**3. Results**

#### *3.1. Sequence Analysis and Antibody Specificity*

The structure of the gene consisted of 13 exons and 12 introns (Figure 1A). Due to the third round of whole-genome duplication in teleost, two paralogues are present in the genome of *N. furzeri*: NUCB2A and NUCB2B. Sequences were obtained from the *Nothobranchius furzeri* Genome Browser–NFINgb (https://nfingb.leibniz-fli.de/): accession number was Nfu\_g\_1\_003870 for NUCB2A and Nfu\_g\_1\_018131 for NUCB2B [34]. Even if the differences between NUCB2A and NUCB2B are very slight, we designed primers to detect only NUCB2B, since it has a higher evolutionary conservation towards mice or humans (Figure 1B). As a result, the primers for generating the ISH probe as well as the qPCR primer bound only to the NUCB2B transcript and the synthetized probe showed 100% identity to NUCB2B (in total 417 nucleotides, transcript position 210 to 626), whereas the identity to NUCB2A was only 36% (in total, 154 nucleotides: transcript position 247 to 341 with 16 mismatches, position 358 to 402 with 6 mismatches, and position 535 to 575 with 5 mismatches).

In *N. furzeri*, like in other fish, Nesf-1 is an 81 amino acid anorexigenic peptide (82 in mammals) encoded in the N-terminal of its translated precursor, nucleobindin-2 (NUCB2). *N. furzeri* Nesf-1 protein sequence showed an overall identity of 78% with Nesf-1 of medaka, 73% with zebrafish, 60% with mouse, and 58% with human (Figure 1C). The antibody specificity was tested by western blot. Currently, there are no commercially available antibodies raised against fish sequences and thus, we employed a polyclonal antibody raised against human Nesf-1 (1–45), recognizing a region highly conserved and corresponding to the bioactive segment of the neuropeptides [47]. The western blot revealed a defined band of −40 kDa in *N. furzeri* brain homogenates of young and old fish (Figure 1D). β-actin, used as an internal marker, showed a band of about 42 kDa.

**Figure 1.** NUCB2B gene structure and Nesfatin–1 peptide analysis: (**A**) NUCB2B intron/exon gene structure of *N. furzeri* and alignment of transcripts from other fish species (downloaded and modified from http://nfingb.leibniz-fli.de/, transcript information for the other fish species can be found at http://www.ensembl.org/). The structure of the two paralogues NUCB2A and NUCB2B were highly similar. In the image, NUCB2B is shown as a template. (**B**) The evolutionary history of NUCB2B peptide sequences was inferred by using the Minimum Evolution method. The phylogenetic tree of amino acid sequences was reconstructed by MEGA X. (**C**) NUCB2B peptide sequence alignments in different species: *Nothobranchus furzeri* (killifish), *Oryzias latipes* (medaka), *Carassius auratus* (goldfish), *Homo sapiens* (human), and *Mus musculus* (mouse). Asterisks mark conserved amino acids (alignment was done with Clustal Omega http://www.ebi.ac.uk/Tools/msa/clustalo/). (**D**) Western blot in the brain of young and old killifish showing an immunoreactive band of about 40 kDa.
