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Gonad maturation is critical for the reproductive success of any organism, and in fish, captivity can significantly affect their reproductive performance, leading to maturation incompetence and spawning failure. The greater amberjack (Seriola dumerili), a fish species recently introduced to aquaculture fails to undergo oocyte maturation, ovulation, and spawning when reared in aquaculture facilities. Since confinement has been shown to influence gonad maturation and completion of the reproductive cycle, investigations into epigenetic mechanisms may shed light on the reasoning behind the reproductive dysfunctions of fish under captivity. Among the known important epigenetic regulators are small non-coding RNAs (sncRNAs), and in particular microRNAs (miRNAs). In this study, immature, maturing (late vitellogenesis), and spent ovaries of captive greater amberjack were collected, and the differential expression of miRNAs in the three different ovarian development stages was examined. Expression patterns of conserved and novel miRNAs were identified, and potential targets of highly differentially expressed miRNAs were detected. Additionally, read length distribution showed two prominent peaks in the three different ovarian maturation stages, corresponding to miRNAs and putative piwi-interacting RNAs (piRNAs), another type of ncRNAs with a germ-cell specific role. Furthermore, miRNA expression patterns and their putative target mRNAs are discussed, in relevance with the different ovarian maturation stages of captive greater amberjack. Overall, this study provides insights into the role of miRNAs in the reproductive dysfunctions observed in fish under captivity and highlights the importance of epigenetic mechanisms in understanding and managing the reproductive performance of economically important fish species., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The greater amberjack Seriola dumerili is a promising candidate for aquaculture production. This study compares the ovary transcriptome of greater amberjack sampled in the wild (WILD) with hatchery-produced breeders reared in aquaculture sea cages in the Mediterranean Sea. Among the seven sampled cultured fish, three were classified as reproductively dysfunctional (DysF group), while four showed no signs of reproductive alteration (NormalF group). The DysF fish showed 1,166 differentially expressed genes (DEGs) compared to WILD females, and 755 DEGs compared to the NormalF. According to gene ontology (GO) analysis, DysF females exhibited enrichment of genes belonging to the biological categories classified as Secreted, ECM-receptor interaction, and Focal adhesion. Protein-protein interaction analysis revealed proteins involved in the biological categories of ECM-receptor interaction, Enzyme-linked receptor protein signaling, Wnt signal transduction pathways, and Ovulation cycle. KEGG pathway analysis showed DEGs involved in 111 pathways, including Neuroactive ligand-receptor interaction, Steroid hormone biosynthesis, Cell cycle, Oocyte meiosis, Necroptosis, Ferroptosis, Apoptosis, Autophagy, Progesterone-mediated oocyte maturation, Endocytosis and Phagosome, as well as Hedgehog, Apelin, PPAR, Notch, and GnRH signalling pathways. Additionally, DysF females exhibited factors encoded by upregulated genes associated with hypogonadism and polycystic ovary syndrome in mammals. This study -which is part of a broader research effort examining the transcriptome of the entire reproductive axis in greater amberjack of both sexes-, enhances our comprehension of the mechanisms underlying the appearance of reproductive dysfunctions when fish are reared under aquaculture conditions., (© 2024. The Author(s).)

We compared the endocrine status of the pituitary-gonad axis of wild and captive-reared greater amberjack (Seriola dumerili) during the reproductive cycle (April - July), reporting on the expression and release of the two gonadotropins for the first time in the Mediterranean Sea. Ovaries from wild females were characterized histologically as DEVELOPING in early May and SPAWNING capable in late May-July, the latter having a 3 to 4-fold higher gonadosomatic index (GSI). SPAWNING capable wild females exhibited an increase in pituitary follicle stimulating hormone (Fsh) content, plasma testosterone (T) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P), while almost a 10-fold increase was observed in pituitary luteinizing hormone (Lh) content. An increasing trend of plasma 17β-estradiol (E 2 ) was also recorded between the two reproductive stages in wild females. Captive-reared females sampled during the reproductive cycle exhibited two additional reproductive categories, with REGRESSED females having extensive follicular atresia and fish in the REGENERATING stage having only primary oocytes in their ovaries. Pituitary content of Fsh and Lh, fshb and lhb expression and plasma levels of Fsh and Lh remained unchanged among the four reproductive stages in captive females, in contrast with plasma E 2 and T that decreased in the REGENERATING stage, and 17,20β-P which increased after the DEVELOPING stage. In general, no significant hormonal differences were recorded between captive-reared and wild DEVELOPING females, in contrast to SPAWNING capable females, where pituitary Lh content, plasma Fsh and T were found to be lower in females in captivity. Overall, the captive females lagged behind in reproductive development compared to the wild ones and this was perhaps related to the multiple handling of the sea cages where all the sampled fish were maintained. Between wild males in the DEVELOPING and SPAWNING capable stages, pituitary Lh content, plasma T and 17,20β-P, and GSI exhibited 3 to 4-fold increases, while an increasing trend of pituitary Fsh content, lhb expression levels and plasma 11-ketotestosterone (11-KT) was also observed, and an opposite trend was observed in plasma Lh. Captive males were allocated to one more category, with REGRESSED individuals having no spermatogenic capacity. During the SPAWNING capable phase, almost all measured parameters were lower in captive males compared to wild ones. More importantly, captive males showed significant differences from their wild counterparts throughout the reproductive season, starting already from the DEVELOPING stage. Therefore, it appears that captivity already exerted negative effects in males prior to the onset of the study and the multiple handling of the cage where sampled fish were reared. Overall, the present study demonstrated that female greater amberjack do undergo full vitellogenesis in captivity, albeit with some dysfunctions that may be related to the husbandry of the experiment, while males, on the other hand, may be more seriously affected by captivity even before the onset of the study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In earlier studies, wild-caught greater amberjack Seriola dumerili (Risso, 1810) males reared in sea cages showed gametogenesis impairment and low sperm production and quality. Here, we (a) examined if F1 hatchery-produced males reared in sea cages also exhibit reproductive dysfunctions and (b) evaluated the effects of gonadotropin releasing hormone agonist (GnRHa) administration through injections (GnRHa inj ) or sustained-release implants (GnRHa impl ), and human chorionic gonadotropin (hGC) injections on spermatogenesis/spermiation enhancement. Fish were given a hormone treatment just prior to the spawning season, and were transferred to land-based tanks, according to an established spawning induction protocol. Blood samples (n = 6) were obtained on Days 0, 7 and 13 after treatment. Testis samples were obtained on Days 0 (n = 4) and 13 (n = 2 per treatment). The fish prior to their transfer from the sea cages to the land-based tanks, exhibited a low gonadosomatic index, altered sex steroid hormone profile and high density of testicular apoptotic cells. After transfer to tanks, there was a general depression of sex steroid plasma levels parallel to an increase in cortisol concentrations. Despite the negative effect on steroidogenesis by the transfer from the sea, the hormonal treatments increased the number of fish from where sperm could be obtained, as well as testis growth, and reduced testicular apoptosis. Treatment with hCG resulted in the most significant changes in spermatogenesis, while GnRHa impl appeared to induce less intense, but likely longer-lasting effects. The study indicated that F1 hatchery-produced males also exhibited reproductive dysfunctions as wild-caught captive-reared greater amberjack, and that the observed positive effects of the hormone treatments on spermiation/spermatogenesis were likely mediated by factors other than sex steroid hormones., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We evaluated the onset of puberty of first-generation (F1) hatchery-produced greater amberjack (Seriola dumerili) reared in sea cages for 5 years. Fish were sampled every year in June, at the expected peak of the spawning period in the Mediterranean Sea. No sexual dimorphism in body weight was observed in the study. The ovaries of 1 and 2-year-old (yo) females consisted of primary oocytes only, while at the age of 3-yo early vitellogenic (Vg) oocytes were also identified, but with extensive follicular atresia. At the age of 4-yo, late Vg oocytes were observed, but again extensive follicular atresia characterized the ovaries of 50 % of females. At the age of 5-yo, follicular atresia of Vg oocytes was very limited. In males, gametogenesis was evident already in 1- and 2-yo fish, and 100 % of sampled 3-yo males produced collectable viable sperm. Plasma testosterone (T), 17β-estradiol (E2), and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) remained similar in 3 - 5-yo females, with T and E2 levels being highest in females in advanced vitellogenesis or with significant follicular atresia, compared to immature females. In males, plasma T declined over the years, while 11-ketotestosterone (11-KT) and 17,20β-P were highest in 4 and 5-yo males, with spermatozoa motility characteristics being improved from the 4th year onwards. The administration of GnRHa implants to 5-yo fish induced only two spawns, albeit no fertilized eggs were obtained. The results indicate that hatchery-produced greater amberjack males mature well and within the same age observed in the wild, however with smaller gonad size. On the contrary, females mature later than in the wild, also with a smaller gonad size. Spawning in response to GnRHa treatment was not effective, suggesting that Mediterranean hatchery-produced broodstocks may be dysfunctional, and further research is needed to document any improvement as the fish get older, or to determine if the results may be related to the specific stock of fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Reproductive dysfunctions have been recently documented in male greater amberjack Seriola dumerili caught from the wild and reared in captivity. In the present study, we compared testis transcriptome in wild fish (WILD), hatchery-produced fish with apparently normal spermatogenesis (Normal Farmed; NormalF) and hatchery-produced fish with evident reproductive dysfunction (Dysfunctional Farmed; DysF). Gene expression analysis identified 2157, 1985 and 74 differentially expressed genes (DEGs) in DysF vs WILD, NormalF vs DysF and NormalF vs WILD comparisons, respectively. In DysF, a dysregulation of several interconnected biological processes, including cell assembly, steroidogenesis and apoptosis was found. Gene enrichment of progesterone-mediated oocyte maturation, oocyte meiosis and cell cycle pathways were identified in the DysF vs NormalF comparison. Most of the DEGs involved in the enriched pathways were downregulated in DysF. The comparison of NormalF vs WILD showed that most of the DEGs were downregulated in NormalF, including a gene that encodes for a regulatory protein with a protective role in apoptosis regulation (ptpn6), indicating that spermatogenesis was dysfunctional also in the apparently "normal" hatchery-produced fish. Hence, rearing of male greater amberjack in captivity, from eggs produced by captive breeders, did not prevent the appearance of reproductive dysfunctions, and these dysfunctions involved several biological processes and metabolic pathways., (© 2023. Springer Nature Limited.)

