The invasive brown seaweed Rugulopteryx okamurae has raised increasing environmental and management concerns along the southern coast of Spain, where it forms massive biomass accumulations on shorelines. In this study, we investigated the seasonal and spatial variability in the biochemical composition of Rugulopteryx okamurae collected from two coastal locations with contrasting environmental conditions. The aim was to identify optimal conditions for valorizing its biomass for agricultural applications. Samples were collected over a twoyear period. The results showed that the total lipid content of Rugulopteryx okamurae was significantly associated with its carbon content and was strongly influenced by the amount of dissolved inorganic carbon in the seawater. By contrast, temperature and ammonium were the main drivers of nitrogen content. The most favorable C/N ratio, sulfur content, and mineral composition were observed during spring and summer 2024, with mineral content being strongly affected by the seawater temperature. Notably, the concentrations and seasonal variability of potentially toxic metals (Pb, As and Hg) remained below the maximum limits established by EU legislation for agricultural fertilizers. Furthermore, the fatty acid methyl ester profile was significantly influenced by dissolved inorganic carbon which modulated the relative proportions of monounsaturated and polyunsaturated fatty acids. In contrast, alginate yield remained relatively stable (10-15%) across seasons and locations. Overall, these findings highlight the combined influence of spatial and seasonal factors on the biochemical composition of Rugulopteryx okamurae. The results indicate that the warmer months are the optimal period for harvesting biomass intended for soil amendment applications.

Global ocean acidification, driven by rising atmospheric CO2, threatens marine ecosystems and biodiversity, with increasing evidence of disruptive effects on fish neurobiology and behaviour. However, the precise mechanisms underlying these impacts remain largely unresolved.Here, we reveal how chronic exposure to future-predicted CO2 levels disrupts brain function in the marine teleost Solea senegalensis. Using an integrative approach combining electrophysiology, immunohistochemistry and transcriptomics, we demonstrate that elevated CO2 induce a complex multifaceted disruption in brain physiology. Contrary to the prevailing GABAA receptor reversal hypothesis, which predicts Cl- loss and heightened excitatory signalling under high CO2, we observed increased Cl- and HCO3- in cerebrospinal fluid and suppressed neural excitability. Immunohistochemistry revealed reduced expression of glial fibrillary acidic protein across multiple brain regions, suggesting glial impairment. Furthermore, transcriptomic profiling of the olfactory bulb uncovered immune modulation, downregulation of neural excitability genes, and upregulation of neuroplasticity, ciliary, and anti-inflammatory pathways, hallmarks of cellular stress adaptation. Notably, genes involved in circadian regulation and thyroid signalling were also dysregulated, pointing to broader neuroendocrine disruption. These findings challenge simplistic models of ocean acidification impact, unveiling a cascading interplay of enhanced GABAergic inhibition, immune shifts, glial dysfunction, and disrupted timekeeping mechanisms, likely contributing to the behavioural impairments under high CO2. Unlike prior studies relying on behavioural assays or direct physiological proxies, our integrative approach, combining direct cerebrospinal fluid ionic measurements, electrophysiology, immunohistochemistry and transcriptomics, unveils a multifactorial physiological cascade. Our work advocated for integrative neurophysiological frameworks to predict marine fish resilience and vulnerability in a rapidly changing ocean.

Flatfish metamorphosis is an abrupt post-embryonic transformation driven by thyroid hormones (THs), in which a bilaterally symmetric pelagic larvae becomes an asymmetric benthic juvenile. While the craniofacial changes associated with eye migration during metamorphosis are well documented, the role of THs in central nervous system (CNS) remodelling remains poorly understood. Here we investigated the role of THs on CNS remodelling during metamorphosis of the flatfish, Solea senegalensis, by integrating high-throughput transcriptomic analysis with experimental manipulation of TH availability using an inhibitor of hormone synthesis, methimazole (MMI) or exogenous T4. Transcriptome profiling revealed 567 differentially expressed gene transcripts associated with TH-levels involved in CNS development, neuronal and glial differentiation, migration, myelination and metabolism. Key CNS-related factors such as klf9, sox9, mbp, and plp were strongly down-regulated in MMI-treated larvae. Cell proliferation assays further demonstrated increased interocular neural proliferation under hypothyroidism, consistent with impaired differentiation. Region-specific analyses of the head and body uncovered distinct patterns of TH signalling involving dio2, dio3, thra, thrb, and mct8, underscoring the spatial complexity of endocrine regulation. These results highlight that THs are crucial for both morphological remodelling and CNS plasticity during flatfish metamorphosis, underscoring their conserved role in vertebrate brain development.

