Following staining with nitro blue tetrazoliumchloride and 5-bromo-4-chloro-3-indolyl-phosphate (Roche, Basel, Switzerland), sections were mounted with Aquatex, an aqueous mounting agent for microscopy (Merck KGaA, Darmstadt, Germany), and examined under bright-field illumination. living under extreme conditions, such as HAE Notothenioidei (Perciform suborder), which represent the prevalent component of the Antarctic fish fauna. During their evolutionary history, Nototheniodei have undergone extraordinary challenges in adapting to the constantly cold marine environment of Antarctica. The Antarctic notothenioid families have been proposed as a superfamily, named Cryonotethenioidea [16], to be distinguished from the non-Antarctic families that are considered the most phyletically basal branch, having remained in peri-Antarctic seawaters under temperate conditions [17,18]. This taxonomic group has long been considered an attractive model to study biochemical, physiological, and morphological adaptations, although poorly investigated at a molecular level. In previous studies, we investigated the genes encoding IgM and IgT isotypes in several cold-adapted and temperate notothenioid species [19,20,21] and highlighted the first evidence of a possible hepato-biliary transport of Ig in the Antarctic species [22]. The most recent advances in collecting omics data from notothenioid fishes provided a source of fundamental information about molecular and genetic features, allowing evolutionary studies on notothenioids in comparison with other perciform species. Thus, the main goal of this study is to investigate the specificities of the gene related to evolutionary adaptation, through a comparative analysis, based on the genomes and transcriptomes available for and 11 Antarctic species belonging to the same suborder as (Notothenoidei). In particular, we extended the analysis to (Nototheniidae family), (Harpagiferidae family), (Bathydraconidae family), (Channichthyidae family), all adapted to live in the extreme environment of Antarctica. Moreover, another notothenioid species, gene was evaluated through q-PCR and in situ hybridization (ISH), which allowed transcript localization. Taken together, these findings underline several peculiar features that may be considered the hallmarks of cold pIgRs and underpin the primary role of pIgR in mucosal immune response and host protection in a cold-adapted teleost species. 2. Results 2.1. Analysis of T. bernacchii pIgR Gene Locus The whole genomic sequence (8310 nt) was retrieved as a single-copy gene from the genome (GenBank assembly accession: “type”:”entrez-nucleotide”,”attrs”:”text”:”NW_022987689″,”term_id”:”1835951677″NW_022987689) by using NCBI Genome Data Viewer. The gene structure consists of eight exons, interrupted by seven introns. The first exon includes the 5 UTR and encodes the leader peptide (245 nt); the second exon (345 nt) encodes the D1 domain; the third exon (82 nt) and the 5 208-nt of the fourth exon encode the D2 domain; the 3 end of the fourth (19 nt) exon, the fifth (88 nt) exon, and the 5-most first end of the sixth (10 nt) exon encode the Extracellular-Membrane Proximal Domain (EMPD); the 5 HAE end of the sixth exon (51 nt) encodes the transmembrane domain (TM); the 3 end of the sixth exon (31 nt), along with the seventh (50 nt) and eighth (81 nt) exons, encode the cytoplasmic region. The terminal sequence is 348 nt, including the stop codon (Figure S1). Along with those found in the 3 UTR, an alternative polyadenylation signal was identified in the fourth intron, and its functionality was assessed. Although this additional site was predicted to have a low confidence score compared to that of the additional two, it might be inferred that there is a possible involvement Flt4 in the transcription of the messenger RNA encoding the secreted HAE form of pIgR. Concerning the gene locus corporation, the gene is definitely flanked from the (5383 nt upstream) and (21,790 nt downstream) genes, as demonstrated in zebrafish and additional teleost varieties [15]. In addition to the gene, a further gene of 6481 nt was recognized in another genomic scaffold (“type”:”entrez-nucleotide”,”attrs”:”text”:”NW_022988066.1″,”term_id”:”1835951300″NW_022988066.1). To investigate the evolution of the gene locus, we performed a comparative analysis by considering two Antarctic varieties belonging to the same Notothenoidei suborder as (Notothenidae family), (Bathydraconidae family), and (Channichthyidae family), as well as the.