Characterization and functional analysis of AgRunx in the blood clam (Anadara granosa): implications for hemocyte proliferation

The Runx family of transcription factors, evolutionarily conserved regulators of vertebrate hematopoiesis, coordinates hematopoietic lineage commitment through DNA-binding Runt domains. While their roles are well-characterized in vertebrates, functional insights into bivalve hemocyte proliferation remain limited. Here, we identified a Runx homolog (AgRunx) in the blood clam Anadara granosa, encoding 556-amino-acid protein with a conserved Runt domain. Spatial expression analysis revealed predominant AgRunx transcription in hemolymph (10.12-fold vs. gonads, p<0.05), with dynamic upregulation peaking during the D-shaped veliger developmental stage. Furthermore, AgRunx was significantly up-regulated at both the mRNA and protein levels in the hemolymph of A. granosa with high total hemocyte count group (HTHC: 32.70±2.36×104 cells/μL), compared to individuals with low hemocyte count group (LTHC: 7.90±0.85×104 cells/μL). Following AgRunx RNA interference (RNAi) showed both the total hemocyte count (THC) and hemoglobin concentration (HGC) were significantly reduced. Moreover, a decrease in the number of EdU-labeled proliferating cells in gill tissues was observed, accompanied by down-regulation of the G0/G1 phase and up-regulation of the G2/M phase in hemocytes. These results conclusively demonstrate AgRunx is involved in the hematopoietic process of A. granosa and plays a critical role in cell proliferation.