The thyroid hormones (THs) are proposed as putative regulators of immune system maturation in developing teleost fish. To gain insight into this process the Senegalese sole (Solea senegalensis) that has a well-characterized TH-driven metamorphosis was used. Differential gene expression analysis was performed across developmental stages (n = 3 per stage): pre-metamorphosis, onset of metamorphosis, metamorphosis, early climax, climax, and post-metamorphic juveniles. Metamorphosis is a massive gene-oriented developmental process, involving the differential expression of 8145 genes. Clustering analysis was used to identify immune-related genes with similar expression patterns to hypothalamus-pituitary-thyroid (HPT) axis-related genes. TH-regulated candidate immune genes were identified (133) and analysis of their promoter region revealed 84 contained putative TH receptor (TR) binding sites (TREs). Two consensus TRE sequences were identified in the candidate genes, 5'-ntgnGntCacan (exclusive to TRα), and 5'-nnntGgtCannn (common to both TRs). TRα-exclusive TREs were less common than those that bound interchangeably TRα and TRβ. In the promoter region, TRα-exclusive TREs were always accompanied by the pan-TRE consensus sequence, never occurring independently.

Water temperature and prey availability are key factors influencing the successful recruitment of early life stages in fish. Understanding how these variables modulate larval growth and survival is essential for modelling larval dynamics. In this study we reared S. senegalensis larvae under controlled laboratory conditions to assess the effects of temperature and feeding frequency on larval development. Three temperatures (17, 20, and 23 °C) and three feeding frequencies (Ff 2.5 fed 2.5 times per week, Ff 4 four times per week, and Ff 6 six times per week) were tested from 12 to 32 days post-hatch (dph) in both individual and group housing systems. Survival, growth, and metamorphosis progress were monitored, and the expression of six genes related to nutrition (tryp1a and apoA4Aa2), cellular stress (hsp90aa and hsp70), endocrine regulation (tgb), and muscle development (myf4) were monitored on S3 and S4 metamorphic larvae. The feeding frequency appeared as the primary driver influencing all investigated traits, while temperature played a less pronounced effect. These data demonstrate the critical role of energy provision in regulating growth, development, and survival, which interacts with temperature, particularly under conditions where metabolic and energy demands cannot be fully fulfilled. Additionally, the Senegalese sole larvae exhibited compensatory genomic adaptive responses to efficiently mobilize nutrients from the gut and adjust the thyroid axis and cellular responses to support metamorphosis transformation and metabolism when food availability was limited or when temperature approached physiological thresholds.

Fish gonadal melatonin production is still unexplored and could contribute to a better understanding of its role in reproduction control, especially for species with reproductive impairments. This study aimed to comprehend if Senegalese sole testes are an extra-pineal production site of melatonin and if it has seasonal and daily variations. Wild and F1 broodstocks were sampled in the breeding season (BS) and out of the reproductive season (OS), at mid-light (ML) and mid-dark (MD) daytimes. Blood plasma melatonin concentration was determined by radioimmunoassay (RIA). The expression of genes involved in melatonin biosynthesis (tph1a, tph2, hiomt1, aanat1a, aanat1b, and aanat2) and melatonin receptors (mel1, mel1c, and mel2) was evaluated in the brain, eye, and testis by quantitative real-time PCR (qPCR). Plasma melatonin concentration in wild sole displayed day/night differences in both seasons (average ML: 36 ± 22 pg/mL, MD: 108 ± 63 pg/mL), whereas differences in the F1 broodstock were only found OS (ML: 100 ± 54 pg/mL, MD: 187 ± 88 pg/mL). Gene expression of mel1 and mel2 receptors, and tph1a, aanat1a, aanat2, and hiomt1 enzymes was detected and quantified in the fish testes. Moreover, daily and seasonal fluctuations in the expression of those genes were found in all tissues and broodstock groups. However, the F1 group showed distinct gene expression patterns compared to the wild type, suggesting a disruption in the circadian system. This study revealed that Senegalese sole testes are a melatonin production site and, at the same time, suggested a dysregulation in the hypothalamus-pituitary-gonad (HPG) axis of F1 males.

