Pessimum range protein network regulation
The energy metabolism protein network associated with the pessimum range consists mainly of mitochondrial proteins. Two of these, cytochrome b-c1 complex subunits 7 and Reiske, where also found to be strongly correlated with blood osmolality, indicating that these proteins may be rate-limiting or particularly sensitive to salinity. Cytochrome b-c1 complex subunit 7 was the most highly upregulated mitochondrial protein and ATP/ADP translocase was also significantly upregulated in O. niloticus gill near the upper salinity tolerance for this species (25 g/kg) (Root et al., 2021a). This observation may indicate that 25 g/kg already represents the pessimum range for the strain of O. niloticus used in this previous study.
Structural changes in the pessimum range include downregulation of ECM proteins including collagen subunits, and proteins forming the connection between the ECM and cell membrane, e.g., integrin-α. Most of the proteins whose abundances were highly correlated with blood osmolality were significantly regulated following extended time in the pessimum range, and these proteins were all involved in determining cellular structure. Within this network of highly correlated proteins is a group of Serpins, as well as multiple proteins involved in focal adhesion connections between the cell membrane and ECM. Serine proteases were the most highly downregulated ontological category in gills ofO. niloticus acclimated to high salinity (Root et al., 2021a), whereas in the present study serine protease inhibitors were downregulated. Clearly serine protease action is important for osmotic regulation of gill protein networks. These proteins are connected by STRING networks to structural and ECM proteins, and they are highly responsive to the intensity of salinity stress rather than being uniformly regulated during hypersaline exposure. Structural proteins were a functional category with a high degree of non-linearity in regulation of mRNA and proteins in O. niloticus , further indicating that cell structure regulation is complex and likely fluctuates in response to internal and external signaling, especially around the critical threshold.
Changes in ionocyte numbers and composition in the gill epithelium lead to changes in overall structure of the tissue. O. mossambicus has dramatically reduced epithelial permeability as salinities increase above SW (Kültz & Onken, 1993). Ionocytes involved in osmoregulation in high salinity environments have unique “deep-hole” apical crypts in comparison with other types of ionocytes (Fridman et al., 2013; Lee et al., 2000). In high salinity, ionocytes form cell specific clusters (Inokuchi & Kaneko, 2012), and the gill epithelium develops a complex microtubule network along the basolateral membrane layer (Karnaky, 1986). The formation of tubulin networks in response to salinity was first noted decades ago in Cyprinodon variegatus andFundulous heteroclitus (Karnaky, 1986; Karnaky et al., 1976). Although they were not found in the STRING network, tubulin α-1A and α-1B chain both decreased in a highly correlated way with increasing blood osmolality (significantly downregulation of both in extended 105g/kg treatment, and of α-1A for extended 85g/kg). On the other hand, tubulin α-1C chain and tubulin β were both significantly upregulated in 85g/kg and 105g/kg treatments at MP and significantly downregulated in the 75g/kg treatment. This is interesting in itself, but also provides context for the highly downregulated uncharacterized protein we have identified as fucolectin-like. While the role of lectins is not fully understood in fish physiology (Elumalai et al., 2019), they likely play an important role in the development of microtubule networks in response to increased salinity. Binding sites have been identified on exposed gill epithelium which interact with the lectins wheat germ agglutinin (WGA), peanut lectin agglutinin (PNA), and concanacalvin A (ConA) (Hirose et al., 2003). WGA and PNA only react with FW specific ionocytes in O. mossambicus (WGA) (Tsai & Hwang, 1998a) andOncorhynchus mykiss (PNA) (Goss et al., 2001). WGA exposure was shown to stimulate Ca+ ion uptake in O. mossambicus and promote microtubule network formation, and binding was more prevalent in O. mossambicus adapted to Ca+deficient water (Tsai & Hwang, 1998b). The uncharacterized protein, along with rhamnose binding lectin which was also one of the most highly downregulated proteins in our data set, may be involved in changing the composition of tubulin-based cell structures, likely to reduce Ca+ uptake. This would help control internal ion concentration and impact cell-cell adhesions through cadherin binding, which is also impacted by the high upregulation of δ-catenin 1.