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Fish oogenesis is characterised by a massive growth of oocytes each reproductive season. This growth requires the stockpiling of certain molecules, such as ribosomal RNAs to assist the rapid ribosomal assembly and protein synthesis required to allow developmental processes in the newly formed embryo. Massive 5S rRNA expression in oocytes, facilitated by transcription factor 3A (Gtf3a), serves as marker of intersex condition in fish exposed to xenoestrogens. Our present work on Gtf3a gene evolution has been analysed in silico in teleost genomes and functionally in the case of the zebrafish Danio rerio. Synteny-analysis of fish genomes has allowed the identification of two gtf3a paralog genes, probably emerged from the teleost specific genome duplication event. Functional analyses demonstrated that gtf3ab has evolved as a gene specially transcribed in oocytes as observed in Danio rerio, and also in Oreochromis niloticus. Instead, gtf3aa was observed to be ubiquitously expressed. In addition, in zebrafish embryos gtf3aa transcription began with the activation of the zygotic genome (similar to 8 hpf), while gtf3ab transcription began only at the onset of oogenesis. Under exposure to 100 ng/L 17 beta-estradiol, fully feminised 61 dpf zebrafish showed transcription of ovarian gtf3ab, while masculinised (100 ng/L 17 alpha-methyltestosterone treated) zebrafish only transcribed gtf3aa. Sex related transcription of gtf3ab coincided with that of cyp19a1a being opposite to that of amh and dmrt1. Such sex dimorphic pattern of gtf3ab transcription was not observed earlier in larvae that had not yet shown any signs of gonad formation after 26 days of oestradiol exposure. Thus, gtf3ab transcription is a consequence of oocyte differentiation and not a direct result of estrogen exposure, and could constitute a useful marker of gonad feminisation and intersex condition.

Fish oogenesis is characterised by a massive growth of oocytes each reproductive season. This growth requires the stockpiling of certain molecules, such as ribosomal RNAs to assist the rapid ribosomal assembly and protein synthesis required to allow developmental processes in the newly formed embryo. Massive 5S rRNA expression in oocytes, facilitated by transcription factor 3A (Gtf3a), serves as marker of intersex condition in fish exposed to xenoestrogens. Our present work on Gtf3a gene evolution has been analysed in silico in teleost genomes and functionally in the case of the zebrafish Danio rerio. Synteny-analysis of fish genomes has allowed the identification of two gtf3a paralog genes, probably emerged from the teleost specific genome duplication event. Functional analyses demonstrated that gtf3ab has evolved as a gene specially transcribed in oocytes as observed in Danio rerio, and also in Oreochromis niloticus. Instead, gtf3aa was observed to be ubiquitously expressed. In addition, in zebrafish embryos gtf3aa transcription began with the activation of the zygotic genome (similar to 8 hpf), while gtf3ab transcription began only at the onset of oogenesis. Under exposure to 100 ng/L 17 beta-estradiol, fully feminised 61 dpf zebrafish showed transcription of ovarian gtf3ab, while masculinised (100 ng/L 17 alpha-methyltestosterone treated) zebrafish only transcribed gtf3aa. Sex related transcription of gtf3ab coincided with that of cyp19a1a being opposite to that of amh and dmrt1. Such sex dimorphic pattern of gtf3ab transcription was not observed earlier in larvae that had not yet shown any signs of gonad formation after 26 days of oestradiol exposure. Thus, gtf3ab transcription is a consequence of oocyte differentiation and not a direct result of estrogen exposure, and could constitute a useful marker of gonad feminisation and intersex condition.

Fish oogenesis is characterised by a massive growth of oocytes each reproductive season. This growth requires the stockpiling of certain molecules, such as ribosomal RNAs to assist the rapid ribosomal assembly and protein synthesis required to allow developmental processes in the newly formed embryo. Massive 5S rRNA expression in oocytes, facilitated by transcription factor 3A (Gtf3a), serves as marker of intersex condition in fish exposed to xenoestrogens. Our present work on Gtf3a gene evolution has been analysed in silico in teleost genomes and functionally in the case of the zebrafish Danio rerio. Synteny-analysis of fish genomes has allowed the identification of two gtf3a paralog genes, probably emerged from the teleost specific genome duplication event. Functional analyses demonstrated that gtf3ab has evolved as a gene specially transcribed in oocytes as observed in Danio rerio, and also in Oreochromis niloticus. Instead, gtf3aa was observed to be ubiquitously expressed. In addition, in zebrafish embryos gtf3aa transcription began with the activation of the zygotic genome (similar to 8 hpf), while gtf3ab transcription began only at the onset of oogenesis. Under exposure to 100 ng/L 17 beta-estradiol, fully feminised 61 dpf zebrafish showed transcription of ovarian gtf3ab, while masculinised (100 ng/L 17 alpha-methyltestosterone treated) zebrafish only transcribed gtf3aa. Sex related transcription of gtf3ab coincided with that of cyp19a1a being opposite to that of amh and dmrt1. Such sex dimorphic pattern of gtf3ab transcription was not observed earlier in larvae that had not yet shown any signs of gonad formation after 26 days of oestradiol exposure. Thus, gtf3ab transcription is a consequence of oocyte differentiation and not a direct result of estrogen exposure, and could constitute a useful marker of gonad feminisation and intersex condition.

