Aspects of Reproductive Biology of the European Hake (Merluccius merluccius) in the Northern and Central Adriatic Sea (GSA 17-Central Mediterranean Sea)

The study focused on the macroscopic, histological, and biometric analysis of European hake females in GSA 17 (Central-North Adriatic Sea). From 2013 to 2015, 976 females were collected and analyzed monthly. Though females in spawning conditions were found during the whole year, the trend of GSI showed a peak of the reproductive season from April to July in 2014 and 2015. HSI and Kn reached the highest values in September, after the spawning peaks. In 2013, the trend of these indices did not highlight an evident peak, probably due to an adverse event that occurred in the previous winter in the Adriatic shelf. The length at first maturity (L50) was estimated by macroscopic and histological approaches, resulting in 30.81 cm for the macroscopical length and 33.73 cm for the histological length; both values are higher than the current catching legal size. For the first time in this area, batch and relative fecundity were estimated. Relative fecundity was similar to the Mediterranean and the Atlantic stocks, whereas batch fecundity values were lower compared to other fishing grounds. Overall, the analysis of reproductive parameters plays a fundamental role in the sustainable management of this resource in an area as overfished as the Central-North Adriatic Sea

Mediterranean swordfish (Xiphias gladius Linnaeus, 1758) population structure revealed by microsatellite DNA: genetic diversity masked by population mixing in shared areas

Background The Mediterranean swordfish stock is overfished and considered not correctly managed. Elucidating the patterns of the Mediterranean swordfish population structure constitutes an essential prerequisite for effective management of this fishery resource. To date, few studies have investigated intra-Mediterranean swordfish population structure, and their conclusions are controversial. Methods A panel of 20 microsatellites DNA was used to investigate fine-scale population structuring of swordfish from six main fishing areas of the Mediterranean Sea. Results This study provides evidence to reject the hypothesis of a single swordfish population within the Mediterranean Sea. DAPC analysis revealed the presence of three genetic clusters and a high level of admixture within the Mediterranean Sea. Genetic structure was supported by significant FST values while mixing was endorsed by the heterozygosity deficit observed in sampling localities indicative of a possible Wahlund effect, by sampling admixture individuals. Overall, our tests reject the hypothesis of a single swordfish population within the Mediterranean Sea. Homing towards the Mediterranean breeding areas may have generated a weak degree of genetic differentiation between populations even at the intra-basin scale

Study of reproductive biology of European hake (Merluccius merluccius) in Adriatic Sea

