Edwardsiella-associated outbreaks are increasingly reported on both marine and freshwater aquaculture setups, accounting for severe financial and biomass losses. E. tarda, E. ictaluri, and E. hoshinae have been the traditional causative agents of edwardsiellosis in aquaculture, however, intensive studies due to the significance of the disease have just recently revealed two more species, E. piscicida and E. anguillarum. Whole genome sequencing that was conducted on the strain EA011113, isolated from farmed Diplodus puntazzo after an edwardsiellosis outbreak in Greece, confirmed it as a new clinical strain of E. anguillarum. Extensive phylogenetic analysis showed that this Greek strain is closely related to an Israeli E. piscicida-like clinical strain, isolated from diseased groupers, Epinephelus aeneus and E. marginatus in Red Sea. Bioinformatic analyses of E. anguillarum strain EA011113 unveiled a wide repertoire of potential virulence factors, the effect of which was corroborated by the mortalities that the strain induced in adult zebrafish, Danio rerio, under different levels of infection intensity (LD₅₀ after 48 h: 1.85 × 10⁴ cfu/fish). This strain was non-motile and according to electron microscopy lacked flagella, a fact that is not typical for E. anguillarum. Comparative genomic analysis revealed a deletion of 36 nt found in the flagellar biosynthetic gene (FlhB) that could explain that trait. Further in silico analysis revealed an intact prophage that was integrated in the bacterial genome. Following spontaneous induction, the phage was isolated, purified, characterized and independently sequenced, confirming its viability as a free, inducible virion as well. Separate genomic analysis of the prophage implies a plausible case of lysogenic conversion. Focusing on edwardsiellosis as a rapidly emerging aquaculture disease on a global scale, this work offers some insight into the virulence, fitness, and potential lysogenic conversion of a of a newly described, yet highly pathogenic, strain of E. anguillarum.

The Hsd11b2 enzyme converts cortisol into its inactive form, cortisone and regulates cortisol levels, in particular in response to stress. Taking advantage of CRISPR/Cas9 technology, we generated a hsd11b2 zebrafish mutant line to evaluate the involvement of this gene in stress response regulation. The absence of a functional Hsd11b2 affects survival of zebrafish, although homozygous hsd11b2^−/− mutants can reach adulthood. Reproductive capability of hsd11b2^−/− homozygous adult males is almost completely abrogated, while that of females is reduced. Interestingly, basal cortisol levels and glucocorticoid-dependent transcriptional activities are not affected by the mutation. In agreement with basal cortisol results, we also demonstrated that basal response to light (LMR-L/D) or mechanical (VSRA) stimuli is not significantly different in wild-type (hsd11b2^+/+) compared to mutant larvae. However, after exposure to an acute stressor, the cortisol temporal patterns of synthesis and release are prolonged in both 5 days post fertilization larvae and one-year-old adult hsd11b2^−/− zebrafish compared to wild-type siblings, showing at the same time, at 5 dpf, a higher magnitude in the stress response at 10 min post stress. All in all, this new zebrafish model represents a good tool for studying response to different stressors and to identify mechanisms that are induced by cortisol during stress response.

Cortisol concentration in fish scales is a novel and reliable indicator of chronic stress. However, until now cortisol in scales has been considered to be accumulated through the circulation and it has not yet been studied whether it can be de novo produced from cells found in the scales. In the current study, scales of European sea bass, Dicentrarchus labrax, were stimulated in-vitro with a range of concentrations of adrenocorticotropic hormone (ACTH) to investigate if they can produce and release cortisol. Moreover, scales were exposed to a combination of ACTH and metyrapone, an inhibitor of cortisol production, to examine whether cortisol was actually produced in the scales. Results from ACTH administration showed that scales increased their cortisol release in a dose-dependent manner. This effect was reversed when scales were co-incubated with ACTH and metyrapone, indicating that cortisol was produced de novo and not released only upon stimulation with ACTH.

