Androgens and Mating Systems in Fish: Intra- and Inter-specific Analysis

One of the predictions of the ‘challenge hypothesis’ (Wingfield et al., 1990) is that androgen patterns during the breeding season should vary among species according to the parenting and mating system. Here we assess this prediction of the challenge hypothesis both at the intra- and at the inter-specific level. To test the hypothesis at the inter-specific level, a literature survey on published androgen pat- terns from teleost fish with different mating systems was carried out. The results confirm the predicted effect of mating system on andro- gen levels. To test the hypothesis at an intra-specific level, a species with flexible reproductive strategies (i.e. monogamy vs. polygyny), the Saint Peter’s fish was studied. Polygynous males had higher 11- ketotestosterone levels. However, males implanted with methyl-tes- tosterone did not became polygynous and the variation of the ten- dency to desert their pair mates was better explained by the repro- ductive state of the female partner. This result stresses the point that the effects of behaviour on hormones cannot be considered without respect to the social context.

Evolution, gene regulation and functional analysis of BMP2 in fish

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.

Identification of novel molecular determinants of tissue mineralization in fish

The identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.

Early nutritional programming in fish: tailoring the metabolic use of dietary carbohydrates

Tese de doutoramento, Ciências da Vida, do Mar, da Terra e do Ambiente (Nutrição), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Identification of novel molecular determinants of tissue mineralization in fish

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Long-term changes in fish communities of the Ria Formosa coastal lagoon (southern Portugal) based on two studies made 20 years apart

Fish communities of the Ria Formosa coastal lagoon (south Portugal) were sampled on a monthly basis with a beach seine at 4 sites, during two different time periods: 1980-1986 and 2001-2002. Community indices, species ranking and multivariate analysis were used in order to identify changes in the fish community between the two time periods. A total of 153,511 fish representing 57 taxa were recorded. Although species composition was very similar for both sampling periods, multivariate analysis performed on annual species abundance in number and weight revealed differences in fish community structure between the two periods. Structural changes in fish community were related mostly to a sharp decrease in the abundance of Mugilidae from 1980-1986 to 2001-2002. These changes were probably associated to a decrease in organic matter contents and nutrients concentrations due to improvements in sewage treatment and better water circulation inside the lagoon. The changes in fish community structure are more evident in the inner areas of the lagoon than near the inlet. The association between changes in sewage patterns and changes in the ichthyofaunal community structure reinforces the importance of fish communities as a biological indicator of human induced changes in marine systems. (c) 2007 Elsevier Ltd. All rights reserved.

Seasonal, tidal and diurnal changes in fish assemblages in the Ria Formosa lagoon (Portugal)

Fish fauna were collected in two different subtidal shallow sites, seagrass and sand, using a small beam trawl in the Ria Formosa lagoon (South Portugal). Samples were taken at low and high tides, during day and night, and in each season. Fish assemblages associated with each site were significantly different, with seagrass site supporting greater fish abundance and higher number of species than sand. These site-related differences in fish assemblages were stronger than any other factor studied. Both sites showed seasonal variations in their fish assemblages, mainly because of recruitment of marine juvenile migrants during spring and summer. No significant tidal or diel changes were observed in the fish assemblages of either site, but there was a significant site-tide interaction, with higher fish abundance in seagrass at low tide. In sand, tide effect was evident only for certain species, with resident species more abundant at high tide and marine species more abundant at low tide. Within the Ria Formosa coastal lagoon, ichthyofaunal composition and structure is mainly influenced by site followed by season. (c) 2005 Elsevier Ltd. All rights reserved.