14 pages, 10 figures, supplementary information https://doi.org/10.1038/s42003-021-01921-9.-- Data availability: All data generated or analyzed during this study are included in this manuscript (and its supplementary information files). The complete alignments shown in Fig. 1 and Supplementary Fig. 1 are provided in Supplementary Data 1. The source data underlying plots shown in figures are provided in Supplementary Data 2. All other data is available from authors upon reasonable request.-- Code availability: Mr Bayes software used for phylogenetic inference is available at https://github.com/NBISweden/MrBayes/releases. MAFFT used for multiple sequence alignments is available at https://mafft.cbrc.jp/alignment/software. PAL2NAL used for codon conversion is available at http://www.bork.embl.de/pal2nal/, This work was supported by grants from the Norwegian Research Council (Grants no. 254872/E40, 204813/E40 and 294768/E40 to R.N.F., and 203516/O30 to F.N.) and the Spanish Ministry of Science and Innovation (MICINN) (Grant no. PID2019-103868RB-I00 to J.C). Participations of M.C. and J.A.S. were funded by the Norwegian Research Council (Grant nos 254872/E40 and 204813/E40, respectively). Participation of F.C. was funded by a “Ramon y Cajal” contract (RYC-2015-17103) from the Spanish MINECO, Peer reviewed

21 pages, 8 figures, 3 tables, supplementary materials https://doi.org/10.3390/ijms21239051, Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain–pituitary–gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis, Research was funded by Project ReproF1 (Programa Operacional Mar2020, MAR-16-02-01-FMP-0059 to E.C.), Portuguese national funds (FCT-Foundation for Science and Technology) through project UIDB/04326/2020, the COST Office (COST Action 16120: EPITRANS), the Spanish Ministry of Science and Innovation (Grant no. PID2019-103868RB-I00 to J.C) and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 683210 to J.M.O.F.). C.O., C.A, I.F. and F.C. acknowledge their contracts, respectively, DL 57/2016/CP1361/CT0007 and SFRH/BD/130910/2017 (FCT-Foundation for Science and Technology), RYC2018-025337-I and RYC-2015-17103 (“Ramón y Cajal” contracts, MICIU and the European Social Fund)

15 pages, 3 figures, supplemental information https://doi.org/10.1016/j.tig.2024.01.004, Genetic adaptations of organisms living in extreme environments are fundamental to our understanding of where life can evolve. Water is the single limiting parameter in this regard, yet when released in the oceans, the single-celled eggs of marine bony fishes (teleosts) have no means of acquiring it. They are strongly hyposmotic to seawater and lack osmoregulatory systems. Paradoxically, modern teleosts successfully release vast quantities of eggs in the extreme saline environment and recorded the most explosive radiation in vertebrate history. Here, we highlight key genetic adaptations that evolved to solve this paradox by filling the pre-ovulated eggs with water. The degree of water acquisition is uniquely prevalent to marine teleosts, permitting the survival and oceanic dispersal of their eggs, This work was supported by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033), the European Regional Development Fund (ERDF) ‘A way of making Europe’ (European Union), Grants no. AGL2016-76802-R and PID2019-103868RB-I00, the Catalan Government (2017 SGR 1042 and 2021 SGR 00068) (to J.C.), and the Norwegian Research Council Grant no. 294768/E40 (to R.N.F.). R.N.F. was also supported by the University of Bergen (Norway). The authors also acknowledge the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S) funded by AEI 10.13039/501100011033, Peer reviewed