Background: In fish, minimally invasive blood sampling is widely used to monitor physiological stress with blood plasma biomarkers. As fish blood cells are nucleated, they might be a source a potential new markers derived from ‘omics technologies. We modified the epiGBS (epiGenotyping By Sequencing) technique to explore changes in genome-wide cytosine methylation in the red blood cells (RBCs) of challenged European sea bass (Dicentrarchus labrax), a species widely studied in both natural and farmed environments.Results: We retrieved 501,108,033 sequencing reads after trimming, with a mean mapping efficiency of 73.0% (unique best hits). Minor changes in RBC methylome appeared to manifest after the challenge test and a family-effect was detected. Only fifty-seven differentially methylated cytosines (DMCs) close to 51 distinct genes distributed on 17 of 24 linkage groups (LGs) were detected between RBCs of pre- and post-challenge individuals. Thirty-seven of these genes were previously reported as differentially expressed in the brain of zebrafish, most of them involved in stress coping differences. While further investigation remains necessary, few DMC-related genes associated to the Brain Derived Neurotrophic Factor, a protein that favors stress adaptation and fear memory, appear relevant to integrate a centrally produced stress response in RBCs.Conclusion: Our modified epiGBS protocol was powerful to analyze patterns of cytosine methylation in RBCs ofD. labraxand to evaluate the impact of a challenge using minimally invasive blood samples.This study is the first approximation to identify epigenetic biomarkers of exposure to stress in fish.

The social behaviour of twenty-one lambs of three sheep breeds (Chios, Karagouniki, and Synthetic) was observed from the age of 2.5–7 months, divided into three equal periods (A, B, C) of 1.5 months each. The frequency of the performed agonistic behaviours was higher (p < 0.01) before four months of age (period A: 11 × 10⁻⁴ ± 2 × 10⁻⁴) compared to the other periods (B: 5 × 10⁻⁴ ± 1 × 10⁻⁴; C: 8 × 10⁻⁴ ± 1 × 10⁻⁴). The same was noticed for affiliative behaviours performed (A: 21 × 10⁻⁴ ± 3 × 10⁻⁴; B: 8 × 10⁻⁴ ± 1 × 10⁻⁴; C: 5 × 10⁻⁴ ± 1 × 10⁻⁴), and for agonistic and affiliative behaviours received (p < 0.001). The lambs had higher levels of wool cortisol (23.398 ± 5.344 pg/mg; p < 0.01) at period C compared to A (7.899 ± 1.19 pg/mg). Breed affected the affiliative behaviours. Karagouniki showed higher frequencies than Synthetic (16 × 10⁻⁴ ± 3 × 10⁻⁴ vs. 6 × 10⁻⁴ ± 1 × 10⁻⁴). Blood cortisol was higher (p < 0.05) in the Synthetic (4.789 ± 0.928 ng/mL) compared to the Chios (1.805 ± 0.417 ng/mL) breed. Overall, lambs’ behaviour displayed changes upon time, with four months of age being a step to a next developmental stage with fewer social interactions and higher levels of cortisol.

There is growing interest about marine bivalve aquaculture globally, not only for the market value of the goods produced, but also for the socio-economic and environmental services that this activity can provide. Arca noae is an endemic Mediterranean bivalve of commercial value, whose previously undescribed population in Elounda Bay we studied in terms of its structure and reproduction, while constructing a timeseries of the basic environmental parameters of the bay, thus, gaining fundamental knowledge for the potential future exploitation of the species in the area. We found a variable spatial distribution of arks in the study area, with local high peaks in the population density, consisting of smaller size individuals, in comparison to other areas. Because of protandry of the species, human pressure on this population could have a strong negative effect, by targeting the limited numbers of large females in the study area. The reproduction pattern was similar to the reports from other Mediterranean locations. The abiotic conditions in Elounda Bay differed from those in the adjacent coastal zone, confirming that the Bay is a unique semi-enclosed marine area in the island of Crete.