Metamorphosis entails hormonally regulated morphological and physiological changes requiring high energy levels. Probiotics as feed supplements generate ameliorative effects on host nutrient digestion and absorption. Thereby, the aim of the present research was to investigate the impact of the probiotic Phaeobacter inhibens as a water additive on cellular signaling pathways in the metamorphosis of greater amberjack ( Seriola dumerili ). Activation of insulin-like growth factor type 1 receptor (IGF-1R), protein kinase B (Akt), mitogen-activated protein kinases (MAPKs) and AMP-activated protein kinase (AMPK), induction of heat shock proteins (Hsps), and programmed cell death were assessed through SDS-Page/immunoblot analysis, while energy metabolism was determined through enzymatic activities. According to the results, greater amberjack reared in P. inhibens -enriched water entered the metamorphic phase with greater body length, while protein synthesis was triggered to facilitate the hypertrophic growth as indicated by IGF-1/Akt activation and AMPK inhibition. Contrarily, MAPKs levels were reduced, whereas variations in Hsps response were evident in the probiotic treatment. Apoptosis and autophagy were mobilized potentially for the structural remodeling processes. Furthermore, the elevated enzymatic activities of intermediary metabolism highlighted the excess energy demands of metamorphosis. Collectively, the present findings demonstrate that P. inhibens may reinforce nutrient utilization, thus leading greater amberjack to an advanced growth and developmental state.

The meagre Argyrosomus regius (Asso, 1801) is a marine fish species that has an increasing aquaculture production in Europe. Lowering the age at maturity of hatchery-produced juveniles would support meagre aquaculture by reducing time between generations in selective breeding programs and reducing industrial costs for broodstock maintenance. The aim of this work was to assess the effects of a treatment with recombinant follicle stimulating hormone (rFsh), produced in ovarian cells of Chinese hamsters, on male germ cell proliferation and apoptosis in sexually immature meagre. The rFsh-treated fish had higher gonadosomatic index, larger seminiferous tubules, more abundant luminal spermatozoa, a lower density of anti-PCNA positive single A spermatogonia, a higher density of anti-PCNA positive spermatocysts and a lower incidence of germ cell apoptosis than control groups. The present study demonstrated the effectiveness of the produced rFsh in stimulating testis development and spermatogenesis in pre-pubertal meagre. Moreover, the rFsh treatment proved to be highly efficient in removing the apoptotic block of spermatogenesis observed in juvenile meagre, allowing spermatogonial survival and progress towards meiosis. The administration of rFsh did not stimulate spermatogonial self-renewal, a process whose control still needs to be elucidated., (© 2023. The Author(s).)

Background: Undergraduate medical curricula often fail to integrate experiential learning methodologies. Thus, a pilot series of interactive pathology lessons was designed and implemented in an attempt to promote experiential learning., Methods: Thirty pre-graduate medical students voluntarily participated in the interactive study groups at the First Department of Pathology of the National and Kapodistrian University of Athens, Medical School. A questionnaire was designed to investigate the satisfaction of students regarding their participation in pathology study groups and to identify the characteristics that shape students' perceptions of the foundations of medical education. Descriptive statistics (mean values) were used to describe the students' evaluations of the pathology study groups, and thematic analysis was conducted to investigate the data collected using open-ended questions., Results: Interactions with the professor and the option of co-observing the slides using dual-view optical microscopes and virtual slides were each evaluated as "Excellent" by ≅ 95% of the students. Four overarching themes were identified regarding the core characteristics of medical education according to the students' perspectives: 1) educational background in medical education, 2) interaction with educators in medical education, 3) educational material in medical education and 4) assessment in medical education., Conclusions: The high rates of acceptance of the pathology study groups reflect the desire and need for active learning methodologies to be implemented in modern medical education. Nearly all the students mentioned the need for practical skill acquisition, the integration of theory into practice and ethics in medical education. The success of these optional pathology study groups highlights the need for similar modalities to be incorporated into the main medical education curriculum., (© 2023. The Author(s).)

The meagre, Argyrosomus regius , has recently become a species of increasing economic interest for the Mediterranean aquaculture and there is ongoing work to boost production efficiency through selective breeding. Access to the complete genomic sequence will provide an essential resource for studying quantitative trait-associated loci and exploring the genetic diversity of different wild populations and aquaculture stocks in more detail. Here, we present the first complete genome for A. regius , produced through a combination of long and short read technologies and an efficient in-house developed pipeline for assembly and polishing. Scaffolding using previous linkage map data allowed us to reconstruct a chromosome level assembly with high completeness, complemented with gene annotation and repeat masking. The 696 Mb long assembly has an N50 = 27.87 Mb and an L50 = 12, with 92.85% of its length placed in 24 chromosomes. We use this new resource to study the evolution of the meagre genome and other Sciaenids, via a comparative analysis of 25 high-quality teleost genomes. Combining a rigorous investigation of gene duplications with base-wise conservation analysis, we identify candidate loci related to immune, fat metabolism and growth adaptations in the meagre. Following phylogenomic reconstruction, we show highly conserved synteny within Sciaenidae. In contrast, we report rapidly evolving syntenic rearrangements and gene copy changes in the sex-related dmrt1 neighbourhood in meagre and other members of the family. These novel genomic datasets and findings will add important new tools for aquaculture studies and greatly facilitate husbandry and breeding work in the species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Papadogiannis, Manousaki, Nousias, Tsakogiannis, Kristoffersen, Mylonas, Batargias, Chatziplis and Tsigenopoulos.)

Cell growth and differentiation signals of insulin-like growth factor-1 (IGF-1), a key regulator in embryonic and postnatal development, are mediated through the IGF-1 receptor (IGF-1R), which activates several downstream pathways. The present study aims to address crucial organogenesis and development pathways including Akt, MAPKs, heat shock response, apoptotic and autophagic machinery, and energy metabolism in relation to IGF-1R activation during five developmental stages of reared Seriola dumerili: 1 day prior to hatching fertilized eggs (D-1), hatching day (D0), 3 days post-hatching larvae (D3), 33 (D33) and 46 (D46) days post-hatching juveniles. During both the fertilized eggs stage and larval-to-juvenile transition, IGF-1R/Akt pathway activation may mediate the hypertrophic signaling, while p44/42 MAPK phosphorylation was apparent at S. dumerili post-hatching processes and juvenile organs completion. On the contrary, apoptosis was induced during embryogenesis and autophagy at hatching day indicating a potential involvement in morphogenetic rearrangements and yolk-sac reserves depletion. Larvae morphogenesis was accompanied by a metabolic turnover with increased substantial energy consumption. The findings of the present study demonstrate the developmental stages-specific shift in critical signaling pathways during the ontogeny of reared S. dumerili., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Environmental effects and, particularly, temperature changes have been demonstrated to influence the activity, function, and well-being of teleosts. Temperature may change seasonally in the wild, and in captivity under aquaculture operations. Moreover, climate change is expected to shift temperature profiles worldwide. MicroRNAs (miRNA) are important temperature-sensitive gene-expression regulators acting at the post-transcriptional level. They are known to be key regulators in development, reproduction, and immune responses. Therefore, early larval development of the European sea bass ( Dicentrarchus labrax ), one of the most extensively cultured species in Mediterranean aquaculture, was investigated at early rearing temperatures, i.e., 15, 17.5, and 20 °C, in regard to the impact of temperatures on miRNAs through sncRNA high-throughput sequencing but also at the phenotypic level in terms of growth, sex, vision, and skeletal deformities. Expression profiling revealed stage- and temperature-specific miRNA expression targeting genes with roles in reproduction and immune response mainly at the flexion and all-fins stages. Similar stage- and temperature-specific results were also observed concerning the number of rod cells and lower jaw elongation. The present work presents for the first time highly promising results on the influence of early rearing temperature at the post-transcriptional level during European sea bass development, with a putative impact on reproduction and immune response, as well as regarding teleost vision and larval development.