[Background] Extremophiles evolved capacities to survive extended exposure to harsh environmental conditions such as complete desiccation (anhydrobiosis) and freezing (cryobiosis). Accumulation of the three-carbon polyhydric alcohol glycerol is commonly observed in anhydrobiotic organisms, although it is considered to preferentially enhance cryobiosis rather than anhydrobiosis., [Results] Here, using dormant stages of the halophilic extremophile crustacean Artemia franciscana, we show that this role is reversed. We find that A. franciscana and related branchiopods evolved co-opted entomoglyceroporin (Eglp)-like aquaporin-type channels previously only characterized in hexapods. Phylogenomic and site-directed mutagenesis analyses indicate that EglpL orthologs likely evolved during the early Cambrian in the common ancestor of the Pancrustacea. RNAi-mediated knockdown experiments show that the A. franciscana EglpL glycerol transporter is subfunctionally co-regulated with canonical aquaglyceroporins (Glps) to mediate glycerol accumulation in the diapause cysts. Termination of diapause using either desiccation or hydrogen peroxide and further exposure of the cysts to freezing suggest that the acquired glycerol plays a more critical role in anhydrobiosis rather than cryobiosis., [Conclusions] These findings uncover the essential role of evolutionary divergent aquaporin-type glycerol channels in the accrual of glycerol in an anhydrobiotic organism and reveal a previously overlooked function of this polyol for desiccation tolerance., This work was supported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/https://doi.org/10.13039/501100011033) and the European Regional Development Fund (ERDF) “A way of making Europe” (European Union), Grant no. PID2019-103868RB-I00 (to J.C.) and PID2019-104483 GB-I00 (to J.L.M.), and by the Agency for Management of University and Research Grants (Government of Catalonia) Grant 2021 SGR 00068 (to J.C.). A.R-A. was supported by a predoctoral contract from Spanish MICIU (PRE2020-092214). R.N.F. was supported by the University of Bergen (Norway). Institutional support to CNAG was from the Spanish MICIU and Generalitat de Catalunya through the Departament de Recerca i Universitats and Departament de Salut. The authors also acknowledge the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S) funded by the Spanish Agencia Estatal de Investigación (AEI) https://doi.org/10.13039/501100011033., With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2019-000928-S)., Peer reviewed

This work was supported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/https://doi.org/10.13039/501100011033) and the European Regional Development Fund (ERDF) “A way of making Europe” (European Union), Grant no. PID2019-103868RB-I00 (to J.C.) and PID2019-104483GB-I00 (to J.L.M.), and by the Agency for Management of University and Research Grants (Government of Catalonia) Grant 2021 SGR 00068 (to J.C.). A.R-A. was supported by a predoctoral contract from Spanish MICIU (PRE2020-092214). R.N.F. was supported by the University of Bergen (Norway). Institutional support to CNAG was from the Spanish MICIU and Generalitat de Catalunya through the Departament de Recerca i Universitats and Departament de Salut. The authors also acknowledge the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S) funded by the Spanish Agencia Estatal de Investigación (AEI) https://doi.org/10.13039/501100011033, Peer reviewed

Extremophiles evolved capacities to survive extended exposure to harsh environmental conditions such as complete desiccation (anhydrobiosis) and freezing (cryobiosis). Accumulation of the three-carbon polyhydric alcohol glycerol is commonly observed in anhydrobiotic organisms, although it is considered to preferentially enhance cryobiosis rather than anhydrobiosis. Here, using dormant stages of the halophilic extremophile crustacean Artemia franciscana, we show that this role is reversed. We find that A. franciscana and related branchiopods evolved co-opted entomoglyceroporin (Eglp)-like aquaporin-type channels previously only characterized in hexapods. Phylogenomic and site-directed mutagenesis analyses indicate that EglpL orthologs likely evolved during the early Cambrian in the common ancestor of the Pancrustacea. RNAi-mediated knockdown experiments show that the A. franciscana EglpL glycerol transporter is subfunctionally co-regulated with canonical aquaglyceroporins (Glps) to mediate glycerol accumulation in the diapause cysts. Termination of diapause using either desiccation or hydrogen peroxide and further exposure of the cysts to freezing suggest that the acquired glycerol plays a more critical role in anhydrobiosis rather than cryobiosis. These findings uncover the essential role of evolutionary divergent aquaporin-type glycerol channels in the accrual of glycerol in an anhydrobiotic organism and reveal a previously overlooked function of this polyol for desiccation tolerance, Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/https://doi.org/10.13039/501100011033) and the European Regional Development Fund (ERDF) “A way of making Europe” (European Union), Grant no. PID2019-103868RB-I00 (to J.C.) and PID2019-104483GB-I00 (to J.L.M.)
Agency for Management of University and Research Grants (Government of Catalonia) Grant 2021 SGR 00068 (to J.C.).
Spanish MICIU (PRE2020-092214).
University of Bergen (Norway).
Institutional support to CNAG was from the Spanish MICIU and Generalitat de Catalunya through the Departament de Recerca i Universitats and Departament de Salut.
“Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S) funded by the Spanish Agencia Estatal de Investigación (AEI)https://doi.org/10.13039/501100011033, Raw Data (FASTQ files) from samples. Count and Normalized expression matrices. DNA sequences. ERX13367886 Illumina NovaSeq 6000 paired end sequencing; ERX13367887 Illumina NovaSeq 6000 paired end sequencing; ERX13367888 Illumina NovaSeq 6000 paired end sequencing; ERX13367889 Illumina NovaSeq 6000 paired end sequencing; ERX13367891 Illumina NovaSeq 6000 paired end sequencing; ERX13367893 Illumina NovaSeq 6000 paired end sequencing; ERX13367894 Illumina NovaSeq 6000 paired end sequencing; ERX13383215 GridION sequencing; ERX13383216 GridION sequencing; ERX13383217 GridION sequencing; ERX13367896 Illumina NovaSeq 6000 paired end sequencing; ERX13367897 Illumina NovaSeq 6000 paired end sequencing; ERX13367890 Illumina NovaSeq 6000 paired end sequencing; ERX13367892 Illumina NovaSeq 6000 paired end sequencing; ERX13367895 Illumina NovaSeq 6000 paired end sequencing, Peer reviewed