Increased carbon dioxide (CO2) in the ocean is changing seawater chemistry. Behavioural alterations in CO2 exposed fish have been linked to changes in the central nervous system (CNS). However, we hypothesise that receptor cells in direct contact with the environment are more susceptible to changes in water chemistry than the CNS. Electrophysiology, histology, and transcriptomics were used to explore the effect of exposure to CO2 acidified water on the olfactory epithelium (OE) of the Senegalese sole (Solea senegalensis). The upper and lower OE of this flatfish detect different odorants and are in contact with different environments. Acute exposure to acidified water decreased olfactory sensitivity more in the upper than in the lower OE. After chronic exposure to high CO2 there were no histological changes in the upper OE; however, in the lower OE, there was a massive infiltration of melanomacrophage (MMC) and tissue disorganization. In addition, in the upper OE, differential expressed gene transcripts (DETs) were related to inflammation and innate immune processes whereas in the lower OE, DETs were related to the adaptative immune response. Differential regulation of genes related to neurogenesis and plasticity occurred in both epithelia. The effects of ocean acidification in sole OE depends on the nostril; however, the occurrence of an exacerbated immune response, OE remodelling and reduced sensitivity indicate that ocean acidification is likely to have significant and unpredictable consequences for behaviour.

Early development is a critical period in fish aquaculture and is influenced by biotic and abiotic factors (e.g., temperature, feed) that can vary significantly between hatcheries, making it difficult to identify core factors determining quality. Many of the existing larval transcriptome studies are small-scale and occur under specific rearing conditions that do not mirror the diversity of larviculture practices at an industrial level. In the present transcriptome study, gilthead seabream at the larval to juvenile transition (metamorphosis) from several hatcheries in Europe (Greece, Italy, and France) were analysed in a large-scale RNA-seq study. The aim was to uncover the most significant molecular modifications occurring during metamorphosis, irrespective of differences in biotic or abiotic factors, to address knowledge gaps associated with critical early developmental stages under industrial hatchery conditions. Commonly modified gene transcripts between larval stages were identified based on the clustering of gene expression profiles of 25 gilthead seabream libraries from different hatcheries in a PCA analysis. When larvae at flexion were compared to larvae at mid-metamorphosis, 2243 differentially expressed genes (DEGs) were identified, and when larvae at early to mid-metamorphosis were compared to mid to late-metamorphosis, 2299 DEGs were identified. Comparative analysis across the developmental stages of gilthead seabream revealed genes of importance for the metamorphic transition and adaptation to rearing conditions, including genes related to the nervous system at flexion (24 days post hatch), enteroendocrine cell differentiation, and lipid homeostasis at early to mid-metamorphosis (46 dph), and enrichment of genes indicative of immune competence at mid to late-metamorphosis (51-54 dph). The differential expression of some endocrine-associated genes, dio1, dio2, cldn1, ing4, Pou3f4, and fgf22, highlights their importance in metamorphosis. Meta-analysis of the transcriptomes from two species, the gilthead seabream and Senegalese sole, that have differing symmetry and ecology uncovered common molecular expression patterns that underlie larvae maturation during metamorphosis, and we propose that these represent core gene markers of metamorphosis in these two fish species.