Q fever is a worldwide spread zoonotic disease, caused by the gram-negative intracellular bacillus Coxiella burnetii. Apart from its most common manifestations, Q fever has been reported to occasionally mimic autoimmune diseases. We herein present a case of acute Q fever in a 69-year-old man, manifesting as prolonged fever with pneumonitis, in whom biopsy of the temporal artery revealed giant cell arteritis. Moreover, PCR testing of the biopsy specimen was positive for Coxiella burnetii, thus further supporting the possibly infectious etiology of some cases of biopsy proven giant cell arteritis, with implications for treatment., (Copyright © 2022 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The histological process of gonadal differentiation, together with the endocrine changes of sex steroid hormones and some of their precursors, was studied in hatchery-produced greater amberjack Seriola dumerili from 101 until 408 days post-hatching (dph), with samplings conducted every 50 days. Histological processing showed that sex differentiation began at 101 dph with the formation of the ovarian cavity in females, while the presumptive males did not yet contain any germ cells in their gonad. At 150 dph, we observed the first germ cells in the developing testes. Sex differentiation in almost all sampled individuals was complete at 408 dph. No size dimorphism was observed between the sexes, and the sex ratio was 1:1, suggesting that there was no influence of early rearing in captivity on sex differentiation. Plasma concentrations of adrenosterone (Ad), androstenedione (Δ4), 11-ketotestosterone (11ΚΤ), testosterone (Τ), estradiol (Ε 2 ), progesterone (P4) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) were measured in males and females with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine their role in the sex differentiation process. From the seven hormones, the only one that exhibited differences between the sexes was 11-KT and the plasma 11-KT concentration was found to be a useful indication of greater amberjack sex. Variations were observed in the mean values of Ad, Δ4, 11-KT, T, P4 and 17,20βP over time in one or both sexes, indicating their involvement in the sex differentiation process., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Atresia of ovarian follicles, that is the degenerative process of germ cells and their associated somatic cells, is a complex process involving apoptosis, autophagy and heterophagy. Follicular atresia is a normal component of fish oogenesis and it is observed throughout the ovarian cycle, although it is more frequent in regressing ovaries during the postspawning period. An increased occurrence of follicular atresia above physiological rates reduces fish fecundity and even causes reproductive failure in both wild and captive-reared fish stocks, and hence, this phenomenon has a wide range of implications in applied sciences such as fisheries and aquaculture. The present article reviews the available literature on both basic and applied traits of oocyte loss by atresia, including its morpho-physiological aspects and factors that cause a supraphysiological increase of follicular atresia. Finally, the review presents the use of early follicular atresia identification in the selection process of induced spawning in aquaculture and the implications of follicular atresia in fisheries management., (© The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

The effect of fasting on spawning performance, maternal, and egg nutrient composition, and on embryo/larval development was monitored in gilthead seabream (Sparus aurata). Two broodstocks were fasted during two consecutive years, for a period of 43 and 54 days within the spawning season, in a preliminary (year 1, 5-year-old breeders) and the main study (year 2, 6-year-old breeders), respectively. Mean daily fecundity showed a declining trend during fasting in the main study only, while fertilization success was high in both years and it was not affected by fasting, as was hatching and 5-day larval survival. There was a loss of 23.5% of maternal body mass due to fasting, and a reduction in gonadosomatic and hepatosomatic indexes, as well as crude protein in maternal muscle and gonads, but not in the liver. After fasting, muscle Σω-6 PUFA and C18:3ω-3 were reduced while C20:4ω-6, 20:5ω-3/20:4ω-6, and C22:6ω-3/20:4ω-6 increased; in the liver, significant reductions were observed in C16:0, C18:3 ω-3, 20:5ω-3/C22:6ω-3 and increases in C18:0, C20:5ω-3, Σω-6 PUFA, and 20:5ω-3/20:4ω-6; in gonads, C15:0, ΣMUFA, 20:5ω-3/C22:6ω-3, 20:5ω-3/20:4ω-6 were increased, while C18:1ω-9 and C20:5ω-3 decreased. Contrary to maternal tissues, the energy density and proximate composition of the eggs did not change due to fasting. The study suggests that fasting of gilthead seabream breeders for 6-8 weeks during the spawning period does not affect spawning performance, egg proximate composition, or embryo and early larval development since maternal nutrient reserves are mobilized to maintain optimal egg nutrient composition., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Sturgeon sperm maturation occurs outside the testes during the transit of testicular spermatozoa (TS) through the kidneys and the Wolffian ducts. A method of in vitro TS maturation in sterlet Acipenser ruthenus was used to investigate the effects of temperature and hormonal stimulation of spermiation on the ability of TS to complete this process. Spermatozoa motility parameters after in vitro maturation of testicular sperm, concentrations of sex steroid hormones and testis morphology were studied in three groups of sterlet: (1) after overwintering in ponds (OW), (2) adapted to spawning temperature (ST), and (3) adapted to spawning temperature with hormonal induction of spermiation (ST-HI). Blood plasma concentrations of testosterone, 11-ketotestosterone and 17,20β-dihydroxy-pregnenolone increased significantly after hormonal induction of spermiation (group ST-HI). In all groups, TS were not motile. After in vitro sperm maturation, motility was up to 60% only in group ST-HI. The data suggest that the ability of TS to be matured in vitro was not related to the environmental temperature, while hormonal stimulation of spermiation during the spawning season was an absolute requirement for optimal in vitro maturation.

A wild-caught stock of greater amberjack Seriola dumerili reared in sea cages in two commercial aquaculture facilities in Greece was administered gonadotropin-releasing hormone agonist (GnRHa), loaded in controlled release implants, and was used in spawning induction experiments throughout the reproductive season. Sperm quality was evaluated using computer-assisted sperm analysis (CASA) before and after GnRHa administration and the extent of the spermiation period was determined. Males were in spermiation throughout the monitoring period from May 30 until July 18, at temperatures between 19 and 24 °C. However, lower sperm motility duration, density, and survival under cold storage were observed from early July onwards. Sperm quality did not recover after the end of spawning induction experiments in tanks and the return of the fish for 14-28 days to the sea cage in mid-July, which could be related to the high temperatures of this period. An improvement trend was observed in the quality of the milt collected on day 7 after a single GnRHa administration, but a significant decrease was observed on day 21 in sperm density, survival under cold storage, and straight line velocity (VSL). On the contrary, a double GnRHa administration spaced 14 days apart maintained the same sperm quality for a longer period of 29 days. Further spermiation enhancement experiments should be conducted in the future to describe in more detail the kinetics of sperm production after GnRHa therapy and its effects on sperm quality.

Passive Acoustic Monitoring (PAM) is a non-intrusive and cost-effective method capable of providing high-resolution, long-term information on the status and health of vocal populations and communities. To successfully monitor the same species over wide geographical and temporal scales, it is necessary to characterise the range of sound variability, as well as the consistency of sound features between populations. The meagre (Argyrosomus regius, Asso 1801) is an interesting case study because recent investigations suggest a wider vocal repertoire than previously described. In this study, meagre vocalizations were recorded and analysed from a variety of settings, ranging from rearing facilities to wild populations to provide a comprehensive characterisation of its vocal repertoire, while investigating the consistency of spawning sound features between populations. All sounds presented a similar acoustic structure in their basic unit (i.e. the pulse), while an important variability was found in the number of pulses; the meagre can emit sounds made of one single pulse or many pulses (up to more than 100). High level of overlap in the Principal Component Analysis made difficult to differentiate sound type clusters. Despite this, two sound types were identifiable: knocks (sounds from 1 to 3 pulses) and long grunts (sounds with more than 29 pulses). Discriminant Analysis carried out on PCA residuals showed that knock had the highest proportion of correct placement (92% of the observations correctly placed) followed by long grunts (80%). All other previously described sound types (intermediate grunt, short grunt and disturbance sounds) could not be separated and presented low levels of correct placement, suggesting that care should be taken when defining these as independent sound types. Finally, acoustic features consistency was found in meagre grunts emitted by different populations during spawning nights; statistical differences could be explained by recording settings and fish conditions. The results of this study provide important information for fostering PAM programs of wild meagre populations, while contributing to the discussion around the definition of fish sound types in vocal fish communities. Studies of this kind, which evaluate both variability and consistency of sound features, are of fundamental importance for maximising PAM efforts in the wild, at both the specific and the community level., Competing Interests: The authors have declared that no competing interests exist.