14 pages, 9 figures, supplementary material https://doi.org/10.6084/m9.figshare.c.8173869.-- Data accessibility: All data are available in either the manuscript or as electronic supplementary material online, Trehalose accumulation is commonly observed in extremophile organisms capable of surviving extended periods of complete desiccation and freezing. However, direct evidence linking the sugar to cryptobiotic survival in metazoans is generally lacking. Here, using the extremophile crustacean Artemia franciscana as a model, we test the anhydrobiotic and cryobiotic roles of trehalose through RNAi-mediated knockdown of its biosynthetic and transport pathways. Transcriptomic and genomic screening uncovered a major expansion of trehalose transporter (tret) genes in branchiopods with up to 27 copies in Artemia sp., but only a single trehalase enzyme (treh), and three trehalose-6-phosphate synthase (tps) genes containing fused synthase and phosphatase domains. Gene expression analysis determined that the tps and selected tret genes are expressed within the diapause-programmed oocytes and embryos, with independent RNAi-mediated knockdown of each reducing cyst trehalose accumulation by approximately 90% and 30%, respectively. Trehalose depletion in diapause cysts critically affects the anhydrobiotic and cryobiotic survival of embryos, a feature confirmed through diapause-termination experiments using either desiccation or H2O2 exposure. These latter data further uncovered the developmental reprogramming of carbohydrate metabolism to cope with low amounts of stored trehalose in the cysts. The findings demonstrate that diapause-induced biosynthesis and transport of trehalose are essential for A. franciscana anhydrobiosis, This work was supported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033) and the European Regional Development Fund (ERDF) ‘A way of making Europe’ (European Union), grant no. PID2019-103868RB-I00 (to J.C.), and by the Agency for Management of University and Research Grants (Government of Catalonia) grant 2021 SGR 00068 (to J.C.). A.R.-A. was the recipient of a predoctoral contract from the Spanish MICIU (PRE2020-092214). R.N.F. was supported by the University of Bergen (Norway). The authors also acknowledge the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S) funded by the Spanish Agencia Estatal de Investigación (AEI) 10.13039/501100011033, Peer reviewed

Supplementary Table S1. Oligonucleotide primers used for RT-qPCR.
Supplementary Table S2. Oligonucleotide primers used for dsRNA synthesis., This work was supported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033) and the European Regional Development Fund (ERDF) ‘A way of making Europe’ (European Union), grant no. PID2019-103868RB-I00 (to J.C.), and by the Agency for Management of University and Research Grants (Government of Catalonia) grant 2021 SGR 00068 (to J.C.). A.R.-A. was the recipient of a predoctoral contract from the Spanish MICIU (PRE2020-092214). R.N.F. was supported by the University of Bergen (Norway). The authors also acknowledge the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S) funded by the Spanish Agencia Estatal de Investigación (AEI) 10.13039/501100011033, Peer reviewed

