phyloBARCODER: A Web Tool for Phylogenetic Classification of Eukaryote Metabarcodes Using Custom Reference Databases.

We developed phyloBARCODER (https://github.com/jun-inoue/phyloBARCODER), a new web tool that can identify short DNA sequences to the species level using metabarcoding. phyloBARCODER estimates phylogenetic trees based on the uploaded anonymous DNA sequences and reference sequences from databases. Without such phylogenetic contexts, alternative, similarity-based methods independently identify species names and anonymous sequences of the same group by pairwise comparisons between queries and database sequences, with the caveat that they must match exactly or very closely. By putting metabarcoding sequences into a phylogenetic context, phyloBARCODER accurately identifies (i) species or classification of query sequences and (ii) anonymous sequences associated with the same species or even with populations of query sequences, with clear and accurate explanations. Version 1 of phyloBARCODER stores a database comprising all eukaryotic mitochondrial gene sequences. Moreover, by uploading their own databases, phyloBARCODER users can conduct species identification specialized for sequences obtained from a local geographic region or those of nonmitochondrial genes, e.g. ITS or rbcL.

Development of branchial ionocytes in embryonic and larval stages of cloudy catshark, Scyliorhinus torazame.

In teleost fish, branchial ionocytes are important sites for osmoregulation and acid-base regulation by maintaining ionic balance in the body fluid. During the early developmental stages before the formation of the gills, teleost ionocytes are localized in the yolk-sac membrane and body skin. By comparing with teleost fish, much less is known about ionocytes in developing embryos of elasmobranch fish. The present study investigated the development of ionocytes in the embryo and larva of cloudy catshark, Scyliorhinus torazame. We first observed ionocyte distribution by immunohistochemical staining with anti-Na+/K+-ATPase (NKA) and anti-vacuolar-type H+-ATPase (V-ATPase) antibodies. The NKA- and V-ATPase-rich ionocytes appeared as single cells in the gill filaments from stage 31, the stage of pre-hatching, while the ionocytes on the body skin and yolk-sac membrane were also observed. From stage 32, in addition to single ionocytes on the gill filaments, some outstanding follicular structures of NKA-immunoreactive cells were developed to fill the inter-filament region of the gill septa. The follicular ionocytes possess NKA in the basolateral membrane and Na+/H+ exchanger 3 in the apical membrane, indicating that they are involved in acid-base regulation like single NKA-rich ionocytes. Three-dimensional analysis and whole-mount immunohistochemistry revealed that the distribution of follicular ionocytes was limited to the rostral side of gill septum. The rostral sides of gill septum might be exposed to faster water flow than caudal side because the gills of sharks gently curved backward. This dissymmetric distribution of follicular ionocytes is considered to facilitate efficient body-fluid homeostasis of catshark embryo.

Functional characterization of follicle-stimulating hormone and luteinizing hormone receptors in cloudy catshark, Scyliorhinus torazame.

The follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in cloudy catshark were cloned, and recombinant FSHR and LHR were expressed for characterization. Ventral lobe extract (VLE) from the pituitary contains homologous FSH and LH, and it stimulated the cAMP signaling of FSHR and LHR dose-dependently. Two transcript variants of LHR (LHR-L with exon 10 and LHR-S without) were identified, and LHR-S was the dominant form with higher basal cAMP activity without VLE stimulation. Among various developmental stages of follicles, FSHR expression was mainly associated with the pre-vitellogenic and early white follicles. When follicles were recruited into vitellogenesis, the expression of FSHR decreased while of LHR was upregulated reciprocally, suggesting that LHR may also be responsible for the control of vitellogenesis in chondrichthyans. The expression of LHR-L was upregulated among maturing follicles before ovulation, indicating LHR-L could have a specific role in receiving the LH surge signal for final maturation. Plasma LH-like activity was transiently increased prior to the progesterone (P4)-surge and testosterone-drop at the beginning of P4-phase, supporting a pituitary control of follicle-maturation via LH signaling in chondrichthyans. The expression of follicular LHR was downregulated during the P4-phase when LH-like activity was high, indicating that the LH-dependent downregulation of LHR is conserved in chondrichthyans as it is in other vertebrate lineages. (213 words).