Streptococcus alactolyticus, a member of the Streptococcus bovis/Streptoccus equinus complex, is primarily hosted in the gastrointestinal tract of animals and rarely of humans, with only scarce reports relating to human disease. We herein present a case of subacute infective endocarditis (IE) caused by S. alactolyticus in a 64-year old male with pre-existing mitral prolapse. Despite a 10-month history of low-grade fever and weight loss, the diagnosis of IE was triggered by left quadrant abdominal pain which revealed splenic infarcts on computed tomography. A definitive diagnosis of IE was subsequently established by four consecutive blood cultures positive for S.alactolyticus plus demonstration of a vegetation on the mitral valve by trans-esophageal ultrasound. Further workup revealed multiple embolic phenomena including brain and spine. A dental abscess was identified as the most probable origin of the bacteraemia, while colonoscopy revealed no evidence of cancer. The patient recovered uneventfully with antibiotic treatment and underwent successful cardiac surgery post-discharge. Although rare, IE caused by S. alactolyticus may be severe and of obscure origin; oral cavity should not be overlooked as a possible origin. Attention should be given in patients with preexisting risk factors.

Meagre (Argyrosomus regius) undergo spermatogenesis and spermiation when reared in captivity, but often produce low milt volumes, sometimes with reduced quality and for a limited time period. In the present study we a) compared the efficiency of gonadotropin releasing hormone agonist (GnRHa) implants versus injections on testicular stimulation and spermiation enhancement, b) investigated the effect of GnRHa on the endocrine spermiation regulation (sex steroid hormones), and c) evaluated a commercial induced spawning simulation scenario. Firstly, males (n = 5) were injected with 15.0 ± 0.2 μg GnRHa kg -1 (Injections) or implanted with 51.0 ± 5.1 μg GnRHa kg -1 (Implant) and compared their sperm production response. Secondly, the best hormonal treatment (Implant) was tested treating males (n = 8) with 57.5 ± 7.5 μg GnRHa kg -1 every 3 weeks for a period of 70 days. Milt production was improved by the GnRHa implants with only minor sperm quality alterations (improved sperm motility percentage). Elevated plasma testosterone (T) and 11-ketotestosterone (11-KT) levels were recorded in response to GnRHa implants, while no significant difference for 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was observed. In the commercial induced spawning simulation, it was shown that meagre females are capable of on-demand induction of spawning at random intervals (5-21 days) using GnRHa injections, over a period of at least 2.5 months. During this period, spermiation enhancement was achieved with GnRHa implants every 3 weeks, producing sperm with stable, in general, quality and motility parameters. Percentage of motile cells, motility duration and density fluctuated significantly, but remained within levels that are considered appropriate for high fertilization success in this species., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Non-coding RNAs (ncRNAs) are involved in several different regulatory pathways including reproduction. In teleost fish, efficacious reproduction is heavily dependent on the completion of the reproductive cycle. The presence of ncRNA, however, and their expression dynamics and putative regulatory role in mature and immature gonads have not yet been extensively explored. Therefore, the abundance of ncRNAs in mature and immature female sharpsnout seabream (Diplodus puntazzo) was investigated. The sharpsnout seabream is a rudimentary hermaphrodite which, in captivity, displays dysfunctions in the gonad maturation process. Our analyses revealed a gonad specific read length distribution with two main peaks representing miRNAs (21-26 nt) and PIWI RNA (27-34 nt). Besides, distinct expression patterns for several ncRNA biotypes including microRNAs (miRNAs), PIWI RNAs (piRNAs), and ribosomal RNAs (rRNAs) were detected. Identified miRNA accounted to 938, corresponding to ~ 13% of obtained transcripts. Among the differential expressed ncRNAs, 10 (~ 7%) were annotated as miRNA, out of which 2 were found in higher abundance in immature gonads (miR-125c and miR-24) and 8 (miR-451, miR-7a, miR-122-1, miR190a, miR129, ENSGACT00000029608, ENSGACT00000029489, and ENSGACT00000029667) were found to be higher expressed in mature gonads. Putative miRNA targets, including long non-coding RNAs (lncRNAs) and genes, are proposed. Target genes are involved in several processes of fish oocyte development, such as steroidogenesis, proteolysis, and apoptosis, and may explain hormone regulation. This study demonstrates a gonad maturation biased ncRNA profile which in turn may support the role of ncRNAs in ovarian physiology and reproductive performance of fish, stressing the specific function of each RNA biotype in oocyte development.

The greater amberjack Seriola dumerili is a new aquaculture fish that may display reproductive dysfunctions. During extensive follicular atresia, which is a common reproductive dysfunction in females during vitellogenesis, part of the reabsorbed yolk returns to the liver to be metabolized and recycled. Melanomacrophage centres (MMCs) are aggregates of macrophage-like cells that play a role in the destruction, detoxification and recycling of endogenous and exogenous materials, and have been associated with systemic stress. Wild and captive-reared greater amberjack were sampled in the Mediterranean Sea during two different phases of the reproductive cycle. The liver of reproductively dysfunctional captive-reared females sampled during the spawning season showed a high density of both MMCs and apoptotic cells. A weak liver anti-cytochrome P450 monooxygenase 1A immunoreactivity was observed, suggesting that the examined fish were not exposed to environmental pollutants. We propose that the observed increase in MMCs and apoptosis in captive-reared fish was related to the hepatic overload associated to the metabolism of yolk proteins reabsorbed during extensive follicular atresia. Since follicular atresia is a frequent physiological and pathological event in teleosts, we suggest that the reproductive state should be always assessed when MMCs are used as markers of exposure to stress or pollutants., (© 2020 John Wiley & Sons Ltd.)

The greater amberjack Seriola dumerili (Risso, 1810) is a large migratory pelagic fish occurring in tropical and temperate waters with a great potential for the world aquaculture industry. Previous studies showed that wild-caught female greater amberjack reared in sea cages and handled during the reproductive season, underwent extensive ovarian atresia. This atresia, however, was not related to an insufficient liver transcription or oocyte uptake of vitellogenin (Vtg). In the present study, the structure of two greater amberjack vitellogenin receptors, namely Vtgr (Lr8-) and Lrp13, was characterized. Moreover, vtgr and lrp13 gene expression and the fatty acid profiles of specific phospholipids and neutral lipids were compared in the ovaries of wild and captive-reared greater amberjack during different phases of the reproductive cycle (i.e. early gametogenesis, advanced gametogenesis and spawning). Ovarian vtgr and lrp13 transcription was more active during early gametogenesis, suggesting that vitellogenin receptor transcripts were synthesized by previtellogenic oocytes and remained in the cellular mRNA pool until oocytes resumed meiosis and entered into secondary growth (i.e. vitellogenesis). Rearing of wild-caught greater amberjack in captivity together with handling during the reproductive season was associated with a reduced vtgr and lrp13 transcription and with a diminished capacity of oocytes in the early phase of gametogenesis (primary oocyte growth) to enter into vitellogenesis. During early gametogenesis, remarkable differences in the fatty acid composition were observed between wild and captive-reared individuals: all phospholipids of captive fish displayed dramatic increases of saturates (16:0 and 18:0) and decreases of arachidonic acid (ARA) and docosahexaenoic acid (DHA). The present study confirms the susceptibility of greater amberjack reproductive function to handling stress and suggests that the consequent extensive atresia of vitellogenic follicles originated during the primary oocytes growth when the capacity of oocytes to synthesize vitellogenin receptors was reduced. The study also suggests that this reduced capacity was associated with an altered oocyte phospholipid fatty acid composition during early gametogenesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nocardia spp. is a genus of Gram-positive bacteria which can cause cutaneous, pleuropulmonary, or disseminated disease. The latter two forms are encountered in immunocompromised patients, with prolonged usage of corticosteroids being a well-recognized risk factor. However, endogenous Cushing's syndrome is less frequently associated with nocardiosis. We report on a 40-year-old woman who presented for further workup of abnormal findings in the chest computed tomography (three lung nodules, one of which being cavitary). She underwent trans-thoracic fine-needle lung aspiration of the cavitary nodule, which led to the diagnosis of lung nocardiosis. Moreover, the identification of cushingoid features from the history and clinical examination initiated further investigation with hormonal laboratory assessment and bilateral inferior petrosal sinus sampling which established the diagnosis of pituitary adrenocorticotropic hormone (ACTH) hypersecretion (Cushing's disease).  We conclude that pulmonary nocardiosis can be an opportunistic infection as well as a presenting manifestation of Cushing's disease.

