Matteo Alessandro Avella

Microbial manipulations to improve fish health and production--a Mediterranean perspective.

The interactions between the endogenous gut microbiota and the fish host are integral in mediating the development, maintenance and effective functionality of the intestinal mucosa and gut associated lymphoid tissues (GALTs). These microbial populations also provide a level of protection against pathogenic visitors to the gastrointestinal (GI) tract and aid host digestive function via the production of exogenous digestive enzymes and vitamins. Manipulation of these endogenous populations may provide an alternative method to antibiotics to control disease and promote health management. Applications of probiotics for Mediterranean teleosts can stimulate immune responses, enhance growth performance, feed utilisation, digestive enzyme activities, antioxidant enzyme activities, gene expression, disease resistance, larval survival, gut morphology, modulate GI microbiota and mediate stress responses. Although considerably less information is available regarding prebiotic applications for Mediterranean teleosts, prebiotics also offer benefits with regards to improving immune status and fish production. Despite the promising potential benefits demonstrated in current literature, obtaining consistent and reliable results is often difficult due to our incomplete understanding of indigenous fish GI microbiota and their subsequent host interactions which mediate and drive both localised and systemic host immunological responses. Additionally, the probiotic and prebiotic (biotics) mechanisms which mediate host benefits at the mucosal interface are poorly understood. Future studies focused on these interactions utilising gnotobiotic techniques should provide a better understanding of how to extract the full potential of biotic applications to promote immune function of Mediterranean teleosts.

Effect of dietary probiotics on clownfish: a molecular approach to define how lactic acid bacteria modulate development in a marine fish

We set out to determine whether probiotic addition would improve larval development in the false percula clownfish Amphiprion ocellaris and to determine what molecular responses could be observed in the larvae following probiotic exposure. We supplied the probiotic bacterial strain Lactobacillus rhamnosus IMC 501 to clownfish larvae from the first day posthatch simultaneously by live prey and with addition to rearing water (group 2) and exclusively by live prey (group 3). We observed twofold higher body weight in both clownfish larvae and juveniles when probiotics were supplied via live prey and added to the rearing water. In addition, development was accelerated with metamorphosis occurring 3 days earlier in fingerlings treated with probiotic. Alteration in molecular biomarkers supported the faster growth observation. There was significantly increased gene expression of factors involved in growth and development (insulin-like growth factors I and II, myostatin, peroxisome proliferator-activated receptors alpha and beta, vitamin D receptor alpha, and retinoic acid receptor gamma) when probiotics were delivered via live prey and added to the rearing water. Moreover, probiotic treatment lessened the severity of the general stress response as exhibited by lower levels of glucocorticoid receptor and 70-kDa heat shock protein gene expression. Furthermore, an improvement of skeletal head development was observed, with a 10-20% reduction in deformities for juveniles treated with probiotic. All data suggest a potent effect on development resulting from the administration of lactic acid bacteria to larval clownfish, and this study provides a preliminary molecular entry path into the investigation of mechanisms responsible for probiotic enhancement in fish development.

Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

Endogenous microbiota play essential roles in the host’s immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host’s development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

Effects of probiotic administration on zebrafish development and reproduction.

As the consumption of probiotics increases worldwide, scientists focus on identifying bacterial strains able to improve human life quality and evidence the biological pathways affected by probiotic treatment. In this review, some recent observations on the effects of changes of microbiota on zebrafish metabolism were discussed. In addition, the effects of Lactobacillus rhamnosus - a component of the human gut microflora - as a diet supplement on Danio rerio were presented. When administered chronically, L. rhamnosus may affect larval development and the physiology of reproductive system in the zebrafish model. It was hypothesized exogenous L. rhamnosus accelerates larval growth and backbone development by acting on insulin-like growth factors-I (igfI) and -II (igfII), peroxisome proliferator activated receptors-α and -β, (pparα,β) vitamin D receptor-α (vdrα) and retinoic acid receptor-γ (rarγ). Gonadal differentiation was anticipated at 6weeks together with a higher expression of gnrh3 at the larval stage when L. rhamnosus was administered throughout development. Moreover, brood stock alimented with a L. rhamnosus-supplemented diet showed better reproductive performances as per follicles development, ovulated oocytes quantification and embryos quality. A plausible involvement of factors such as leptin, and kiss1 and 2 in the improvements was concluded. The observations made on the physiology of female reproduction were correlated with the gene expression of a gigantic number of factors as the aromatase cytochrome p 19 (cyp19a), the vitellogenin (vtg) and the α isoform of the E2 receptor (erα), luteinizing hormone receptor (lhr), 20-β hydroxysteroid dehydrogenase (20β-hsd), membrane progesterone receptors α and β, cyclin B, activinβA1, smad2, transforming growth factor β1 (tgfβ1), growth differentiation factor9 (gdf9) and bone morphogenetic protein15 (bmp15.) A model in which the exogenous L. rhamnosus in the digestive tract of zebrafish from the first days of life through sexual maturation positively influences the physiological performances of zebrafish was evidenced and a number of pathways that might be influenced by the presence of this human probiotic strain were proposed.