Growth performance is a complex genetic trait that interacts with several environmental variables and life-history attributes, most notably sex and reproduction. Thus, unveiling the genetic architecture of growth remains as a major challenge particularly in flatfish in which sexual dimorphism seems to be a common feature. In this study, we investigated the gene expression profiles and genomic windows associated with growth in the Senegalese sole (Solea senegalensis), which exhibits a female-biased size dimorphism. For this purpose, three fast-growing (FG) and two slow-growing (SG) genetic families were selected according to their breeding values for weight at harvest. Principal component analysis (PCA) using 17 morphometric traits showed that main variation (92.5%) was explained by size and shape. While phenotypic variation clearly separated the FG and SG groups, some differences associated with sex were still observable. Females exhibited greater weight, higher ventral body heights and less elliptical body shapes than males supporting sexual dimorphism both in size and shape. RNA-seq analysis identified 693, 12,645 and 1059 differentially expressed transcripts (DETs) between FG and SG in muscle, liver, and brain, respectively. It should be noted a major effect of sex in muscle and liver with 7428 and 15,715 DETs, respectively. In these two tissues, >50% of DETs for growth were also co-regulated by sex. Functional analysis using DETs for growth and sex unveiled intricate interactions between vitellogenesis, reproductive status, and growth-related transcriptional networks, particularly in muscle and liver tissues. Main transcriptional enriched pathways associated with growth were closely related to energy provision, cell cycle and signaling. Analysis of sex-specific and growth×sex interacting DETs between males and females highlighted lipid metabolism, macrophagy regulation, cell cycle, DNA and RNA metabolism, ribosome biogenesis, energy provision and maintainance of cellular homeostasis as the most relevant pathways driving sexual dimorphism. A variants analysis using RNA pool-seq data identified eleven genomic windows associated with growth distributed across the genome. Main gene candidates (pptc7, taz, abcd1, tfe3, nfix, ptcd2, prkaa1), whose expression was highly modified by growth, were involved in mitochondrial homeostasis, regulation of mitochondrial activity, energy production and regulation of musculoskeletal system. All these data improve our understanding of the intricate genetic architecture governing growth in sole.

Tenebrio molitor (TM) is considered as one of the most promising protein sources for replacing fish meal in aquafeeds, among other things because it is rich in protein, a good source of micronutrients and has a low carbon footprint and land use. However, the main drawback of TM is its fatty acid profile, in particular its low content of n-3 PUFA. This study evaluates the effects of partially replacing plant or marine-derived with full-fat TM meal at two different levels on growth performance and lipid profiles of Senegalese sole (Solea senegalensis). For this purpose, a control diet (CTRL) and four experimental isoproteic (53%) and isolipidic (16%) diets were formulated containing 5 and 10% TM meal replacing mostly fish meal (FM5 and FM10), or 10 and 15% TM meal replacing mostly plant meal (PP10 and PP15). Fish (215 g) were fed at 1% of their body weight for 98 days. The final body weight of fish fed the experimental diets containing TM meal was not different from that of fish fed the CTRL diet (289 g). However, the inclusion of TM meal resulted in a gradual improvement in growth rate and feed efficiency in both cases (replacement of fish or plant meals), and significant differences in specific growth rate (SGR) were observed between fish fed the CTRL diet (SGR = 0.30% day−1) and those fed diets with the highest TM meal content (PP15; SGR = 0.35% day−1). The experimental groups did not show any differences in the protein content of the muscle (19.6% w/w). However, significant differences were observed in the total lipid content of the muscle, with the FM10, PP10, and PP15 groups having the lowest muscle lipid contents (2.2% ww). These fish also showed the lowest neutral lipid content in muscle (6.6% dw), but no differences were observed in the total phospholipid content (2.6% dw). Regarding the fatty acid profile, fish fed FM10, PP10 and PP15 had lower levels of linoleic acid (18:2n-6) and higher levels of oleic acid (18:1n-9) in liver and muscle compared to fish fed CTRL. However, no differences were found between fish fed CTRL and TM-based diets for docosahexaenoic acid (22:6n-3) and total n-3 PUFA in liver and muscle. In conclusion, our study demonstrated that full-fat TM inclusion up to 15% in S. senegalensis diets had no negative effects or even some positive effects on fish survival, growth performance, nutrient utilization and flesh quality.