Bisphenol A (BPA), a known endocrine disrupting chemical (EDC), was administered by diet to gilthead sea bream ( Sparus aurata ) in order to study its effects on the endocannabinoid system (ECS) and gonadal steroidogenesis. 2-year-old male gilthead sea bream were fed with two different concentrations of BPA (LOW at 4 and HIGH at 4000 µg/kg body weight for 21 days during the reproductive season. Exposure to 4000 µg BPA/kg bw/day (BPA HIGH) reduced sperm motility and altered the straight-line velocity (VSL) and linearity (LIN). Effects on steroidogenesis were evident, with testosterone (T) being up-regulated by both treatments and 11-ketotestosterone (11-KT) down-regulated by BPA HIGH. Plasma levels of 17β-estradiol (E 2 ) were not affected. The Gonadosomatic Index (GSI) increased in the BPA HIGH group. Interestingly, the levels of endocannabinoids and endocannabinoid-like compounds were significantly reduced after both treatments. Unpredictably, a few changes were noticed in the expression of genes coding for ECS enzymes, while the receptors were up-regulated depending on the BPA dose. Reproductive markers in testis (leptin receptor ( lepr ), estrogen receptors ( era , erb ), progesterone receptors ( pr ) and the gonadotropin releasing hormone receptor ( gnrhr )) were up-regulated. BPA induced the up-regulation of the hepatic genes involved in oogenesis (vitellogenin ( vtg ) and zona pellucida 1 ( zp1 )).

In this study, adult gilthead seabream (Sparus aurata) were exposed for 21 days to Di-iso-nonylphthalte (DiNP at 15 and 1500 μg kg -1  bw day -1 ) via the diet. This plastic additive has been recently introduced to replace the di-(2-ethylhexyl)phthalate, the toxicity of which has been demonstrated conclusively both in vivo and in vitro trials. An analysis of a set of biomarkers involved in stress and immune response provides evidence of hepatic toxicity by DiNP in the present study. Both hsp70 and gr mRNA levels were upregulated significantly by DiNP, while plasma cortisol increased only in fish fed with the lowest DiNP dose. The oxidative stress markers g6pdh, glut red, gpx1 and CAT were upregulated by DiNP; gst mRNA was induced by the high dose and gck mRNA was downregulated significantly by the low dose. The mRNA levels of genes involved in the immune response, such as pla2, 5-lox, tnfa and cox2, were upregulated significantly only by the high dose of DiNP, while il1 mRNA increases in both doses. These molecular evidences were complemented with features obtained by Fourier Transform Infrared Imaging (FTIRI) analysis regarding the hepatic distribution of the main biological macromolecules. The FTIRI analysis showed an alteration of biochemical composition in DiNP samples. In particular, the low dose of DiNP induced an increase of saturated and unsaturated lipids and phosphorylated proteins, and a decrease of glycogen levels. The levels of caspase did not change significantly in the study, suggesting that DiNP does not activate apoptosis. Finally, the results also suggested the onset of hepatic oxidative stress and the activation of immune response, adding new knowledge to the already described hepatic DiNP toxicity.

Lambari-do-rabo-amarelo Astyanax altiparanae in the wild reproduce during spring and summer, but females undergo vitellogenesis throughout the year, including the non-spawning winter period when water temperatures are low. The present study investigated the physiological role of temperature modulation on the hypothalamus-pituitary-gonads axis of lambari during winter, as well as the effects of gonadotropin releasing hormone agonist (GnRHa) therapy. Captive females were exposed to two different temperatures (20 °C and 27 °C) and were injected weekly with GnRHa for 21 days during winter (Control, CTR; Low dose; LD and high dose of GnRHa, HD). At the end of the 21-days period gonadosomatic index (GSI), oocyte stage of development and theoretical fecundity were evaluated, together with plasma levels of 17β-estradiol (E 2 ). Gene expression of the two pituitary gonadotropins follicle-stimulating hormone (fshβ) and luteinizing hormone (lhβ), as well as hepatic vitellogenin-A (vtgA) expression were also analyzed. At the end of the experimental period, females from the six different experimental conditions were induced to spawn using human chorionic gonadotropin (hCG). Spawning performance parameters and plasma levels of the maturation inducing steroid (MIS) were analyzed. Gene expression of fshβ did not change with temperature manipulation, but females exposed to 27 °C and supplemented with a HD of GnRHa exhibited an increased fshβ gene expression, associated with higher E 2 levels. The higher water temperature alone was able to increase E 2 levels. At both water temperatures GnRHa injections induced a decrease in E 2 levels. GnRHa injected females had a lower vtgA gene expression levels at 20 °C. Even with differences in the gene expression of gonadotropins among the various temperature/GnRHa treatments, GSI and oocyte diameter did not change, but GnRHa enhanced the number of vitellogenic oocytes at 20 °C. The reproductive performance of lambari induced to spawn with hCG was better after the combined treatment with GnRHa and summer temperature., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The greater amberjack (Seriola dumerili) is one of the most promising finfish species for aquaculture, due to its cosmopolitan distribution and acceptability, high growth rates and large size. However, lack of reproduction control has been one of the main bottlenecks for its commercialization. The study examined two endocrine methods for the induction of oocyte maturation/ovulation and spawning, which are based on the induction of endogenous Luteinizing Hormone (LH) release from the pituitary, through the use of Gonadotropin Releasing Hormone agonist (GnRHa) either in the form of sustained-release delivery systems (implants) or injections (acute release). The stock (n = 28) consisted of wild fish captured in 2011 and each GnRHa administration method was conducted in two replicates, with 6 and 8 fish per tank, respectively, at a 1:1 sex ratio. Fish were given a GnRHa injection once a week (three administrations), or a GnRHa implant every 2 weeks (two administrations). Mean daily relative fecundity was significantly higher (t 1.3  = -5.24, P = 0.012) in the implanted fish (15,170 ± 2,738 eggs kg -1 female day -1 ) compared to the injected fish (6,119 ± 2,790 eggs kg -1 female day -1 ). Total relative fecundity was also significantly higher (t 1.3  = -9.93, P = 0.003) in the implanted fish (102,402 ± 20,337 eggs kg -1 female) compared to the injected ones (26,517 ± 9,938 eggs kg -1 female), but there were no differences in the quality of eggs in terms of fertilization, 24-h embryo survival, hatching and 5-d larval survival. The number of females with fully vitellogenic oocytes eligible for induction of spawning decreased from the initial to the final sampling, from 7 to 6 females for the GnRHa implant treatment and from 7 to 3 females for the GnRHa injection treatment. In addition to apparently promoting the proper endocrine changes leading to multiple cycles of oocyte maturation, ovulation and spawning, and thus producing larger numbers of eggs; the use of GnRHa implants may be more appropriate in greater amberjack than multiple injections because (a) it also stimulates vitellogenesis and (b) involves less handling of the fish., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In the last years, an increasing number of studies reported that food pollution represents a significant route of exposure to environmental toxicants, able to cause mild to severe food illnesses and health problems, including hormonal and metabolic diseases. Pollutants can accumulate in organisms and biomagnify along the food web, finally targeting top consumers causing health and economic problems. In this study, adults of gilthead sea bream, Sparus aurata, were fed with diets contaminated with Bisphenol A (BPA) (4 and 4000 μg BPA kg -1  bw day -1 ) and Di-isononyl phthalate (DiNP) (15 and 1500 μg DiNP kg -1  bw day -1 ), to evaluate the effects of the contamination on the muscle macromolecular composition and alterations of its texture. The analysis conducted in the muscle using infrared microspectroscopy, molecular biology and biochemical assays, showed, in fish fed BPA contaminated diets, a decrease of unsaturated lipids and an increase of triglycerides and saturated alkyl chains. Conversely, in fish fed DiNP, a decrease of lipid content, caused by a reduction of both saturated and unsaturated chains and triglycerides was measured. Protein content was decreased by both xenobiotics evidencing a novel macromolecular target affected by these environmental contaminants. In addition, in all treated groups, proteins resulted more phosphorylated than in controls. Calpain and cathepsin levels, orchestrating protein turnover, were deregulated by both xenobiotics, evidencing alterations of muscle composition and texture. In conclusion, the results obtained suggest the ability of BPA and DiNP to modify the muscle macromolecular building, advising this tissue as a target of Endocrine-Disrupting Chemicals (EDCs) and providing a set of biomarkers as possible monitoring endpoints to develop novel OEDC test guidelines., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Diisononyl phthalate (DiNP) is a plasticizer used to improve plastic performance in a large variety of items which has been reported as an endocrine-disrupting chemical (EDC) in several organisms. The endocannabinoid system (ECS) is a cellular signaling system, whose functionality is tightly involved with reproductive function. The aim of the present study was the assessment of the effects of DiNP on the gonadal ECS and on the reproductive function of male gilthead sea bream Sparus aurata, an important marine aquacultured species in Europe, during the reproductive season. Fish were fed for 21 days with two diets contaminated with different nominal concentrations of DiNP (DiNP LOW at 15 µg DiNP kg -1 bw day -1 and DiNP HIGH at 1500 µg DiNP kg -1 bw day -1 ), based on the tolerable daily intake (TDI) ruled by the European Food Safety Authority for humans. The transcription of several genes related to the ECS was affected by the DiNP. Specifically, DiNP reduced the levels of endocannabinoids and endocannabinoid-like mediators, concomitant with the increase of fatty acid amide hydrolase (FAAH) activity. At the histological level, DiNP LOW induced the highest occurrence of individuals with regressed testes. Steroidogenesis was affected significantly, since plasma 11-ketotestosterone (11-KT), the main active androgen in fish, was significantly decreased by the DiNP HIGH treatment, while plasma 17β-estradiol (E 2 ) levels were raised, associated with an increase of the gonadosomatic index (GSI). Additionally, the level of testosterone (T) was significantly increased in the DiNP LOW group, however, the same DiNP concentration reduced the levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P). The production of sperm was in general not affected, since spermiation index, sperm density, survival and the duration of forward motility did not exhibit any changes compared to controls. However, computer-assisted sperm analysis (CASA) showed that DiNP reduced the percentage of motile cells. The results clearly suggest a negative effect of DiNP via the diet on the male endocrine system of gilthead sea bream during the reproductive season.

