Functional characterisation of TNFAIP3 (A20) in the pearl oyster Pinctada fucata martensii: RNA interference reveals a conserved negative regulator of NF-κB signalling and a modulator of apoptosis.

Introduction. The pearl oyster Pinctada fucata martensii underpins a high-value marine-pearl industry increasingly threatened by mass mortalities, yet the negative regulators that restrain its innate immune signalling remain almost unknown. In mammals, TNFAIP3 (A20) is the prototypical ubiquitin-editing brake on NF-κB, but its role in molluscs has never been tested. We report, to our knowledge, the first functional characterisation of TNFAIP3 in a pearl oyster.

Methods. We cloned PmTNFAIP3 and resolved its domain architecture, and then silenced it in vivo with gene-specific double-stranded RNA. Over a 96 h time course, we profiled canonical NF-κB-pathway genes (TNF-α, NF-κB, IKK, TRAF-3, IRAK-1) in the hepatopancreas by qRT-PCR, measured innate immune enzymes (CAT, ACP, AKP), and assessed apoptosis by TUNEL, against RFP-dsRNA and PBS controls.

Results. PmTNFAIP3 encodes a 1,120-amino-acid A20 orthologue with an N-terminal OTU deubiquitinase domain and seven C2/C2 zinc fingers. Silencing was maximal at 6 h with de-repressed NF-κB signalling: TNF-α rose to 6.1-fold and NF-κB (4.2-fold) and IKK increased (24–48 h), whereas the upstream adaptors TRAF-3 and IRAK-1 decreased (TRAF-3 to 0.18-fold), consistent with compensatory feedback. Catalase and acid phosphatase activities increased while alkaline phosphatase was unchanged, and TUNEL revealed elevated hepatopancreatic apoptosis, peaking at 6–24 h.

Conclusions. TNFAIP3 functions as a negative regulator of NF-κB and a modulator of apoptosis in P. fucata martensii, indicating that the A20 NF-κB negative-feedback axis is conserved in molluscs. These findings provide a mechanistic basis for managing immune balance and health in cultured pearl oysters.