Body shape is a complex and plastic trait with a high impact on fish performance and commercialization. Shape is particularly relevant in flatfish with highly asymmetric bodies. In this study we investigated the skeleton features, transcriptomic profiles and genomic regions in two Senegalese sole groups from a full-sib family with positive (named as HE) or negative (LE) breeding values for body ellipticity (ELL). Soles from HE group were heavier and displayed more elongated bodies than LE. A skeleton analysis showed that HE had a higher number of vertebrae, longer vertebral bodies and intervertebral spaces and a lower incidence of skeletal fusions than LE. With respect to the gender effect, females showed a higher height of ventral body and some differences in the ratio urostyle to skeleton length than males for similar ELL values. The PCA analysis using 28 dimensionless morphological traits indicated that PC1 and PC2 explained 51.3% of the total variance and they were mainly associated with ELL and gender variation, respectively. Expression analysis using two factors (ELL and gender) revealed a major effect of gender in muscle, pterygiophores and liver with 6764, 6506 and 7559 differentially expressed transcripts (DETs) between sexes, respectively. In brain, only 16 DETs were identified. When HE and LE were compared controlling by gender, we identified the subset of DETs with conserved responses in both sexes or those regulated in a sex-specific manner in each tissue. A comparison across tissues identified 31 ubiquitous transcripts as differentially expressed in at least two of them. Functional analysis showed that liver and pterygiophores had the highest number of enriched gene ontology categories (24 and 14, respectively). Some of these enriched pathways were related to regulation of cell cycle, cell structure and shape, locomotory behavior, immune system, hormonal responses and homeostasis. A BSR-Seq analysis confirmed five significant genomic windows associated with ELL by using the tricube-smoothed G' and Fst statistics. Windows located in linkage groups (LG) 5 and LG14 included the malt1 and vtg3 genes, respectively, that were also detected within the subset of ubiquitous DETs. Moreover, malt1 appeared located very close to the bmpr1b gene within the significance hotspot of LG5 indicating that mediators of the inflammatory responses and BMP signaling pathway could be responsible for the differences found in the skeleton features and body ellipticity.

Body shape is an important morphological trait in aquaculture. This study investigates the use of Elliptic Fourier descriptors (EFDs) analysis and ellipse fitting estimators to assess shape variation in Senegalese sole and their genetic variance components. A total of 2271 fish were individually photographed and body contours reconstructed using 20 Elliptic Fourier harmonics. Principal component analysis identified four symmetric components that explained the 68.6% of the total variation. They were mainly related to the adjustment of the body to an ellipse (PC1s and PC3s) and caudal fin morphological features (PC2s, PC3s, PC4s). Moreover, four asymmetric components related to caudal fin (PC1a, PC2a, PC3a) and head orientation (PC4a) that explained 24.5% of the total variation were also found. In addition to EFDs, body perimeter and five ellipse fitting estimators including the maximum body height to caudal peduncle height ratio (MBH/CPH), body aspect ratio (AR), ellipticity (ELL), AR from theoretical ellipse (ARe) and solidity (SOL) were calculated. PC1s was strongly correlated with ARe, AR and ELL (≥0.75), PC2s with SOL (−0.79) and PC3s with AR and ELL (0.48–0.49). Males appeared more elliptic than females as revealed by all ellipse descriptors with a tendency to caudal fins with obtuse angles (PC3a) and heads orientated toward the abdominal cavity (PC4a). Overall, heritabilities for symmetric components were higher than for asymmetric. Heritabilities for body ellipse fitting estimators including PC1s were high or very high with the highest values for AR (0.80) and ARe (0.78) for body and whole-body, respectively. Heritability for MBH/CPH was high (0.51) with low genetic correlation with other morphological traits. Heritabilities for symmetrical and asymmetrical features of caudal fin were moderate or low and indirect selection using other highly correlated traits such as PC2s through SOL and PC3a through PC4a or PC1s would facilitate progress in the selection. Perimeter showed a moderate heritability and it was more genetically correlated with growth than shape-related traits. The data provided represent a valuable tool to assess body shape in sole and in the design of genetic breeding programs in Senegalese sole.

