We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 microns in length can be clearly visualized. The method is suitable for both ontogenic studies during early stages of skeletal development in most marine fishes (e.g., Sparus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.

A microencapsulated feed additive composed by garlic, carvacrol and thymol essential oils (EOs) was evaluated regarding its protective effect in gills parasitized by Sparicotyle chrysophrii in Sparus aurata. A nutritional trial (65 days) followed by a cohabitation challenge with parasitized fish (39 days) were performed. Transcriptomic analysis by microarrays of gills of fish fed the EOs diet showed an up-regulation of genes related to biogenesis, vesicular transport and exocytosis, leukocyte-mediated immunity, oxidation–reduction and overall metabolism processes. The functional network obtained indicates a tissue-specific pro-inflammatory immune response arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant and anti-inflammatory responses. The histochemical study of gills also showed an increase of carboxylate glycoproteins containing sialic acid in mucous and epithelial cells of fish fed the EOs diet, suggesting a mucosal defence mechanism through the modulation of mucin secretions. The outcomes of the in vivo challenge supported the transcriptomic results obtained from the nutritional trial, where a significant reduction of 78% in the abundance of S. chrysophrii total parasitation and a decrease in the prevalence of most parasitic developmental stages evaluated were observed in fish fed the EOs diet. These results suggest that the microencapsulation of garlic, carvacrol and thymol EOs could be considered an effective natural dietary strategy with antiparasitic properties against the ectoparasite S. chrysophrii.

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BackgroundOrganophosphate pesticides-OP-, like malathion, can alter the normal functioning of neuro-endocrine systems (e.g., hypothalamus-pituitary-thyroid-HPT- axis), and to interfere on the thyroidal homeostasis. Through direct interactions with thyroid receptors, an/or indirectly via up-stream signalling pathways, from the HPT axis (i.e., negative feedback regulation), malathion possess the ability to affect integrity of thyroidal follicular tissue, and it can also block or delay its hormonal functioning. This insecticide can alter the majority of the ontogenetic processes, inducing several deformities, and also provoking decreases in the growth and survival patterns. The present study has been performed to determine the sublethal effects of malathion during the first month of life of the Senegalese sole, Solea senegalensis, and it is mainly focused on the metamorphosis phase. Different transcript expression levels (i.e. thyroid receptors, matrix and bone -Gla-proteins) and immunohistochemical patterns (i.e. thyroid hormones, osteocalcin, cell proliferation) have been analysed during the most critical phases of the flatfish metamorphosis, that is, through differentiation of thyroid system and skeletal development, migration of the eye, and further adaptation to benthic behaviours.ResultsIn early life stages of the Senegalese sole, the exposure to the highest concentration of malathion (6.25 μg/L) affected to the growth patterns, showing the exposed individuals, a reduction around 60 and 92% of the total length and the dry weigth, respectively. In paralell, a significant reduction of the thyroid follicles (i.e., size and number) it was also been recorded, in a dose-dependent way. Abnormal phenotypes induced in the exposed larvae, did not complete the process of metamorphosis, and displayed several morphological abnormalities and developmental disorders, which were mainly associated with the eye migration process, and with thyroidal and skeletal disorders (i.e., transcriptional and protein changes of thyroid hormones and receptors, and of matrix and bone Gla proteins distribution), that conduced to an inadequate adaptation to the benthic life.ConclusionsIn the Senegalese sole, the majority of the ontogenetic alterations induced by the exposure to malathion were mainly associated to the metamorphosis period, which is a thyroid-driven proccess. In fact, most crucial and transitional ontogenic events, appeared notably disturbed, for e.g., thyroid gland differentiation and functioning, migration of eye, skeletal development and benthonic behaviors.

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins’ glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.

Vitamin A (VA) and retinoid derivatives are known morphogens controlling vertebrate development. Despite the research effort conducted during the last decade, the precise mechanism of how VA induces post-natal bone changes, and particularly those operating through crosstalk with the thyroid hormones (THs) remain to be fully understood. Since effects and mechanisms seem to be dose and time-dependent, flatfish are an interesting study model as they undergo a characteristic process of metamorphosis driven by THs that can be followed by external appearance. Here, we studied the effects of VA imbalance that might determine Senegalese sole (Solea senegalensis) skeletogenetic phenotype through development of thyroid follicles, THs homeostasis and signaling when a dietary VA excess was specifically provided during pre-, pro- or post-metamorphic stages using enriched rotifers and Artemia as carriers. The increased VA content in enriched live prey was associated to a higher VA content in fish at all developmental stages. Dietary VA content clearly affected thyroid follicle development, T3 and T4 immunoreactive staining, skeletogenesis and mineralization in a dose and time-dependent fashion. Gene expression analysis showed that VA levels modified the mRNA abundance of VA- and TH-specific nuclear receptors at specific developmental stages. Present results provide new and key knowledge to better understand how VA and TH pathways interact at tissue, cellular and nuclear level at different developmental periods in Senegalese sole, unveiling how dietary modulation might determine juvenile phenotype and physiology.