral line) or ulcers in two different trials. Our results showed that the healing process was faster below the lateral line than above it. Furthermore, immune parameters analysed in the skin mucus and serum showed variations depending on the wound site. Gene expression of several immune-relevant genes (il1b, il6, tgfb, ight, grhl, krt1) was also evaluated, showing variations depending on the location of the wound. Regarding experimental ulcers, the abundance of terminal carbohydrates and some immune parameters evaluated in skin mucus showed variations in the ulcerated skin compared to the non-ulcerated. Concomitantly, the analysis of the skin microbiota was performed and demonstrated that skin ulcers provide microenvironments that disturb the mucus composition and the microbial biodiversity of the skin. In another experiment, the fluorescein test was used as an easy and inexpensive tool to detect incipient skin lesions on experimentally damaged skin of rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). In Part 2, the application of real-time ultrasonography and X-ray micro computed tomography (micro-CT) to study gilthead seabream and European sea bass demonstrated that the skin thickness varied depending on the body region. Furthermore, a complete segmentation of gilthead seabream body was carried out by micro-CT showing density values for the whole fish in the range of -1,000 to +2,500 HU (Hounsfield Units), while values for skin and subcutaneous fat were between -400 and -50 HU. In addition, fat density range was stablished between -115 to +50 HU allowing us to identify segmented areas, which topographically coincided with fat depots in gilthead seabream. In conclusion, our study demonstrates that the skin and its mucus may be affected by several external factors present in everyday aquaculture practices. On the other hand, the predisposition of skin for colonization by pathogens, the effect of the diet on the enhancement of the immune response and the influence of physical damage on the skin and its mucus was demonstrated. Hence, it is necessary to improve husbandry conditions, as well as the implementation of preventive tools to ensure and assess an adequate health status in the farmed fish. Finally, the application of imaging techniques to study fish morphometry, provided novel results that allow us to evaluate body changes in situ providing valuable information to be used for the in vivo experimentation, thus seeking to apply the principle of the 3Rs on animal ethics.