Álvaro J. Arana

Aquaculture is the world’s fastest-growing food production sector, yet it faces major challenges, including limited understanding of the biology of farmed species. In this context, cell lines are becoming increasingly relevant for studying complex traits and their underlying genetic basis. The in vitro application of molecular tools—such as RNA interference and genome editing—can help disentangle the molecular basis of target traits. However, the lack of protocols for in vitro manipulation is limiting progress in the field. Here, we optimized RNA transfection protocols in SaB-1 cells derived from gilthead seabream, one of the most important farmed species in Mediterranean aquaculture. Despite its importance, in vitro research on this species has been scarce to date. Our results show that RNA delivery in this cell line can be achieved using either electroporation or FUGENE^®-SI-based chemical transfection, although electroporation resulted in lower survival (around 35% versus 76% for chemical transfection). Finally, as a proof-of-concept, both delivery strategies were tested for CRISPR/Cas9-mediated gfap gene editing, which was achieved with efficiencies of around 10%. This study provides useful methodological advances for functional genomics in aquaculture, offering RNA delivery that enables genome-editing strategies and can accelerate molecular research and trait improvement in gilthead seabream.

The light–dark test (DLT) is a useful paradigm to assess the light-dependent modulation of locomotor activity during development. Here, we characterized the ontogeny of the DLT response in Senegalese sole larvae (Solea senegalensis) from 72 to 192 hpf, and we compared it with the dynamics of an endogenous autofluorescence signal observable at early stages. Analysis of integrated activity (actinteg) suggested an ontogenetic trajectory, with activity high at early stages and apparently maximal at 96 hpf, followed by a decrease towards 192 hpf. In parallel, phase-dependent organisation became more stereotyped towards late stages under the light–dark stimulus used here. Because early stages were represented by a single independent recording (R = 1), early-stage patterns are considered exploratory. Autofluorescence observable without exogenous staining was stronger at 96–168 hpf and decreased by 192 hpf; spectral or multi-filter controls are needed to confirm fluorophore identity and to exclude exogenous/background contributions. Overall, under fixed detection settings, we observed a descriptive transition from higher detected activity at early stages to lower detected activity at 192 hpf, alongside stronger light-dependent structuring. Since the DLT quantifies a locomotor response driven by illumination, these findings should be interpreted as a functional readout of light–locomotion coupling, rather than as a direct measure of neurosensory/visual maturation.

Invasive fungal infections, especially those caused by Candida spp., have been classified as a serious global threat. The emergence of species intrinsically resistant to current drugs, along with the increase in acquired resistance, places significant pressure on the need to develop novel and more effective antifungal agents. A limited number of studies have shown the potential of palladium organometallic complexes as promising antifungal alternatives. Although the mechanism of antifungal activity of these complexes remains unaddressed, the findings support the idea that designing palladium (II) complexes could represent the next generation of antifungals. In this work, we synthesized four cyclopalladated complexes, 1a , 1b , 2a , and 2b, from Schiff base–amine phosphanes and evaluated their antifungal potential. Specifically, we assessed their spectrum of activity against several medically relevant Candida spp., their capacity to overcome resistance to current antifungal drugs, antibiofilm properties, uptake by fungal cells, in vivo toxicity, and intracellular effects. The most promising complexes, 1b and 2b , induce strong oxidative stress and lipid peroxidation, inhibit lipolysis, and disrupt vacuole integrity. Moreover, the rational design of the complexes allowed us to infer important structure–activity relationships. Our findings highlight the potential of palladium complexes as promising scaffolds for future antifungal therapeutic strategies and open new horizons for further development.

The CRISPR/Cas9 system is a powerful genome-editing tool widely used in molecular biology and gene therapy, whose efficiency strongly depends on the physicochemical properties of the Cas9 ribonucleoprotein complex. Optimizing Cas9 activity remains essential for reliable genome- editing applications, yet the factors limiting its in vitro cleavage efficiency are not fully understood. Among these, protein aggregation has been suggested to critically impair Cas9 functionality, although its role has not been systematically analysed. Here, we investigate Cas9 aggregation under different environmental conditions and evaluate its impact on in vitro DNA cleavage efficiency. Using fluorescently labelled Cas9 and single-molecule fluorescence techniques, we quantify aggregation as a function of buffer composition, ionic strength, salt concentration and sgRNA presence, and relate these properties to cleavage activity. Our results show that Cas9 aggregation significantly reduces DNA cleavage efficiency, with higher aggregation levels consistently correlating with lower activity. In contrast, buffers with higher ionic strength or stabilizing components reduce aggregation and enhance Cas9 performance. Overall, this study demonstrates that Cas9 aggregation plays a critical role in determining in vitro cleavage efficiency and highlights the importance of controlling protein aggregation to optimize CRISPR/Cas9-based genome-editing applications and delivery strategies. GRAPHICAL