Sex-biased gene expression is the mode through which sex dimorphism arises from a nearly identical genome, especially in organisms without genetic sex determination. Teleost fishes show great variations in the way the sex phenotype forms. Among them, Sparidae, that might be considered as a model family displays a remarkable diversity of reproductive modes. In this study, we sequenced and analyzed the sex-biased transcriptome in gonads and brain (the tissues with the most profound role in sexual development and reproduction) of two sparids with different reproductive modes: the gonochoristic common dentex, Dentex dentex , and the protandrous hermaphrodite gilthead seabream, Sparus aurata . Through comparative analysis with other protogynous and rudimentary protandrous sparid transcriptomes already available, we put forward common male and female-specific genes and pathways that are probably implicated in sex-maintenance in this fish family. Our results contribute to the understanding of the complex processes behind the establishment of the functional sex, especially in hermaphrodite species and set the groundwork for future experiments by providing a gene toolkit that can improve efforts to control phenotypic sex in finfish in the ever-increasingly important field of aquaculture.

The increasing manufacture of plastics and their mismanagement has turned plastic into a ubiquitous waste in the marine environment. Among all the substances conforming the plastic items, the effects of a dietary Bisphenol A (BPA) and Di-isononyl phthalate (DiNP) have been evaluated in adult male gilthead sea bream, focusing on their effects in the modulation of the Endocannabinoid System (ECS). In zebrafish, the ECS has been recently chosen as a new target for the activity of some Endocrine Disrupting Chemicals (EDC), since it represents a complex lipid signaling network essential for the well-being of the organisms. The results obtained in gilthead seabream showed that BPA and DiNP altered the structure and the biochemical composition of liver, increasing the presence of lipids and triglycerides and decreasing the glycogen and phospholipids. Moreover, the addition of BPA or DiNP in the gilthead sea bream diet altered the levels of endocannabinoids (EC) and EC-like mediators in the liver. These alterations were also associated to changes at the transcriptomic level of genes involved in lipid biosynthesis and ECS metabolism. At the central level, both BPA and DiNP reduced the expression of the endocannabinoid receptor type I (cnr1) and the neuropeptide Y (npy) as well as the levels of the endocannabinoid Anandamide (AEA), suggesting a downregulation of appetite. The results herein reported highlighted the negative effects of chronic dietary exposure to DiNP or BPA on ECS functions and lipid metabolism of male gilthead sea bream liver, showing a similar disruptive activity of these contaminants at metabolic level. Moreover, the novelty of the biomarkers used evidenced possible innovative endpoints for the development of novel OEDCS test guidelines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata , a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata , first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes., Competing Interests: The authors declare no competing interests.

Sex differentiation is a puzzling problem in fish due to the variety of reproductive systems and the flexibility of their sex determination mechanisms. The Sparidae, a teleost family, reflects this remarkable diversity of sexual mechanisms found in fish. Our aim was to capture the transcriptomic signature of different sexes in two protogynous hermaphrodite sparids, the common pandora Pagellus erythrinus and the red porgy Pagrus pagrus in order to shed light on the molecular network contributing to either the female or the male phenotype in these organisms. Through RNA sequencing, we investigated sex-specific differences in gene expression in both species' brains and gonads. The analysis revealed common male and female specific genes/pathways between these protogynous fish. Whereas limited sex differences found in the brain indicate a sexually plastic tissue, in contrast, the great amount of sex-biased genes observed in gonads reflects the functional divergence of the transformed tissue to either its male or female character. Α common "crew" of well-known molecular players is acting to preserve either sex identity of the gonad in these fish. Lastly, this study lays the ground for a deeper understanding of the complex process of sex differentiation in two species with an evolutionary significant reproductive system.

Background: Teleosts of the genus Seriola, commonly known as amberjacks, are of high commercial value in international markets due to their flesh quality and worldwide distribution. The Seriola species of interest to Mediterranean aquaculture is the greater amberjack (Seriola dumerili). This species holds great potential for the aquaculture industry, but in captivity, reproduction has proved to be challenging, and observed growth dysfunction hinders their domestication. Insights into molecular mechanisms may contribute to a better understanding of traits like growth and sex, but investigations to unravel the molecular background of amberjacks have begun only recently., Findings: Illumina HiSeq sequencing generated a high-coverage greater amberjack genome sequence comprising 45 909 scaffolds. Comparative mapping to the Japanese yellowtail (Seriola quinqueriadiata) and to the model species medaka (Oryzias latipes) allowed the generation of in silico groups. Additional gonad transcriptome sequencing identified sex-biased transcripts, including known sex-determining and differentiation genes. Investigation of the muscle transcriptome of slow-growing individuals showed that transcripts involved in oxygen and gas transport were differentially expressed compared with fast/normal-growing individuals. On the other hand, transcripts involved in muscle functions were found to be enriched in fast/normal-growing individuals., Conclusion: The present study provides the first insights into the molecular background of male and female amberjacks and of fast- and slow-growing fish. Therefore, valuable molecular resources have been generated in the form of a first draft genome and a reference transcriptome. Sex-biased genes, which may also have roles in sex determination or differentiation, and genes that may be responsible for slow growth are suggested., (© The Authors 2017. Published by Oxford University Press.)

The greater amberjack, (Risso, 1810), is a promising candidate for the diversification of European aquaculture production, but inconsistent reproduction in captivity prevents commercial production. Recent studies showed that greater amberjack confined in sea cages exhibited scarce gonad development and early interruption of gametogenic activity during the reproductive season. The aim of the present study was to improve our understanding of the observed impairment of spermatogenesis. Adult wild and captive-reared males were sampled during 3 different phases of the reproductive cycle: early gametogenesis (EARLY; late April to early May), advanced gametogenesis (ADVANCED; late May to early June), and spawning (SPAWNING; late June to July). Spermatogonial stem cells and proliferating germ cells were identified through the immunohistochemical localization of and proliferating cell nuclear antigen, respectively. Apoptotic germ cells were identified throughout the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling method. Sperm quality of captive-reared fish was evaluated using computer-assisted sperm analysis. Captive-reared males exhibited seminiferous lobules of a smaller diameter, a precocious and progressive decrease of spermatogonial mitosis, and a high level of apoptosis at the beginning of the reproductive season, concomitant with a many-fold higher 17β-estradiol plasma concentration. The motile spermatozoa percentage of captive greater amberjack was lower than in other teleosts, and a drastic decrease of spermatozoa motility duration, velocity, and ATP content occurred along the reproductive season. An abnormal increase of sperm concentration as well as an increase of dead spermatozoa occurred during the SPAWNING phase, probably because of lack of sperm hydration and ejaculation and consequent sperm ageing. The present study demonstrates the extreme susceptibility of greater amberjack to rearing stress and underscores the need for improvement of the rearing and handling procedures to ameliorate gametogenesis dysfunctions in commercial aquaculture production.