The present study aims at identifying the lowest number of fish (European seabass) that could be used for monitoring and/or experimental purposes in small-scale fish facilities by quantifying the effect that the number of individuals has on the Shannon entropy (SE) of the trajectory followed by the shoal's centroid. Two different experiments were performed: (i) one starting with 50 fish and decreasing to 25, 13, and 1 fish, and (ii) a second experiment starting with one fish, adding one new fish per day during 5 days, ending up with five fish in the tank. The fish were recorded for 1h daily, during which time a stochastic event (a hit in the tank) was introduced. The SE values were calculated from the images corresponding to three arbitrary basal (shoaling) periods of 3.5 min prior to the event, and to the 3.5 min period immediately after the event (schooling response). Taking both experiments together, the coefficient of variation (CV) of the SE among measurements was largest for one fish systems (CV 37.12 and 17.94% for the daily average basal and response SE, respectively) and decreased concomitantly with the number of fish (CV 8.6-10% for the basal SE of 2 to 5 fish systems and 5.86, 2.69, and 2.31% for the basal SE of 13, 25, and 50 fish, respectively). The SE of the systems kept a power relationship with the number of fish (basal: R-2 = 0.93 and response: R-2 = 0.92). Thus, 5-13 individuals should be the lowest number for a compromise between acceptable variability (< 10%) in the data and reduction in the number of fish. We believe this to be the first scientific work made to estimate the minimum number of individuals to be used in subsequent experimental (including behavioral) studies using shoaling fish species that reaches a compromise between the reduction in number demanded by animal welfare guidelines and a low variability in the fish system's response.

The present study aims at identifying the lowest number of fish (European seabass) that could be used for monitoring and/or experimental purposes in small-scale fish facilities by quantifying the effect that the number of individuals has on the Shannon entropy (SE) of the trajectory followed by the shoal's centroid. Two different experiments were performed: (i) one starting with 50 fish and decreasing to 25, 13, and 1 fish, and (ii) a second experiment starting with one fish, adding one new fish per day during 5 days, ending up with five fish in the tank. The fish were recorded for 1h daily, during which time a stochastic event (a hit in the tank) was introduced. The SE values were calculated from the images corresponding to three arbitrary basal (shoaling) periods of 3.5 min prior to the event, and to the 3.5 min period immediately after the event (schooling response). Taking both experiments together, the coefficient of variation (CV) of the SE among measurements was largest for one fish systems (CV 37.12 and 17.94% for the daily average basal and response SE, respectively) and decreased concomitantly with the number of fish (CV 8.6-10% for the basal SE of 2 to 5 fish systems and 5.86, 2.69, and 2.31% for the basal SE of 13, 25, and 50 fish, respectively). The SE of the systems kept a power relationship with the number of fish (basal: R-2 = 0.93 and response: R-2 = 0.92). Thus, 5-13 individuals should be the lowest number for a compromise between acceptable variability (< 10%) in the data and reduction in the number of fish. We believe this to be the first scientific work made to estimate the minimum number of individuals to be used in subsequent experimental (including behavioral) studies using shoaling fish species that reaches a compromise between the reduction in number demanded by animal welfare guidelines and a low variability in the fish system's response.

The present study aims at identifying the lowest number of fish (European seabass) that could be used for monitoring and/or experimental purposes in small-scale fish facilities by quantifying the effect that the number of individuals has on the Shannon entropy (SE) of the trajectory followed by the shoal's centroid. Two different experiments were performed: (i) one starting with 50 fish and decreasing to 25, 13, and 1 fish, and (ii) a second experiment starting with one fish, adding one new fish per day during 5 days, ending up with five fish in the tank. The fish were recorded for 1h daily, during which time a stochastic event (a hit in the tank) was introduced. The SE values were calculated from the images corresponding to three arbitrary basal (shoaling) periods of 3.5 min prior to the event, and to the 3.5 min period immediately after the event (schooling response). Taking both experiments together, the coefficient of variation (CV) of the SE among measurements was largest for one fish systems (CV 37.12 and 17.94% for the daily average basal and response SE, respectively) and decreased concomitantly with the number of fish (CV 8.6-10% for the basal SE of 2 to 5 fish systems and 5.86, 2.69, and 2.31% for the basal SE of 13, 25, and 50 fish, respectively). The SE of the systems kept a power relationship with the number of fish (basal: R-2 = 0.93 and response: R-2 = 0.92). Thus, 5-13 individuals should be the lowest number for a compromise between acceptable variability (< 10%) in the data and reduction in the number of fish. We believe this to be the first scientific work made to estimate the minimum number of individuals to be used in subsequent experimental (including behavioral) studies using shoaling fish species that reaches a compromise between the reduction in number demanded by animal welfare guidelines and a low variability in the fish system's response.

Small variations in signalling pathways have been linked to phenotypic diversity and speciation. In vertebrates, teeth represent a reservoir of adaptive morphological structures that are prone to evolutionary change. Cyprinid fish display an impressive diversity in tooth number, but the signals that generate such diversity are unknown. Here, we show that retinoic acid (RA) availability influences tooth number size in Cyprinids. Heterozygous adult zebrafish heterozygous for the cyp26b1 mutant that encodes an enzyme able to degrade RA possess an extra tooth in the ventral row. Expression analysis of pharyngeal mesenchyme markers such as dlx2a and lhx6 shows lateral, anterior and dorsal expansion of these markers in RA-treated embryos, whereas the expression of the dental epithelium markers dlx2b and dlx3b is unchanged. Our analysis suggests that changes in RA signalling play an important role in the diversification of teeth in Cyprinids. Our work illustrates that through subtle changes in the expression of rate-limiting enzymes, the RA pathway is an active player of tooth evolution in fish.

In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8+ memory T cells while downregulating CD4+ memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM+ B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM+ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10.