In the present study, we conducted interspecies comparisons of head kidney proteomes between one icefish (CH) and two red-blooded notothenioids (TB and NC) by using TMT labeling and LC-MS/MS. We compared the expression profiles and described the differences of 123 upregulated proteins and 95 downregulated proteins. The comparison of the expression patterns of the teleost head kidney proteomes showed that the expression of erythrocyte-related proteins such as β-Spectrin, tfr1a, and hemoglobin was significantly downregulated in icefish; however, lymphoid and megakaryocytic lineage marker proteins, including CD9 and ITGB2, were significantly upregulated. An interesting point to note is that the lymphoid and megakaryocytic lineage marker proteins were not affected by the absence of erythrocytes and they were even upregulated in the icefish head kidney (Table 1). It is known that CD9 and ITGB2 play important roles in the development of B cells (Wang et al. 2020; Yang et al. 2020), and B lymphocytes are a subgroup of white blood cells that can differentiate into antibody-secreting cells. CD9 promotes the recruitment of CD10 (a protease involved in the maturation and migration of B cells (Wang et al. 2020)) in exosomes (Navarro-Hernandez et al. 2020). CD9 is not expressed in primitive or memory B cells in mammals, but is expressed in plasma cell lines (Yoon et al. 2013) and in “innate-like” B lymphocytes (Won and Kearney 2002).
CD9 has also been found in multiple aquatic species, including rainbow trout and Atlantic salmon (Fujiki et al. 2002), red stingray (Zhu et al. 2006), shrimp (Wang et al. 2010), and lamprey (Wu et al. 2012). The fact that CD9 is constitutively expressed in trout primitive B cells indicates that a high proportion of primitive fish B cells is the equivalent of mammalian innate B cells (Zhu et al. 2014). ITGB2 is also an important immune effector that plays a vital role in protecting fish from a variety of pathogens (Wang et al. 2020). CD9 also plays an important role in platelet generation (Kaprielian et al. 1995; Aurora et al. 2018). Consistent with our results, Consistent with our results, Xu et al. (2015) has been reported that genes related to erythropoiesis and heme biogenesis are downregulated in the kidney of ice fish. While genes related to leukocyte differentiation and development, platelet activation, aggregation, and formation are upregulated (Xu et al., 2015). The proteomics analysis of the total protein in the head kidney of three Antarctic fish further confirmed that this general expression trend may cause a severe reduction in red blood cell differentiation of CH, while B cells and platelet cells are relatively up-regulated.
The major function of erythrocytes is not only to deliver oxygen to the organs, but they are also involved in innate immune responses, as they can capture specific immune complexes such as certain pathogens and bacteria, partly through membrane electrostatic attraction, and then kill them in the liver and spleen by presenting the pathogens to Kupffer cells and antigen-presenting cells (APCs)(Ukidve et al. 2020). In our study, the number of erythrocytes in the icefish was found to be severe reduced probably due to changes in the immune response; however, at present, we are unsure whether this change is more harmful or more beneficial. A comparison of the mucus microbial communities between white-blooded CH and red-blooded TB individuals sampled from the same locations showed much higher levels of bacterial species diversity in the CH mucus samples (data not published). To our knowledge, the fish skin mucus is a viscoelastic, adhesive gel that covers the exposed skin. The skin and mucus layer of fish are regarded as the first line of defense and play an important role in preventing the entry of pathogenic bacteria and similar harmful substances (Guellec et al. 2003; Laura et al. 2012).The low temperature of the surrounding environment, however, results in inhibition of the inflammatory responses (Finn and Nielsen 2010),including phagocytosis and acquired immune responses. Therefore, it remains unclear whether the increase in the number of mucus microorganisms indicates the increased susceptibility of the CH immune system to attack by microorganisms or whether it represents the ways through which the fish improves its acquired immunity; further studies are needed to confirm this aspect.
The present study mainly discusses the DEPs from total proteins of head kidney of three Antarctic fish species by comparing the significant differentially expressed proteins of red-blooded TB and NC and white-blooded CH. This study found 218 DEPs that could be used for further analysis of their related genes to provide a basis for further studies on the occurrence of red blood cells in icefish. Because there are almost no red blood cells in the Antarctic icefish, we found that among the three fish species, the expression of proteins is important role in B-cell production and platelet cell development in the hematopoietic cell lineage was significantly upregulated CH. The results of this study can serve as good references for further research and exploration of the immunity and hematopoiesis of the Antarctic icefish.