A wide range of changes in the MW/pI of Ni-Sp185/333 proteins can occur over time and in response to multiple challenges with different types of microbes.

<p>(A) Prior to immune challenge, sea urchin 107 shows two major trains of Ni-Sp185/333 proteins of ~80 and ~150 kDa (arrows). (B) After the first challenge with <i>V</i>. <i>diazotrophicus</i>, the majority of the proteins in the trains shift to more basic (arrows), with an expansion of acidic proteins within the same trains (black arrowheads). (C) Prior to challenge with <i>Bacillus sp</i>, sea urchin 107 shows a wide repertoire of Sp185/333 proteins, particularly those of ~60 to 80 kDa and pI of ~7 to 8 (white box). (D) After challenge with <i>Bacillus sp</i>, there is a slight decrease in the intensity of the Ni-Sp185/333 proteins, particularly those of ~60 to 80 kDa/pI ~6 to 8 (white box). (E) The second challenge with <i>Bacillus sp</i> results in a decrease in the intensity of the repertoire of Sp185/333 proteins. (F) The third challenge with <i>Bacillus sp</i> further decreases the repertoire and intensity of the Ni-Sp185/333 proteins. (G) Two weeks after challenge with <i>Bacillus sp</i> the Ni-Sp185/333 protein repertoire shows a further decrease in the number of spots and their intensity. (H) Subsequent challenge with <i>V</i>. <i>diazotrophicus</i> does not induce an increase in the Ni-Sp185/333 protein repertoire, but shows further decreases in diversity. Images (A, B, E-H) were cropped at the bottom because they do not show spots of less than 40 kDa.