This protocol describes a versatile in vivo zebrafish embryo infection model with Candida albicans that enables precise microinjection and monitoring of defined infection readouts to compare experimental conditions (e.g., injection site and incubation temperature). The core workflow consists of quantitative microinjection of DiI-labeled C. albicans into the hindbrain ventricle of 30-48 hpf embryos, incubation at 23 deg C, and daily survival scoring to evaluate condition-dependent mortality. We calibrate the inoculum by hemocytometer counts and by plating the same injection suspension to determine colony-forming units (CFU) per pulse, allowing CFU per embryo to be reported and experiments to be compared across laboratories. Survival is analyzed with Kaplan-Meier curves and, when groups are compared, Cox models. DiI fluorescence verifies successful delivery, excludes mis-injections, and permits semi-quantitative burden comparisons between injection sites and temperatures at 24-48 hpi; beyond ~48 hpi, signal becomes unreliable due to dye dilution and redistribution. The protocol is designed to be reproducible without transgenic reporters or confocal microscopy and includes practical guidance on randomization, blinding, sample-size planning, and reporting.

Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies—electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)—for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific genomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate editing (~25%) in DLB-1 but yielded only minimal editing in SaB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluorescence correlation spectroscopy suggested that nuclear localization and Cas9 aggregation may influence editing success, highlighting the importance of intracellular trafficking in CRISPR/Cas9 delivery. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture.

Given the central role of neurogenesis in building a functional nervous system, we recently developed a zebrafish-based drug-screening protocol to uncover molecules and signalling pathways regulating spinal cord neurogenesis. In this study, we have expanded this drug screen and discovered a previously unknown role of deacetylase sirtuin 2 (SIRT2) in promoting the generation of serotonergic interneurons in the spinal cord. Treatments with specific SIRT2 inhibitors reduced the generation of serotonergic neurons in the spinal cord, which led to locomotor deficits. Our data suggest that SIRT2 regulates mitotic activity in progenitor cells to promote the generation of serotonergic neurons in developing animals. Together, our results uncover SIRT2 as a key regulator of spinal cord neurogenesis and position it as a promising target for strategies aimed at neural repair in spinal cord disorders.

Neurogenesis plays a fundamental role in the development of a functional nervous system. Seeking to uncover molecules and signalling pathways involved in spinal cord neurogenesis, we recently designed a drug screening protocol in developing zebrafish (González-Llera et al., 2024). Here, we have expanded this drug screen and discovered a previously unknown role of the deacetylase sirtuin 2 (SIRT2) in promoting the generation of serotonergic interneurons in the spinal cord. Treatments with specific SIRT2 inhibitors reduced the generation of serotonergic neurons in the spinal cord, which led to locomotor deficits. Our data suggest that SIRT2 regulates mitotic activity in progenitor cells to promote the generation of serotonergic neurons in developing animals. Together, our results uncover SIRT2 as a key regulator of spinal cord neurogenesis and position it as a promising target for strategies aimed at neural repair in spinal cord disorders.

The African turquoise killifish is a short-lived vertebrate that is gaining interest as a model for understanding degenerative processes associated with aging, including neurodegeneration. However, due to its relatively recent adoption in laboratory research, detailed studies on the adult neuroanatomy of the killifish, particularly regarding the organization of neurotransmitter systems and their age-related changes, remain limited. Here, we study the organization of serotonergic cell populations in the brain and spinal cord of juvenile/young adult (1–3-months-old) and geriatric (5–6-months-old) killifish using anti-serotonin immunofluorescence. Our findings reveal that the overall organization of serotonergic cell populations in killifish closely resembles that of most teleosts, including pineal, pretectal, hypothalamic, rapheal and spinal cord neuronal populations. Of note, adult killifish also present a population of serotonergic neurons in the dorsolateral isthmus, which has been only described in a few other fish species. Aged killifish showed a similar organization of serotonergic cell populations to that of juveniles/young adults, with the major difference being the loss of serotonin immunoreactivity in pretectal cells of 6-month-old fish. Our study provides a neuroanatomical framework for future investigations into the serotonergic system in this emerging vertebrate model of aging.