Spatiotemporal changes in branchial ionocyte distribution were investigated following transfer from seawater (SW) to freshwater (FW) in Japanese seabass. The mRNA expression levels of cystic fibrosis transmembrane conductance regulator (CFTR) and Na/K/2Cl cotransporter 1a (NKCC1a) in the gills rapidly decreased after transfer to FW, whereas Na/H exchanger 3 (NHE3) and Na/Cl cotransporter 2 (NCC2) expression were upregulated following the transfer. Using quadruple-color whole-mount immunofluorescence staining with anti-Na/K-ATPase, anti-NHE3, anti-CFTR and T4 (anti-NKCC1a/NCC2) antibodies, we classified ionocytes into one SW type and two FW types: NHE3 cell and NCC2 cell. Time course observation after transfer revealed an intermediate type between SW-type and FW-type NHE3 ionocytes, suggesting functional plasticity of ionocytes. Finally, on the basis of the ionocyte classification of Japanese seabass, we observed the location of ionocyte subtypes on frozen sections of the gill filaments stained by triple-color immunofluorescence staining. Our observation indicated that SW-type ionocytes transformed into FW-type NHE3 ionocytes and at the same time shifted their distribution from filaments to lamellae. However, FW-specific NCC2 ionocytes appeared mainly in the filaments. Taken together, these findings indicate that ionocytes originated from undifferentiated cells in the filaments and expanded their distribution to the lamellae during FW acclimation.

A B S T R A C TAutotomy is an adaptive response in which animals escape from predators by shedding their own appendages. It is made possible by the presence of an efficient mechanism for regeneration. Decapod crustaceans frequently exhibit excellent abilities to regenerate complete pereopods in just a few molts following autotomy. The molecular basis of regeneration pereopods in decapods remains unclear. We identified the primary structure of Baboon (Babo), a type I TGF-β superfamily receptor involved in the activin pathway, in the crayfish, Procambarus fallax f. virginalis Martin, Dorn, Kawai, Heiden and Scholtz, 2010. Molecular cloning revealed that babo possesses three splice variants. The expression levels of the functional babo transcript did not show increases during regeneration. RNA interference (RNAi) targeting a common region of the babo sequence, however, caused a reduction in regenerated pereopod lengths. No loss or reduction in a specific article was observed. Instead, the regenerated legs were smaller but retained the morphology and proportions of regenerated legs from control animals. Babo thus appears to control the growth, but not the pattern, of legs during the regeneration process in decapod crustaceans.

The CRH family of neuropeptides, including CRH and urocortins, plays pivotal roles in the regulation of physiological and behavioral stress responses in vertebrates. In this study, we identified a previously undescribed member of the CRH family of peptides in a teleost fish species (medaka; Oryzias latipes) and named this peptide teleocortin (Tcn). Medaka Tcn is a 41-amino acid polypeptide derived from the C terminus of a larger precursor protein that is encoded by a 2-exon gene, thus sharing common structural features with known CRH family peptides. tcn was found exclusively in teleost fish. Phylogenetic analysis suggested that tcn probably has an ancient origin but was lost from the tetrapod lineage shortly after the divergence of the teleost and tetrapod lineages. In the medaka brain, tcn was expressed in nuclei of the telencephalon, preoptic area, hypothalamus, tegmentum, and isthmic region. Because none of these nuclei have been implicated in the control of ACTH secretion from the pituitary, Tcn may exert its effects centrally in the brain rather than via stimulation of the pituitary-adrenal/interrenal axis. Most, if not all, tcn-expressing neurons also expressed crh, suggesting that Tcn and Crh share common physiological functions. Moreover, Tcn activated Crh receptors 1 and 2 with equivalent or slightly higher potency than Crh, further suggesting that these peptides share common functions. Taken together, these data identified Tcn as a novel, teleost-specific member of the CRH family of peptides that may act centrally with Crh to regulate physiological and behavioral stress responses.

Members of the phylum Arthropoda, comprising over 80% of total animal species, have evolved regenerative abilities, but little is known about the molecular mechanisms mediating this process. Transforming growth factor β (TGF‐β) signaling mediates a diverse set of essential processes in animals and is a good candidate pathway for regulation of regeneration in arthropods. In this study we investigated the role of activin signaling, a TGF‐β superfamily pathway, in limb regeneration in the crayfish. We identified and cloned a downstream transcription factor in the activin pathway, Smox, and characterized its function with regard to other elements of the activin signaling pathway. Gene knockdown of Smox by RNAi induced regeneration of complete but smaller pereopods after autotomy. This indicates that activin signaling via Smox functions in regulation of pereopod growth and size. The expression levels of both Smox and the activin receptor babo were closely correlated with molting. The expression level of Smox increased when babo was knocked down by RNAi, indicating that Smox and babo transcription are linked. Our study suggests that the Babo‐Smox system in activin signaling is conserved in decapods, and supports an evolutionary conservation of this aspect of molecular signaling during regeneration between protostomes and deuterostomes.

In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka (Oryzias latipes) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1β (IL-1β), complement 1q subunit C (C1qc), transferrin a (Tfa), and G-type lysozyme (LyzG), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB (pla2g1b), and elastase-1-like (CELA1), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the Okamura et al. Role of IL-17A/F1 in Teleost Intestineintestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome.