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The diverse physiography of the Portuguese land and marine territory, spanning from continental Europe to the Atlantic archipelagos, has made it an important repository of biodiversity throughout the Pleistocene glacial cycles, leading to a remarkable diversity of species and ecosystems. This rich biodiversity is under threat from anthropogenic drivers, such as climate change, invasive species, land use changes, overexploitation, or pathogen (re)emergence. The inventory, characterisation, and study of biodiversity at inter- and intra-specific levels using genomics is crucial to promote its preservation and recovery by informing biodiversity conservation policies, management measures, and research. The participation of researchers from Portuguese institutions in the European Reference Genome Atlas (ERGA) initiative and its pilot effort to generate reference genomes for European biodiversity has reinforced the establishment of Biogenome Portugal. This nascent institutional network will connect the national community of researchers in genomics. Here, we describe the Portuguese contribution to ERGA’s pilot effort, which will generate high-quality reference genomes of six species from Portugal that are endemic, iconic, and/or endangered and include plants, insects, and vertebrates (fish, birds, and mammals) from mainland Portugal or the Azores islands. In addition, we outline the objectives of Biogenome Portugal, which aims to (i) promote scientific collaboration, (ii) contribute to advanced training, (iii) stimulate the participation of institutions and researchers based in Portugal in international biodiversity genomics initiatives, and (iv) contribute to the transfer of knowledge to stakeholders and engaging the public to preserve biodiversity. This initiative will strengthen biodiversity genomics research in Portugal and fuel the genomic inventory of Portuguese eukaryotic species. Such efforts will be critical to the conservation of the country’s rich biodiversity and will contribute to ERGA’s goal of generating reference genomes for European species.

Abstract Background Energy allocation between growth and reproduction determines puberty onset and fertility. In mammals, peripheral hormones such as leptin, insulin and ghrelin signal metabolic information to the higher centres controlling gonadotrophin-releasing hormone neurone activity. However, these observations could not be confirmed in lower vertebrates, suggesting that other factors may mediate the energetic trade-off between growth and reproduction. A bioinformatic and experimental study suggested co-regulation of the circadian clock, reproductive axis and growth-regulating genes in zebrafish. While loss-of-function of most of the identified co-regulated genes had no effect or only had mild effects on reproduction, no such information existed about the co-regulated somatostatin, well-known for its actions on growth and metabolism. Results We show that somatostatin signalling is pivotal in regulating fecundity and metabolism. Knock-out of zebrafish somatostatin 1.1 (sst1.1) and somatostatin 1.2 (sst1.2) caused a 20–30% increase in embryonic primordial germ cells, and sst1.2 −/− adults laid 40% more eggs than their wild-type siblings. The sst1.1 −/− and sst1.2 −/− mutants had divergent metabolic phenotypes: the former had 25% more pancreatic α-cells, were hyperglycaemic and glucose intolerant, and had increased adipocyte mass; the latter had 25% more pancreatic β-cells, improved glucose clearance and reduced adipocyte mass. Conclusions We conclude that somatostatin signalling regulates energy metabolism and fecundity through anti-proliferative and modulatory actions on primordial germ cells, pancreatic insulin and glucagon cells and the hypothalamus. The ancient origin of the somatostatin system suggests it could act as a switch linking metabolism and reproduction across vertebrates. The results raise the possibility of applications in human and animal fertility.

Abstract Background Antarctica harbors the bulk of the species diversity of the dominant teleost fish suborder—Notothenioidei. However, the forces that shape their evolution are still under debate. Results We sequenced the genome of an icefish, Chionodraco hamatus, and used population genomics and demographic modelling of sequenced genomes of 52 C. hamatus individuals collected mainly from two East Antarctic regions to investigate the factors driving speciation. Results revealed four icefish populations with clear reproduction separation were established 15 to 50 kya (kilo years ago) during the last glacial maxima (LGM). Selection sweeps in genes involving immune responses, cardiovascular development, and photoperception occurred differentially among the populations and were correlated with population-specific microbial communities and acquisition of distinct morphological features in the icefish taxa. Population and species-specific antifreeze glycoprotein gene expansion and glacial cycle-paced duplication/degeneration of the zona pellucida protein gene families indicated fluctuating thermal environments and periodic influence of glacial cycles on notothenioid divergence. Conclusions We revealed a series of genomic evidence indicating differential adaptation of C. hamatus populations and notothenioid species divergence in the extreme and unique marine environment. We conclude that geographic separation and adaptation to heterogeneous pathogen, oxygen, and light conditions of local habitats, periodically shaped by the glacial cycles, were the key drivers propelling species diversity in Antarctica.