Research on recruitment variability has gained momentum in the last years, undoubtedly due to the many unknowns related to climate change impacts. Knowledge about recruitment—the process of small, young fish transitioning to an older, larger life stage—timing and success is especially important for commercial fish species, as it allows predicting the availability of fish and adapting fishing practices for its sustainable exploitation. Predicting tools for determining the combined effect of temperature rise and food quality and quantity reduction (two expected outcomes of climate change) on early-life history traits of fish larvae are valuable for anticipating and adjusting fishing pressure and policy. Here we use a previously published and validated dynamic energy budget (DEB) model for the common sole (Solea solea) and adapt and use the same DEB model for the Senegalese sole (S. senegalensis) to predict the effects of temperature and food availability on Solea spp. early life-history traits. We create seven simulation scenarios, recreating RCP 4.5 and 8.5 Intergovernmental Panel on Climate Change (IPCC) scenarios and including a reduction in food availability. Our results show that temperature and food availability both affect the age at metamorphosis, which is advanced in all scenarios that include a temperature rise and delayed when food is limited. Age at puberty was also affected by the temperature increase but portrayed a more complex response that is dependent on the spawning (batch) period. We discuss the implications of our results in a climate change context.

Sustainability enhancement is one of the main challenges of aquaculture. Since feeds represent one of the major costs from an environmental point of view, it is priority to find sustainable alterna-tive ingredients for aquaculture diet. Insect meals have some advantages as ingredients for aqua-culture, like its sustainability and nutritional value. However, the biggest drawback of full-fat in-sect meal is its fatty acid profile. The objective of this work was to assess the effects of partial plant or marine-derived ingredients replacements with full-fat Tenebrio molitor meal (TM) at two differ-ent levels on growth performance and fatty acids profiles of Solea senegalensis. For this purpose, a control diet and four experimental diets were tested, two of them contained 5 and 10% w/w TM that replaced mostly fish meal. Two other experimental diets included 10 and 15% w/w TM that replaced mostly plant meals. The inclusion of insect meal resulted in an improvement in growth rate and feed efficiency in both cases. Moreover, dietary inclusion of insect meal increased muscle total protein and decreased total lipid, without changes in phospholipids, and maintaining the relative level of n-3 PUFA. In conclusion, our study demonstrated that full-fat TM inclusion up to 15% in S. senegalensis diets had no negative effects or even positive effects on fish survival, growth performance, nutrient utilization and flesh quality.

Despite the growing importance of aquaculture over recent years, and its key role as an alternative to sea overexploitation, there are still a considerable number of challenges that must be faced by the marine aquaculture sectors, especially those regarding conservation approaches. In spite of the aquaculture impacts, such as organic and nutrient enrichment in the water column and sediments, several strategies to reduce negative impacts have emerged, such as integrated multitrophic aquaculture (IMTA), recirculating aquaculture systems (RAS), or the use of new resources (e.g., amphipod crustaceans) to minimize the impact of eutrophication associated with aquaculture facilities. In order to ensure the sustainable development of fish aquaculture, an improvement of the existing management strategies is necessary as a starting point, preventing and reducing the potential environmental impacts of escapees on coastal ecosystems, as well as their indirect socioeconomic consequences. This chapter also addresses two aspects of concern related to aquaculture expansion: the use of alien and locally absent species in aquaculture and the application of genetic improvement technologies. The impact of farmed fish viral infections on the environment and biodiversity has been scarcely studied, and this chapter provides an update on the present knowledge about viral pathogens. The relevance of ornamental species aquaculture for marine conservation, including social implications and psychological perspective, together with an adequate trade in species is mandatory for conservation aquaculture. Aquaculture opportunities to help protect endangered aquatic species, both by reducing stress on wild populations and by enhancing these populations, are also discussed in this chapter.