*2.4. Phylogenetics Analysis*

Orthologs of Nfu NT-6 were recovered from Genbank by querying Genbank translated nucleotide sequences with the translated cDNA of NfuNT-6. *D. rerio* glial derived neurotrophic factor (GDNF) sequence was selected as outgroup. All phylogenetic analyses were performed using MEGA X [28]. The analysis involved 17 amino acid sequences of different fish species (differently named as neurotrophin-6/7-like and nerve growth factor-like) and *Homo sapiens* and *Mus musculus*. The most appropriate amino acid substitution model was selected based on Akaike Information Criterion (AIC). Phylogenetic tree was reconstructed by maximum likelihood analysis using a partial deletion (80%) setting, JTT with gamma function and invariant sites as substitution model, and bootstrap analysis.

#### *2.5. Quantitative Real Time-PCR*

NfuNT6 primers were designed with Primer3 tool [29]. According to the sequence information, one set of primers was designed to quantize NfuNT-6 cDNA: left 5- -GCATTCGTTGAAGTCTGGCT-3- ; right 5- -ATCAGGAAGAGCAGGACCAG-3- . Reactions were performed in 20 μL volume containing <sup>1</sup> <sup>μ</sup>L of diluted cDNA, using BrightGreen 2<sup>×</sup> qPCR MasterMix kit (abm®, Richmond, VA, Canada) following the manufacturer's instructions. Reactions were performed in triplicate and negative control (water) was always included.