Lo scopo del lavoro è stato quello di valutare lo stato attuale dello stock di nasello (Merluccius merluccius, L. 1758) nel mare Adriatico e, più in generale, incrementare la conoscenza sulla biologia riproduttiva di questa specie di importanza commerciale. Per valutare il ciclo riproduttivo, è stato valutato l'andamento degli indici morfometrici per tre anni, il fattore di condizione di Le Cren (Kn), l'indice hepatosomatico (HSI) e l'indice gonadosomatico (GSI). Inoltre, è stata valutata la taglia dei pesci alla prima maturità sia per i maschi che per le femmine. Per la prima volta in Adriatico, è stata calcolata la fecondità, stimata in fecondità di lotto e fecondità relativa, che ha mostrato valori più bassi rispetto ad altre aree del Mediterraneo e Atlantico. Oltre all'analisi macroscopica, è stata eseguita anche l'analisi istologica delle ovaie e testicoli per convalidare la scala macroscopica utilizzata. Infine, per completare lo scenario della fisiologia della riproduzione di questa importante specie commerciale, sono stati sequenziati e caratterizzati gli ormoni gonadotropici (follicle-stimulating hormone, fsh, e luteinizing hormone, lh) e i loro recettori recettori. Nelle femmine, è stata misurata l'espressione di mRNA delle gonadotropine nel cervello e dei recettori negli oociti a differenti classi di oociti, durante il ciclo naturale di riproduzione. Per le gonadotropine, è stato studiata anche la loro localizzazione molecolare nella ghiandola pituitaria delle femmine. Infine, sono stati eseguiti studi tossicologici, attraverso l'analisi dell'espressione molecolare dei geni che indicano una contaminazione da distruttori endocrini, quali vitellogenina a e b e il recettore alpha degli estrogeni nel fegato dei maschi. Tutte le analisi effettuate in questo progetto sono risultate essenziali per prevenire il collasso di questa specie sovrasfruttata.The aim of work was to assess the current state of the stock of European hake (Merluccius merluccius, L. 1758) in Adriatic Sea and overall, to increase the knowledge about its reproductive biology. To evaluate the reproductive cycle, the trend of morphometric indices, Le Cren’s condition factor (Kn), hepatosomatic index (HSI) and gonadosomatic index (GSI) were calculated for three years and the size at first maturity was evaluated in male and female specimens. Furthermore, for the first time in Adriatic Sea, the fecundity, estimated as batch and relative fecundity, were obtained, showing lower values than other Mediterranean and Atlantic areas. In addition to macroscopic analysis, also the histological analysis of the ovaries and testis were performed to validate the used macroscopic scale. Finally, to complete the scenario of the reproductive physiology of this relevant commercial species, the gonadotropin hormones (follicle-stimulating hormone, fsh and luteinizing hormone, lh) and their relative receptors were sequenced, characterized. The mRNA expression of gonadotropins and the receptors was measured in brain and in oocyte, respectively, at different maturity stages of the female specimens during natural reproductive cycle. For the gonadotropins, also their molecular localization was investigated in pituitary gland. Finally, toxicological studies were performed through the analysis of molecular expression of the toxicologically relevant genes (vitellogenin a and b and alpha estrogen receptor), involved in endocrine disruptor metabolism, in male liver, evidencing the contamination in water. All analysis performed in this project are essential to prevent the collapse of overfished species and they should be included in the annual monitoring programs

Reproductive Biology of Male European Hake (Merluccius merluccius) in Central Mediterranean Sea: An Overview from Macroscopic to Molecular Investigation

Simple Summary The vast majority of the world's commercial fish stocks are considered overfished. Among demersal species, in the Mediterranean Sea, the European hake (Merluccius merluccius) is experiencing high fishing pressure, resulting in a critical overexploitation status. The reproductive pattern of the male sex is poorly investigated. In order to provide scientific advice for its sustainable management, this study gave, for the first time in the Central Mediterranean Sea, an overview of the reproductive biology of male European hakes, by using a multidisciplinary approach. The sex ratio throughout the three-year period and by length class showed that males dominated the population from February to June and up to 24 cm, respectively. The reproduction occurred all year round with a seasonal peak in spring/summer. The macroscopic and histological sizes at first maturity were 18.6 cm and 15.4 cm, respectively. The entire reproductive cycle was unequivocally identified by five histological developmental stages of the testis. Finally, the molecular approach allowed us to investigate endocrine regulation from the onset of puberty to the adult lifecycle. The improvement of scientific knowledge about overexploited fishery resources allow us to provide scientific advice for their management and stock protection. By using a multidisciplinary approach, the aim of the study was to characterize, for the first time in the Central Mediterranean Sea (GSA 17), the reproductive biology of males of M. merluccius, currently highly exploited. A multi-year sampling from January 2017 to December 2019 was performed to exhaustively evaluate the sex ratio of the stock, while the 2018 annual sampling was selected to investigate the reproductive pattern of males. Individuals in spawning conditions were found every month, proving that M. merluccius is an asynchronous species, reproducing all year round, with a seasonal reproductive peak in spring/summer, as indicated by GSI. Five gonadal development stages were defined to fully describe the reproductive cycle of males. The macroscopic and histological L-50, respectively 18.6 cm and 15.4 cm, were both below the Minimum Conservation Reference Size (MCRS). According to the mRNA levels, fsh and lh played a significant role during spermiation, whereas the gnrhr2a was involved at the beginning of sexual maturity. In the testis, fshr and lhr reached maximum expression levels before the spermiation. The hormonal stimuli of 11-ketotestosterone and its receptor were significantly higher when the specimen was in reproductive activity