Modulation of cortisol levels, endocannabinoid receptor 1A, proopiomelanocortin and thyroid hormone receptor alpha mRNA expressions by probiotics during sole (Solea solea) larval development.

In the present study, we investigated whether the use of Enterococcus faecium IMC 511 as a probiotic can modulate neuroendocrine system responses during the larval rearing of Solea solea; to this end, the gene expression patterns of proopiomelanocortin (POMC), endocannabinoid receptor 1A (CB1A), and thyroid receptor alpha (TRα) were quantified, and whole-body cortisol levels were measured. Probiotic treatment up-regulated transcription of all selected genes and cortisol concentrations on day 10 post hatch (ph), while on day 30 ph experimental groups showed significantly lower levels of both POMC and CB1A compared to those of the control group. These changes were no longer evident on day 60 ph, when POMC, CB1A, TRα gene expression and cortisol titers were found to be similar in all experimental groups. Our results suggest that metabolic responses to probiotic treatment can be modulated through the activation of genes selected for functional interaction between the hypothalamic-pituitary-thyroid (HPT) axis and the melanocortin and the endocannabinoid systems. Furthermore, the observed (30 ph) down-regulation of both POMC and CB1A gene expression coupled with up-regulation of TRα mRΝΑ levels suggest the activation of a compensatory mechanism that promotes growth and development and perhaps modulates food intake.

Effects of Probiotics on Zebrafish Reproduction

Recent studies have demonstrated that probiotics can influence reproductive performance, however, the mechanisms influencing oogenesis remain poorly explored. In this study, the effects of dietary probiotic Lactobacillus rhamnosus IMC 501 ® administration was investigated on female zebrafish ( D. rerio ) reproductive performance and ovarian development (both growth and maturation phases). After feeding the zebrafish experimental diets for 10 days, real-time PCR and culture based methods revealed alterations on gut microbiota, characterised by high levels of L. rhamnosus IMC 501 ® in the gut of probiotic treated zebrafish, which were absent in the control. The microbial modulation in the gut was related with a significant increase of the gonadosomatic-index (GSI) associated, at the molecular level, to a significant increase of ovarian expression of cytochrome p 19 (cyp19a), hepatic vitellogenin (vtg) and the α isoform of the estradiol receptor (erα) genes evidencing a positive role of probiotic administration on the ovarian growth phase. Concomitantly, a significant increase of signals which positively control the maturation phase such as, cyclin B, activinβA1 and small mother against decapentaplegic 2 (smad2) genes in the ovary led us to confirm the beneficial role of the probiotic on maturation processes. Next to the enhancement of oocyte development processes by probiotic administration, an improvement of fecundity was evidenced which was associated with the increase of cyclooxygenase 2a (cox2a) gene expression in the ovary. The significance of the results obtained herein underlines the importance of diet and gut microbes in the reproductive processes, supporting the hypothesis that feed additives could improve fecundity.

Welfare improvement using alginic acid in rainbow trout (Oncorhynchus mykiss) juveniles

The present study explored alternative strategies to improve the health status and growth metabolism in cultured juvenile fish. In this context the effects of alginic acid administration on the welfare of rainbow trout juveniles were investigated. Both the hepatic gene expression of heat shock protein 70 and the cortisol plasma levels were evaluated in order to estimate the possible beneficial role of alginic acid in improving animal well-being. At the same time the body weight was measured and was correlated with the hepatic expression of insulin-like growth factor I, myostatin, and two isoforms of the thyroid receptor. Finally, improvement of the immune system was investigated by monitoring the expression of innate immune-related genes, such as interleukin1β, interleukin8 and toll-like receptor3, in the spleen. The data obtained demonstrated an improvement in the welfare of rainbow trout juveniles, revealed by growth and innate immune response enhancement by alginic acid administration. These results may be of great importance for the further development of more sustainable aquaculture practices, and can be considered as a biotechnological application to produce a safer product for human health.