Prostate cancer is one of the most prevalent tumors in men, accounting for about 7.3% of cancer deaths. Although there are several strategies for diagnosing prostate cancer, these are only accurate when the tumor is already at a very advanced stage, so early diagnosis is essential. Stanniocalcin 1 (STC1) is a secreted glycoprotein, which has been suggested as a tumor marker as its increased expression is associated with the development and/or progression of different types of malignant tumors. In this work, an electronic tongue (ET) prototype, based on a set of four sensors prepared from thin films that included STC1 antibodies for detecting prostate cancer, was developed. In the preparation of the thin films, polyelectrolytes of polyallylamine hydrochloride, polystyrene sulfonate of sodium and polyethyleneimine, and the biomolecules chitosan, protein A, and STC1 antibody were used. These films were deposited on quartz lamellae and on solid supports using layer-on-layer and self-assembly techniques. The deposition of the films was analyzed by ultraviolet-visible spectroscopy, and the detection of STC1 in aqueous solutions of PBS was analyzed by impedance spectroscopy. The impedance data were statistically analyzed using principal component analysis. The ETs formed by the four sensors and the three best sensors could detect the antigen at concentrations in the range from 5 × 10−11 to 5 × 10−4 M. They showed a linear dependence with the logarithm of the antigen concentration and a sensitivity of 5371 ± 820 and 4863 ± 634 per decade of concentration, respectively. Finally, the results allow us to conclude that this prototype can advance to the calibration phase with patient samples.

Tetrodotoxin (TTX) and its analogues are naturally occurring toxins historically responsible for human poisoning fatalities in Eastern Asia. It is typically linked to the consumption of pufferfish and, to a lesser extent, marine gastropods and crabs. In the scope of a comprehensive project to understand the prevalence of emergent toxins in edible marine organisms, we report, for the first time, the detection of TTX analogues in the soft tissues of edible crabs, the European fiddler crab (Afruca tangeri) and green crab (Carcinus maenas), harvested in southern Portugal. No TTX was detected in the analyzed samples. However, three TTX analogues were detected—an unknown TTX epimer, deoxyTTX, and trideoxyTTX. These three analogues were found in the European fiddler crab while only trideoxyTTX was found in the green crab, suggesting that the accumulation of TTX analogues might be influenced by the crabs’ different feeding ecology. These results highlight the need to widely monitor TTX and its analogues in edible marine species in order to provide adequate information to the European Food Safety Authority and to protect consumers.

Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and are an ancient and well-conserved group of pattern recognition receptors (PRRs). The isolation of the Antarctic continent and its unique teleost fish and microbiota prompted the present investigation into Tlr evolution. Gene homologues of tlr members in teleosts from temperate regions were present in the genome of Antarctic Nototheniidae and the non-Antarctic sister lineage Bovichtidae. Overall, in Nototheniidae apart from D. mawsoni, no major tlr gene family expansion or contraction occurred. Instead, lineage and species-specific changes in the ectodomain and LRR of Tlrs occurred, particularly in the Tlr11 superfamily that is well represented in fish. Positive selective pressure and associated sequence modifications in the TLR ectodomain and within the leucine-rich repeats (LRR), important for pathogen recognition, occurred in Tlr5, Tlr8, Tlr13, Tlr21, Tlr22, and Tlr23 presumably associated with the unique Antarctic microbiota. Exposure to lipopolysaccharide (Escherichia coli O111:B4) Gram negative bacteria did not modify tlr gene expression in N. rossii head–kidney or anterior intestine, although increased water temperature (+4°C) had a significant effect.

Saxitoxin and its more than 50 analogues are a group of naturally occurring neurotoxins collectively designated as paralytic shellfish toxins (PSTs). PSTs are toxic to humans and maximum legal limits in seafood have been implemented by regulatory authorities worldwide. In the European Union, monitoring of PSTs is performed using the AOAC Official Method 2005.06, based on liquid chromatography coupled with fluorescence detection (LC- FLD). However, this method has been suggested to not effectively detect the emerging C-11 hydroxyl (M-toxins) and benzoate (GC-toxins) analogues, with these analogues currently not being surveyed in monitoring programs. In this study, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was used to search for these emerging PSTs in mussels, Mytilus galloprovincialis, contaminated following an intense Gymnodinium catenatum bloom in the Tagus estuary (Lisbon, Portugal). Five M-toxins (M1, M2, M6, dcM6, and dcM10), but no GC-toxins, were detected in the mussels’ whole-soft body tissue. Moreover, the classical PSTs (C1 to C4, GTX 4 to GTX6, dcGTX1 to dcGTX4, dcSTX, dcNEO, and STX) were also found and comprised the largest fraction of the PSTs’ profile. The presence of unregulated PSTs in edible mussel samples suggests potential seafood safety risks and urges further research to determine the frequency of these analogues in seafood and their contribution to toxicity.