The osteological development of the vertebral column and fins in shi drum Umbrina cirrosa was studied in order to improve knowledge for its introduction in Mediterranean aquaculture. The osteological development was studied in 171 individuals, of total length (L T ) from 2·7 to 30·2 mm that were reared under the mesocosm technique. Vertebral ontogeny starts at 3·4 and 4·0 mm L T , with the formation of the first cartilaginous neural and haemal arches, and spines, respectively, and is completed with the full attainment of epicentrals (12·5 mm L T ). The formation of vertebral centra occurs between 4·1 and 7·4 mm L T . Pectoral supports are the first fin elements to develop (3·0 mm L T ), followed by those of the caudal fin (3·8 mm L T ), pelvic fin (3·9 mm L T ) and finally by those of the dorsal and anal fins (4·5 mm L T ). The caudal fin is the first to develop fin rays and attain the full count of principal fin rays (4·5-6·8 mm L T ), but the last to be fully completed with the formation of procurrent fin rays (6·9-17·5 mm L T ). The next fins starting to present rays are the dorsal (5·3 mm L T ) and the pectoral fins (5·6 mm L T ), while the anal and pelvic fins are the last (5·7 mm L T ). Following the caudal principal fin rays (6·8 mm L T ), the dorsal, anal (6·9 mm L T ), pelvic (7·4 mm L T ) and pectoral fins (9·8 mm L T ) are the next with fully completed ray counts. Aggregation of qualitative changes, such as the appearance of cartilages, the beginning and the complement of the ossification process and the full complement of elements in U. cirrosa were measured as cumulative frequency counts. These measurements reveal three ontogenetic intervals: one very developmentally active period during early life stages (from 3 to 5·9 mm L T ), a second slower developmental period (from 6·0 to 8·9 mm L T ) and finally a period of ontogeny more focused on structure refinement up to metamorphosis and settlement (>9·0 mm L T )., (© 2017 The Fisheries Society of the British Isles.)

Background: Elucidating the patterns of the Atlantic Bluefin tuna [ABFT, Thunnus thynnus (Linnaeus, 1758)] population structure constitutes a challenging task of great importance. Most of the unique challenges stem from its biology, as well as the attributes of the marine realm in which it disperses. Accurate information is urgently needed for stock assessment, and the identification of critical features to the persistence and adaptation of populations in order to formulate and adopt effective strategies for ABFT conservation and management. Conclusions of a great number of ABFT genetic studies on the Mediterranean Sea stock structure are rather controversial and not yet conclusive. In this study, ABFT genomic diversity was investigated in the Mediterranean Sea, which is the most important area for the species' reproduction., Results: Analyzing genome-wide SNPs and microsatellites from ABFT samples collected throughout the Mediterranean Sea did not provide strong evidence of genetic structure, pointing towards the existence of a single panmictic unit. An alternative view would recognize a failure to reject the null hypothesis of a panmictic unit as an effect of the study's sampling design, the type of markers used, and the effectiveness/suitability of analysis methods in respect to the species biological characteristics or any combination of the above., Conclusions: Unravelling the drivers of ABFT population diversity would require the consideration of important aspects of the species spawning behavior for the determination of the appropriate sampling design. Novel approaches and methods of analysis that will bring together experts in genetics/-omics, ecology and oceanography are deemed necessary. Analyzing ABFT genetic data under the discipline of seascape genetics could provide the analysis framework under which major abiotic and biotic forces controlling ABFT recruitment could be identified, elucidating the complicated population dynamics of the species, while multiple and continuous fisheries monitoring should in all cases be considered as a prerequisite in order to achieve efficient and long-term ABFT conservation.

The greater amberjack Seriola dumerili is a large teleost fish with rapid growth and excellent flesh quality, whose domestication represents an ambitious challenge for aquaculture. The occurrence of reproductive dysfunctions in greater amberjack reared in captivity was investigated by comparing reproductive development of wild and captive-reared individuals. Wild and captive-reared breeders were sampled in the Mediterranean Sea during three different phases of the reproductive cycle: early gametogenesis (EARLY, late April-early May), advanced gametogenesis (ADVANCED, late May-early June) and spawning (SPAWNING, late June-July). Fish reproductive state was evaluated using the gonado-somatic index (GSI), histological analysis of the gonads and determination of sex steroid levels in the plasma, and correlated with leptin expression in the liver and gonad biochemical composition. The GSI and sex steroid levels were lower in captive-reared than in wild fish. During the ADVANCED period, when the wild greater amberjack breeders were already in spawning condition, ovaries of captive-reared breeders showed extensive atresia of late vitellogenic oocytes and spermatogenic activity ceased in the testes of half of the examined males. During the SPAWNING period, all captive-reared fish had regressed gonads, while wild breeders still displayed reproductive activity. Liver leptin expression and gonad proximate composition of wild and captive greater amberjack were similar. However, the gonads of captive-reared fish showed different total polar lipid contents, as well as specific lipid classes and fatty acid profiles with respect to wild individuals. This study underlines the need for an improvement in rearing technology for this species, which should include minimum handling during the reproductive season and the formulation of a specific diet to overcome the observed gonadal decrements of phospholipids, DHA (22:6n-3) and ARA (20:4n-6), compared to wild breeders., Competing Interests: The authors have declared that no competing interests exist.

Common pandora (Pagellus erythrinus) is a benthopelagic marine fish belonging to the teleost family Sparidae, and a newly recruited species in Mediterranean aquaculture. The paucity of genetic information relating to sparids, despite their growing economic value for aquaculture, provides the impetus for exploring the genomics of this fish group. Genomic tool development, such as genetic linkage maps provision, lays the groundwork for linking genotype to phenotype, allowing fine-mapping of loci responsible for beneficial traits. In this study, we applied ddRAD methodology to identify polymorphic markers in a full-sib family of common pandora. Employing the Illumina MiSeq platform, we sampled and sequenced a size-selected genomic fraction of 99 individuals, which led to the identification of 920 polymorphic loci. Downstream mapping analysis resulted in the construction of 24 robust linkage groups, corresponding to the karyotype of the species. The common pandora linkage map showed varying degrees of conserved synteny with four other teleost genomes, namely the European seabass (Dicentrarchus labrax), Nile tilapia (Oreochromis niloticus), stickleback (Gasterosteus aculeatus), and medaka (Oryzias latipes), suggesting a conserved genomic evolution in Sparidae. Our work exploits the possibilities of genotyping by sequencing to gain novel insights into genome structure and evolution. Such information will boost the study of cultured species and will set the foundation for a deeper understanding of the complex evolutionary history of teleosts., (Copyright © 2016 Manousaki et al.)

An updated second linkage map was constructed for the gilthead sea bream, Sparus aurata L., a fish species of great economic importance for the Mediterranean aquaculture industry. In contrast to the first linkage map which mainly consisted of genomic microsatellites (SSRs), the new linkage map is highly enriched with SSRs found in Expressed Sequence Tags (EST-SSRs), which greatly facilitates comparative mapping with other teleosts. The new map consists of 321 genetic markers in 27 linkage groups (LGs): 232 genomic microsatellites, 85 EST-SSRs and 4 SNPs; of those, 13 markers were linked to LGs but were not ordered. Eleven markers (5 SSRs, 5 EST-SSRs and 1 SNP) are not assigned to any LG. The total length of the sex-averaged map is 1769.7cM, 42% longer than the previously published one, and the number of markers in each LG ranges from 2 to 30. The inter-marker distance varies from 0 to 75.6cM, with an average of 5.75cM. The male and female maps have a length of 1349.2 and 2172.1cM, respectively, and the average distance between markers is 4.38 and 7.05cM, respectively. Comparative mapping with the three-spined stickleback (Gasterosteus acuulatus) chromosomes and scaffolds showed conserved synteny with 132 S. aurata markers (42.9% of those mapped) having a hit on the stickleback genome., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Teleosts are characterized by a remarkable breadth of sexual mechanisms including various forms of hermaphroditism. Sparidae is a fish family exhibiting gonochorism or hermaphroditism even in closely related species. The sparid Diplodus puntazzo (sharpsnout seabream), exhibits rudimentary hermaphroditism characterized by intersexual immature gonads but single-sex mature ones. Apart from the intriguing reproductive biology, it is economically important with a continuously growing aquaculture in the Mediterranean Sea, but limited available genetic resources. Our aim was to characterize the expressed transcriptome of gonads and brains through RNA-Sequencing and explore the properties of genes that exhibit sex-biased expression profiles., Results: Through RNA-Sequencing we obtained an assembled transcriptome of 82,331 loci. The expression analysis uncovered remarkable differences between male and female gonads, while male and female brains were almost identical. Focused search for known targets of sex determination and differentiation in vertebrates built the sex-specific expression profile of sharpsnout seabream. Finally, a thorough genetic marker discovery pipeline led to the retrieval of 85,189 SNPs and 29,076 microsatellites enriching the available genetic markers for this species., Conclusions: We obtained a nearly complete source of transcriptomic sequence as well as marker information for sharpsnout seabream, laying the ground for understanding the complex process of sex differentiation of this economically valuable species. The genes involved include known candidates from other vertebrate species, suggesting a conservation of the toolkit between gonochorists and hermaphrodites.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females. In 2008, an estimated 1.2 million people were diagnosed with and 608,700 people died of CRC. Besides diagnosis and treatment, prognosis is an important matter for cancer patients. Today, clinicopathological correlations have many applications in cancer prognostication. Examples include the prediction of the medium patient survival and the screening for patients suitable for specific therapeutic approaches. Apart from traditional prognostic factors, such as tumor stage and grade, new markers may be useful in clinical practice. Possible markers may result from the study of basement membranes (BMs). BM seems to play a role in the pathogenesis of colorectal cancer, so BM alterations may have prognostic significance as well. The purpose of this review is to briefly describe BMs and their relationship with CRC, in the aspect of clinicopathological correlations.

