Fig. 4: The digestion of shell CaCO₃ by the conchocelis and the function of extracellular carbonic anhydrase.

a Boreholes in the calcareous matrix of mollusk shells occupied by Pyropia conchocelis. b Microscopic image of conchocelis living in mollusk shells. The red arrow indicates the conchocelis in the borehole in shell calcareous matrix. Microscopic observation in a and b were repeated independently on multiple shell conchocelis (n > 10) showing similar structure. c The variation of Ca²⁺ release from conchocelis-dwelling shell w/o inhibitors of carbonic anhydrase or P-type transporter. Triplets were done for each condition. Ca²⁺ concentrations in control medium, growth medium, CA inhibitory medium, and transporter inhibitory medium were plotted in gray, red, blue, and yellow, respectively. Time refers to the duration (in days) since the addition of inhibitors. The following comparisons were done: growth vs control, growth vs CA inhibitory or P-type ATPase inhibitory medium. Vertical bars represent the SD (n = 3 biological independent samples). P value in each comparison was calculated by two-sided independent t test in SPSS. Variations with P < 0.05 were considered to be significant as indicated by asterisks. One asterisk: 0.01 < P < 0.05; two asterisks: P < 0.01. d The abundance of ¹³C isotope in organic acids of TCA cycle in conchocelis co-cultured with ¹²C-CaCO₃ or ¹³C-CaCO₃. Columns and vertical bars represent the mean and SD, respectively (n = 3 biological independent samples). Statistical analysis was done by two-sided independent t test in SPSS. Two asterisks indicated P < 0.01. e A working model for the dissolution of shell CaCO₃ and utilization of generated carbon flux in conchocelis. Only the apical conchocelis cell and part of its adjacent cell are shown in this model. Extracellular CA was represented by purple ovens. Transporters or channels of bicarbonate, CO₂, Ca²⁺, and protons are indicated by green, light green, blue, and gray cylinders, respectively, across the cell membrane. The P-type calcium transporter is indicated by the dark blue circle. Source data underlying Fig. 4c, d are provided as a Source data file.