The environmental conditions and isolation in the Antarctic have driven the evolution of a unique biodiversity at a macro to microorganism scale. Here, we investigated the possible adaptation of the teleost Nototheniacoriiceps immune system to the cold environment and unique microbial community of the Southern Ocean. The fish immune system was stimulated through an intraperitoneal injection of lipopolysaccharide (LPS 0111:B4 from E. coli) and the tissue transcriptomic response and plasma biochemistry were analyzed 7 days later and compared to a sham injected control. Gene transcription in the head-kidney, intestine and skin was significantly modified by LPS, although tissues showed different responsiveness, with the duodenum most modified and the skin the least modified. The most modified processes in head-kidney, duodenum and skin were related to cell metabolism (up-regulated) and the immune system (comprising 30% of differentially expressed genes). The immune processes identified were mostly down-regulated, particularly interleukins and pattern recognition receptors (PRRs), nucleotide-binding oligomerization domain-like receptors and mannose receptors, unlike the toll-like receptors response commonly described in other teleost fish. The modified transcriptional response was not mirrored by a modified systemic response, as the circulating levels of enzymes of innate immunity, lysozyme and antiproteases, were not significantly different from the untreated and sham control fish. In conclusion, while the N.coriiceps immune system shares many features with other teleosts there are also some specificities. Further studies should better characterize the PRRs and their role in Antarctic teleosts, as well as the importance of the LPS source and its consequences for immune activation in teleosts.

The complement system comprises a large family of plasma proteins that play a central role in innate and adaptive immunity. To better understand the evolution of the complement system in vertebrates and the contribution of complement to fish immunity comprehensive in silico and expression analysis of the gene repertoire was made. Particular attention was given to C3 and the evolutionary related proteins C4 and C5 and to one of the main regulatory factors of C3b, factor H (Cfh). Phylogenetic and gene linkage analysis confirmed the standing hypothesis that the ancestral c3/c4/c5 gene duplicated early. The duplication of C3 (C3.1 and C3.2) and C4 (C4.1 and C4.2) was likely a consequence of the (1R and 2R) genome tetraploidization events at the origin of the vertebrates. In fish, gene number was not conserved and multiple c3 and cfh sequence related genes were encountered, and phylogenetic analysis of each gene generated two main clusters. Duplication of c3 and cfh genes occurred across the teleosts in a species-specific manner. In common, with other immune gene families the c3 gene expansion in fish emerged through a process of tandem gene duplication. Gilthead sea bream (Sparus aurata), had nine c3 gene transcripts highly expressed in liver although as reported in other fish, extra-hepatic expression also occurs. Differences in the sequence and protein domains of the nine deduced C3 proteins in the gilthead sea bream and the presence of specific cysteine and N-glycosylation residues within each isoform was indicative of functional diversity associated with structure. The diversity of C3 and other complement proteins as well as Cfh in teleosts suggests they may have an enhanced capacity to activate complement through direct interaction of C3 isoforms with pathogenic agents.

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.

Abstract Androgens, namely 11-ketotestosterone (11KT), have a central role in male fish reproductive physiology and are thought to be involved in both aggression and social signalling. Aggressive encounters occur frequently in social species, and fights may cause energy depletion, injury and loss of social status. Signalling for social dominance and fighting ability in an agonistic context can minimize these costs. Here, we test the hypothesis of a ‘chemical diplomacy’ mechanism through urinary signals that avoids aggression and evokes an androgen response in receiver males of Mozambique tilapia (Oreochromis mossambicus). We show a decoupling between aggression and the androgen response; males fighting their mirror image experience an unresolved interaction and a severe drop in urinary 11KT. However, if concurrently exposed to dominant male urine, aggression drops but urinary 11KT levels remain high. Furthermore, 11KT increases in males exposed to dominant male urine in the absence of a visual stimulus. The use of a urinary signal to lower aggression may be an adaptive mechanism to resolve disputes and avoid the costs of fighting. As dominance is linked to nest building and mating with females, the 11KT response of subordinate males suggests chemical eavesdropping, possibly in preparation for parasitic fertilizations.