Probiotic Administration Mitigates Bisphenol A Reproductive Toxicity in Zebrafish

Although the use of bisphenol A (BPA) has been banned in a number of countries, its presence in the environment still creates health issues both for humans and wildlife. So far, BPA toxicity has been largely investigated on different biological processes, from reproduction to development, immune system, and metabolism. In zebrafish, Danio rerio, previous studies revealed the ability of environmentally relevant concentrations of this contaminant to significantly impair fertility via epigenetic modification. In addition, several studies demonstrated the ability of different probiotic strains to improve organism health. This study provides information on the role of the probiotic mixture SLAb51 to counteract adverse BPA effects on reproduction. A 28-day trial was set up with different experimental groups: BPA, exposed to 10 µg/L BPA; P, receiving a dietary supplementation of SLAb51 at a final concentration of 109 CFU/g; BPA+P exposed to 10 µg/L BPA and receiving SLAb51 at a final concentration of 109 CFU/g and a C group. Since oocyte growth and maturation represent key aspects for fertility in females, studies were performed on isolated class III (vitellogenic) and IV (in maturation) follicles and liver, with emphasis on the modulation of the different vitellogenin isoforms. In males, key signals regulating spermatogenesis were investigated. Results demonstrated that in fish exposed to the combination of BPA and probiotic, most of the transcripts were closer to C or P levels, supporting the hypothesis of SLAb51 to antagonize BPA toxicity. This study represents the first evidence related to the use of SLAb51 to improve reproduction and open new fields of investigation regarding its use to reduce endocrine disrupting compound impacts on health

Gonadotropin characterization, localization and expression inthe European hake Merluccius merluccius

In vertebrates, the regulation of gametogenesis is under the control of gonadotropins (Gth), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). In fish, the physiological role of Gths is not fully understood, especially in species with asynchronous ovarian development. In order to elucidate the role of Gths in species with asynchronous ovary, we studied European hake (Merluccius merluccius) during the reproductive season. For this aim, we first cloned and sequenced both hormones. Then, we characterized their amino acid sequence and performed phylogenetic analyses to verify the relationship to their orthologues in other species. In addition, the quantification of gene expression during their natural reproductive season was analyzed in wild-caught female hake. Our results revealed that fshb peaked during the vitellogenic phase, remaining high until spawning. This is in contrast to the situation in species with synchronous ovary. lhb, on the other hand, peaked during maturation as is common also in species with synchronous ovarian development. Finally, combining double-labeling fluorescent in situ hybridization (FISH) for Gth mRNAs with immunofluorescence for Lh protein, we evidenced the specific expression of fshb and lhb in different cells within the proximal pars distalis (PPD) of the pituitary. In addition to gonadotrope cells specific to expression of either fshb or lhb, some cells showed co-expression of both genes. This suggests either that gonadotropes with co-expression are not yet specified, or they could have a plasticity that permit changes from one cell phenotype to another during certain life stages and in turn during different physiological states

Insights on the seasonal variations of reproductive features in the eastern Atlantic bluefin tuna

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as β and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences

Reducing the Use of Antibiotics in European Aquaculture with Vaccines, Functional Feed Additives and Optimization of the Gut Microbiota

This review presents several alternatives to replace antibiotic therapy and make the European aquaculture industry more friendly and environmentally sustainable. The first part of this review highlights the growing importance of the aquaculture industry worldwide for its ability to supply low-cost proteins and lipids. The second part discusses different strategies for these replacements, from recombinant vaccines to diets with low environmental impact and rich in bioactive molecules that can benefit other species. Specifically, the beneficial effects of bioactive compounds present within insect meals are discussed. In addition, particular focus is placed on the importance of adopting sustainable protocols for fish farming, including supplements such as probiotics, prebiotics, and synbiotics capable of modulating the gut microbiota as the second brain. Those feed additives can stimulate European farmed species’ immunological systems, growth, and welfare