CBFA2T3 affects red blood phenotype in the blood clam Tegillarca granosa by regulating hemocyte proliferation

Abstract

The red blood phenotype of blood clam Tegillarca granosa, is tightly linked to its health conditions and nutritional benefits, making it an economically important trait. Despite its significance, the molecular regulatory mechanisms responsible for red blood phenotype variation in blood clams still poorly understood. Our previous study shows that hemocyte proliferation may affect hemoglobin concentration (HGC) and cause the red blood phenotype variation in T. granosa. Thus, we defined total hemocyte counts (THC) and HGC as red blood-related traits. In this research, we discovered that blood clams with higher THC exhibited elevated HGC and a darker blood color. To gain insights into the genes associated with THC in T. granosa, we conducted RNA-Seq analysis. Our transcriptome analysis identified 682 genes exclusively highly expressed in high THC clams (HTHC:THC ≥ 15.5 × 104 cells/mL), and 709 genes highly expressed in low THC clams (LTHC:THC ≤ 6.5 × 104 cells/mL). Furthermore, the weighted gene co-expression network analysis (WGCNA) revealed that the grey60 module exhibited significant positive correlations with THC and HGC, implying an association of this module with the red-blood phenotype. Subsequently, the red blood-related module exhibited a significant positive correlation with gill tissue, providing an alternative perspective to support the hypothesis that gills serve as the primary hematopoietic organ in T. granosa. By employing differential expression analysis and WGCNA, CBFA2T3, TAL1, and FLI1 are potentially key genes involved in the regulation of hemocyte proliferation and differentiation. RNA interference of CBFA2T3 gene led to a remarkable decrease in HGC and THC in T. granosa, resulting in a lighter blood color. EdU staining further confirmed the decrease of cell proliferation activity in the gills of T. granosa after knockdown of CBFA2T3. This study enhances our apprehending of the molecular mechanisms in red blood phenotype variation in T. granosa, and potential contributes to the selection breeding of high THC blood clams.