A comprehensive understanding of how bacterial community abundance changes in fishes during their lifecycle and the role of the microbiota on health and production is still lacking. From this perspective, the egg bacterial communities of two commercially farmed species, the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata), from different aquaculture sites were compared, and the potential effect of broodstock water microbiota and disinfectants on the egg microbiota was evaluated. Moreover, 16S ribosomal RNA gene sequencing was used to profile the bacterial communities of the eggs and broodstock water from three commercial hatcheries. Proteobacteria were the most common and dominant phyla across the samples (49.7% on average). Vibrio sp. was the most highly represented genus (7.1%), followed by Glaciecola (4.8%), Pseudoalteromonas (4.4%), and Colwellia (4.2%), in eggs and water across the sites. Routinely used iodine-based disinfectants slightly reduced the eggs’ bacterial load but did not significantly change their composition. Site, species, and type of sample (eggs or water) drove the microbial community structure and influenced microbiome functional profiles. The egg and seawater microbiome composition differed in abundance but shared similar functional profiles. The strong impact of site and species on egg bacterial communities indicates that disease management needs to be site-specific and highlights the need for species- and site-specific optimization of disinfection protocols.

The non-specific immunity can induce iron deprivation as a defense mechanism against potential bacterial pathogens, but little information is available as to its role in Antarctic fish. In this study the response of iron metabolism related genes was evaluated in liver and head kidney of the Antarctic notothenoids Notothenia coriiceps and Notothenia rossii 7 days after lipopolysaccharide (LPS) injection. Average plasma Fe2+ concentration was unaffected by treatment in any of the species. The gene expression response to LPS varied between tissues and species, being stronger in N. coriiceps and more prominent in the head kidney than liver. The reaction to LPS was marked by increased individual variability in most genes analyzed, even when the change in expression was not statistically significant, suggesting different individual sensitivity and coping responses in these wild fish. We found that iron related genes had an attenuated and homogenous response to LPS but there was no detectable relationship between plasma Fe2+ and gene expression. However, overall in both tissues and species LPS exposure set a multilevel response that concur to promote intracellular accumulation of iron, an indication that Antarctic Notothenoids use innate nutritional immunity as a resistance mechanism against pathogens.

Invasive species are recognized as a major cause of biodiversity decline. Legal regulations relating to the prevention, control, or eradication of invasive species should always be up-to-date, as the failure to recognize the problem, lack of adequate scientific information, or long legal intervals required to prepare the legislation may result in irreversible, possibly catastrophic, outcomes. This implies constant monitoring of the species distribution and levels of establishment, as well as detailed knowledge about its biology to predict dissemination and viability under changing environmental conditions. Pre-screening kits for potential invasive species are valuable tools for policy makers, as they provide information about if and how management measures should be taken. The Freshwater Fish Invasiveness Scoring Kit (FISK) and the Aquatic Species Invasiveness Screening Kit (AS-ISK) have been suggested as reliable tools to assess the potential risk of a species becoming invasive. The present study highlights the spread of the non-native chameleon cichlid Australoheros facetus in several streams of the major river drainages in southern Portugal and compares the fish assemblages and ecological indices in two selected sites in the Vascão and Odelouca rivers. We reviewed the current knowledge on the distribution, physiology, and behavior of A. facetus, and applied the toolkits FISK v2 and AS-ISK to this species to evaluate whether the species should be classified as invasive in Portugal. Field data show high abundance of the species in most streams and dominance in specific hotspots. The scores reached by the kits (FISK v2: 23; AS-ISK: 37) places A. facetus as a species with high potential of invasiveness and support the recent inclusion of this species in the invasive species list in Portugal (Decree-Law 92/2019), but, most of all, highlights the importance of frequent updates in both the field monitoring and the legal regulation and watch lists of invasive organisms.

The acquisition of reproductive competence requires the activation of the brain-pituitary-gonad (BPG) axis, which in most vertebrates, including fishes, is initiated by changes in photoperiod. In the European sea bass long-term exposure to continuous light (LL) alters the rhythm of reproductive hormones, delays spermatogenesis and reduces the incidence of precocious males. In contrast, an early shift from long to short photoperiod (AP) accelerates spermatogenesis. However, how photoperiod affects key genes in the brain to trigger the onset of puberty is still largely unknown. Here, we investigated if the integration of the light stimulus by clock proteins is sufficient to activate key genes that trigger the BPG axis in the European sea bass. We found that the clock genes clock, npas2, bmal1 and the BPG genes gnrh, kiss and kissr share conserved transcription factor frameworks in their promoters, suggesting co-regulation. Other gene promoters of the BGP axis were also predicted to be co-regulated by the same frameworks. Co-regulation was confirmed through gene expression analysis of brains from males exposed to LL or AP photoperiod compared to natural conditions: LL fish had suppressed gnrh1, kiss2, galr1b and esr1, while AP fish had stimulated npas2, gnrh1, gnrh2, kiss2, kiss1rb and galr1b compared to NP. It is concluded that fish exposed to different photoperiods present significant expression differences in some clock and reproductive axis related genes well before the first detectable endocrine and morphological responses of the BPG axis.