27 pages, 9 figures, supplementary data https://doi.org/10.1083/jcb.202512040.-- Data availability: Data are available in the article itself and its supplementary materials. The amino acid alignments and phylogenetic trees are available upon request, Unorthodox AQP12-type channels are poorly understood intracellular aquaporins localized in the endoplasmic reticulum and zymogen granules (ZGs) of pancreatic acinar cells. Despite connections to major diseases, their biophysical properties and intracellular trafficking regulation remain largely unknown. Here, we show that heterologously expressed plant and metazoan AQP12-related channels specifically localize to the intracellular yolk platelet (YP) membrane of frog oocytes, a feature that is recapitulated in vivo for invertebrate and vertebrate orthologs. Using native YP membranes, we show that the vertebrate channels are mercury-sensitive polytransporters with intracellular trafficking regulated by Ca2+ and cAMP signaling pathways. We identify a novel pan-vertebrate C-terminal YP-targeting domain (YPD) in AQP12, which also drives orthodox aquaporin chimeras and truncated channels to YPs and ZGs. In cultured pancreatic cells, the YPD and Ca2+-induced AQP12 N-terminal phosphorylation coregulate secretagogue-triggered channel transport to the ZGs for enzyme secretion. These findings uncover conserved signaling mechanisms for AQP12 trafficking to intracellular protein storage vesicles, and open unexpected avenues for targeted delivery systems, This work was supported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033) and the European Regional Development Fund “A way of making Europe” (European Union) (PID2019-103868RB-I00 to J. Cerdà), and by the Agency for Management of University and Research Grants (2021 SGR 00068 to J. Cerdà). R. N. Finn was supported by the University of Bergen (Norway). The authors acknowledge the “Severo Ochoa Centre of Excellence” accreditation (CEX2024-001494-S) funded by the Spanish State Agency of Research (10.13039/501100011033), Peer reviewed

Data availability: Data are available in the article itself and its supplementary materials Chauvigné, François; Catalán-García, Marc; Daura, Xavier; Finn, Roderick N.; Cerdà, Joan. A pan-vertebrate signaling motif controls the molecular function of intracellular AQP12. Journal of Cell Biology 225(8): e202512040 (2026). https://doi.org/10.1083/jcb.202512040. The amino acid alignments and phylogenetic trees are available upon request, This work was supported by the Spanish Ministry of Science, Innovation and Universities (MICIU/AEI/10.13039/501100011033) and the European Regional Development Fund “A way of making Europe” (European Union) (PID2019-103868RB-I00 to J. Cerdà), and by the Agency for Management of University and Research Grants (2021 SGR 00068 to J. Cerdà). R. N. Finn was supported by the University of Bergen (Norway). The authors acknowledge the “Severo Ochoa Centre of Excellence” accreditation (CEX2024-001494-S) funded by the Spanish State Agency of Research (10.13039/501100011033), Peer reviewed

Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain-pituitary-gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis.

Genetic adaptations of organisms living in extreme environments are fundamental to our understanding of where life can evolve. Water is the single limiting parameter in this regard, yet when released in the oceans, the single-celled eggs of marine bony fishes (teleosts) have no means of acquiring it. They are strongly hyposmotic to seawater and lack osmoregulatory systems. Paradoxically, modern teleosts successfully release vast quantities of eggs in the extreme saline environment and recorded the most explosive radiation in vertebrate history. Here, we highlight key genetic adaptations that evolved to solve this paradox by filling the pre-ovulated eggs with water. The degree of water acquisition is uniquely prevalent to marine teleosts, permitting the survival and oceanic dispersal of their eggs.

Transmembrane conductance of small uncharged solutes such as glycerol typically occurs through aquaglyceroporins (Glps), which are commonly encoded by multiple genes in metazoan organisms. To date, however, little is known concerning the evolution of Glps in Crustacea or what forces might underly such apparent gene redundancy. Here, we show that Glp evolution in Crustacea is highly divergent, ranging from single copy genes in species of pedunculate barnacles, tadpole shrimps, isopods, amphipods and decapods to up to 10 copies in diplostracan water fleas although with monophyletic origins in each lineage. By contrast the evolution of Glps in Copepoda appears to be polyphyletic, with surprisingly high rates of gene duplication occurring in a genera- and species-specific manner. Based upon functional experiments on the Glps from a parasitic copepod (Lepeophtheirus salmonis), we show that such lineage-level gene duplication and splice variation is coupled with a high rate of neofunctionalization. In the case of L. salmonis, splice variation of a given gene resulted in tissue- or sex-specific expression of the channels, with each variant evolving unique sites for protein kinase C (PKC)- or protein kinase A (PKA)-regulation of intracellular membrane trafficking. The combined data sets thus reveal that mutations favouring a high fidelity control of intracellular trafficking regulation can be a selection force for the evolution and retention of multiple Glps in copepods., info:eu-repo/semantics/publishedVersion

Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain–pituitary–gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis., info:eu-repo/semantics/publishedVersion