As part of the endeavor aiming at the domestication of Atlantic bluefin tuna (BFT; Thunnus thynnus), first sexual maturity in captivity was studied by documenting its occurrence and by characterizing the key hormones of the reproductive axis: follicle stimulating hormone (FSH) and luteinizing hormone (LH). The full length sequence encoding for the related hormone β-subunits, bftFSHβ and bftLHβ, were determined, revealing two bftFSHβ mRNA variants, differing in their 5' untranslated region. A quantitative immuno-dot-blot assay to measure pituitary FSH content in BFT was developed and validated enabling, for the first time in this species, data sets for both LH and FSH to be compared. The expression and accumulation patterns of LH in the pituitary showed a steady increase of this hormone, concomitant with fish age, reaching higher levels in adult females compared to males of the same age class. Conversely, the pituitary FSH levels were elevated only in 2Y and adult fish. The pituitary FSH to LH ratio was consistently higher (>1) in immature than in maturing or pubertal fish, resembling the situation in mammals. Nevertheless, the results suggest that a rise in the LH storage level above a minimum threshold may be an indicator of the onset of puberty in BFT females. The higher pituitary LH levels in adult females over males may further support this notion. In contrast three year-old (3Y) males were pubertal while cognate females were still immature. However, it is not yet clear whether the advanced puberty in the 3Y males was a general feature typifying wild BFT populations or was induced by the culture conditions. Future studies testing the effects of captivity and hormonal treatments on precocious maturity may allow for improved handling of this species in a controlled environment which would lead to more cost-efficient farming., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Spermiation and changes in androgen (testosterone, T and 11-ketotestosterone, 11-KT) levels were studied in sterlet (Acipenser ruthenus) treated with GnRH agonist implants (DAla(6)-Pro(9)-LHRHa) at 25 and 75 μg kg(-1) b.w. and compared with those males treated with 4 mg kg(-1) b.w. of carp pituitary extract (CPE) and 3 pellets of Ovopel kg(-1) b.w., which contains DAla(6)-Pro(9)NEt-mGnRH and metoclopramide. Sperm quality (sperm mass, spermatozoa concentration and sperm motility and velocity) was evaluated 24, 48 and 72 h after hormonal treatments. Males did not release sperm in the control group injected with physiological solution, while sperm could not be collected 7 days after treatments in all hormonally treated groups. Spermiation rates were 100 % in the CPE and Ovopel groups and 25-50 % in the GnRHa-treated groups. Sperm production was significantly lower in the GnRHa-treated groups than in the CPE and Ovopel groups and decreased 72 h after hormonal treatment. Sperm motility and velocity were higher in the Ovopel and GnRHa (75 μg) groups compared to the CPE and GnRHa (25 μg) groups and decreased 72 h after hormonal treatment. Androgens were only affected in spermiating males and changed in the Ovopel and GnRHa (75 μg) after hormonal treatment. Significant correlations were observed between sperm production, sperm motility and sperm velocity, but not androgens. The present study suggests involvement of dopamine in sturgeon spawning. Additionally, better sperm quality observed in the Ovopel group and particularly sperm motility in the GnRHa (75 μg) suggests enhancement of sperm quality in sturgeon treated with GnRHa. Therefore, further study is needed to induce fully spermiation using GnRHa implants in combination with a dopamine inhibitor.

The objective of the study was to acclimatise wild-caught meagre (Argyrosomus regius) to captivity to produce viable eggs for aquaculture production. Twelve meagre (3 males and 9 females, mean weight = 20 ± 7 kg) were caught and transported to a land-based facility on 26 October 2006. During, March to June 2007, all three males were spermiating and five of the nine females were in vitellogenesis with mean maximum oocyte diameter ≥550 μm. No spontaneous spawning was observed. Two hormone treatments, either a single injection of gonadotropin-releasing hormone agonist (GnRHa, 20 μg kg(-1) for females and 10 μg kg(-1) for males) or a slow-release implant loaded with the same GnRHa (50 μg kg(-1) for females and 25 μg kg(-1) for males), were used to induce spawning on three different dates on 26 March 2007, 4 May 2007 and 18 April 2008. From each spawning event, the following parameters were determined: fecundity, number of floating eggs, egg size, fertilisation and hatching success, unfed larval survival, and proximal composition and fatty acid profile of the eggs. In 2007, two females that were injected on 26 March and 4 May spawned a total of 5 times producing 9,019,300 floating eggs and a relative fecundity of 198,200 eggs kg(-1) and two different females that were implanted on the same dates spawned 14 times producing 12,430,000 floating eggs and a relative fecundity of 276,200 eggs kg(-1). In 2008, a pair that was implanted spawned five times producing a total of 10,211,900 floating eggs and a relative fecundity of 527,380 eggs kg(-1). The latency period was 48-72 h. Parameters were compared between hormone treatments, date of hormone induction and parents determined by microsatellites. Percentage hatch and egg size were 70 ± 0.3% and 0.99 ± 0.02 mm, respectively, for GnRHa-implanted fish and were significantly higher (P < 0.05) compared to 30 ± 0.3% and 0.95 ± 0.03 mm, respectively, for injected fish. Few differences were observed in proximal composition and fatty acid profile and for all spawns mean (% dry weight) lipid content was 17.3 ± 3.0%, carbohydrate was 4.4 ± 1.9% and protein was 31.5 ± 6.4% and the essential fatty acids: Arachidonic acid (ARA, 20:4n-6) ranged between 0.9 and 1% (of total fatty acids), eicosapentaenoic acid (EPA 20:5n-3) 7.7-10.4% and docosahexaenoic acid (DHA 22:6n-3), 28.6-35.4%. All good quality spawns were obtained in the second and/or third spawn after GnRHa treatment, whereas all bad quality spawns were obtained either on the first spawn or after the fifth spawn. Both spawning protocols gave commercially viable (1,000,000+) numbers of good quality eggs that could form the basis of a hatchery production.

The cDNA sequences of vitellogenin receptor proteins (VgR(+) and VgR(-)), containing or lacking the O-linked sugar domain, were determined in Atlantic bluefin tuna (Thunnus thynnus L.). VgR(-) gene expression in the ovary was compared in captive-reared and wild Atlantic bluefin tuna during the reproductive cycle. Gonad samples from adult fish were sampled from 2008 to 2010 from stocks reared in captivity at different commercial fattening operations in the Mediterranean Sea and from wild individuals caught either by traditional tuna traps during their migration towards the spawning grounds in the Mediterranean Sea or by the long-line artisanal fishery. In addition, juvenile male and female Atlantic bluefin tuna were sampled from a farming facility, to obtain baseline information and pre-adulthood amounts of VgR(-). The total length of VgR(+) cDNA was 4006 nucleotides (nt) and that of VgR(-) was 3946 nt. Relative amounts of VgR(-) were greater in juvenile females and in those adults having only previtellogenic oocytes (119 ± 55 and 146 ± 26 folds more than juvenile males, respectively). Amounts of VgR(-) were less in individuals with yolked oocytes (ripening stage, May-June) and increased after spawning in July (92 ± 20 and 113 ± 13 folds more than juvenile males in ripening and post-spawning fish, respectively). These data suggest that regulation of VgR(-) is not under oestrogen control. During the ripening period, greater VgR(-) gene expression was observed in wild fish than in fish reared in captivity, possibly because of (a) differences in water temperature exposure and/or energy storage, and/or (b) an inadequate diet in reared Atlantic bluefin tuna., (Copyright © 2012 Elsevier B.V. All rights reserved.)