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.