The paired-box 3 (Pax3) is a transcription factor and it plays an important part in melanin synthesis. In this study, a new Pax3 gene was identified from Pteria penguin (Röding, 1798) (P. penguin) by RACE-PCR (rapid-amplification of cDNA ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpPax3 was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed PpPax3 shared the highest (69.2%) identity with Pax3 of Mizuhopecten yessoensis. Tissue expression profile showed that PpPax3 had the highest expression in mantle, a nacre-formation related tissue. The PpPax3 silencing significantly inhibited the expression of PpPax3, PpMitf, PpTyr and PpCdk2, genes involved in Tyr-mediated melanin synthesis, but had no effect on PpCreb2 and an increase effect on PpBcl2. Furthermore, the PpPax3 knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase (Tyr) knockdown. These data indicated that PpPax3 played an important regulating role in melanin synthesis by Tyr pathway in P. penguin.

MKK6 is a key protein kinase that regulates inflammatory response under pathogen infection by activating MAPK signal pathway. However, the function of MKK6 remained unclear within oysters. Our research recognized a novel KK6 gene from C. hongkongensis , which is named ChMKK6 . The cDNA of ChMKK6 gene was 2,760 bp in length, containing an ORF of 1,032 bp encoding 344 amino acids, with a conserved S-TKc region and a DVD region. As illustrated by amino acid alignment and phylogenetic tree, ChMKK6 shared the maximal (99.0%) identity showing MKK6 of Crassostrea gigas. As indicated by tissue expression analysis, ChMKK6 possessed the maximal expression within hepatopancreas, and was markedly increased in all tissues following Vibrio Parahaemolyticus ( V. Parahaemolyticus ) challenge. As stated by the qRT-PCR analysis, ChMKK6 silencing significantly suppressed IL17 transcription, suggesting its involvement in innate immunity by regulating IL17 -mediated inflammatory response. Moreover, ChMKK6 silencing led to the suppression of key immune-related enzyme activities (ACP, AKP, CAT, LZM, and SOD), while raising the MDA content in gill. In addition, the antimicrobial viability of hemolymph supernatant was markedly reduced by ChMKK6 silencing. These results collectively demonstrate that ChMKK6 is a key player in immunity defense of C. hongkongensis from V. Parahaemolyticus.