Halophilic and halotolerant organisms are capable of adapting to high salinity waters and maintaining their internal osmotic balance in order to thrive in harsh environments. In general, the salt-in and salt-out processes are regarded as two distinct osmoregulation strategies. Salt-in organisms typically utilize potassium as the main intracellular cation in high salinity waters, while salt-out organisms accumulate organic solutes (e.g., betaines, glycerol, ectoine, sucrose, and mannitol) as the main osmolytes [17, 59]. In addition, salt-in organisms characteristically contain acidic proteins with a negative charge, which are not observed in the salt-out organisms, to avoid protein aggregation [17, 60]. In the present study, Artemia (class Branchiopoda) may be considered a salt-in organism due to increased Na+/K+-ATPase activity in high salinity water (230 psu). However, the notable absence of acidic protein profiles in the brine shrimp Artemia was identical to other crustaceans in seawater. Thus, our transcriptomic analysis confirms that A. salina is a strict hyporegulator [24–26, 61].
In crustaceans, the enzyme Na+/K+-ATPase serves to maintain sodium and potassium ion homeostasis across cell membranes [62–65]. Several studies have used the silver staining method to demonstrate that the metepipodites, digestive gut, and maxillary glands in A. salina exhibit high Na+/K+-ATPase activity under highly saline conditions [24, 26, 61]. This confirms that the enzyme Na+/K+-ATPase plays an essential role in A. salina osmoregulation at the genetic level. Interestingly, genes related to beta-mannosidase and other enzymes involved with mannose metabolism were up-regulated in high salinity media. This result implies that A. salina may accumulate mannose or mannose derivatives as organic osmolytes. Mannose and mannitol are widely recognized as organic osmolytes in diatoms, brown algae, green algae, and terrestrial plants [66, 67]. However, most marine animals accumulate betaine, taurine, trimethylamine oxide, glycine, alanine, proline, homarine, or arginine as organic osmolytes [68]. Thus, the accumulation of mannose and mannose derivatives at high salinities in A. salina is particularly interesting. Alternatively, considering that glucose and galactose-related activities were suppressed at high salinities, mannose may be the primary sugar involved in the glycolytic pathway under such conditions. Furthermore, A. salina down-regulated UDP-glucose:hexose-1-phosphate uridylyltransferase (synonym: galactose-l-phosphate uridylyltransferase), which converts galactose-1-phosphate into glucose-1-phosphate, at high salinities. This result further supports the theory that mannose is the main energy source used by A. salina in highly saline environments. Bunn & Higgins [69] reported that organisms that accumulate high concentrations of aldohexoses with unstable ring structures (i.e., mannose and galactose) may experience enzymatic malfunctions as a result of covalent modifications to individual proteins. Therefore, the covalent modification of A. salina proteins may also be detected in high salinity waters. Furthermore, Horst [70] noted that mannose and mannose derivatives in A. salina are substantially involved in glycoprotein catabolic processes, suggesting that exposure to increased salinity may result in an increased activity of mannose-related processes. Investigation of the primary role of mannose and mannose derivatives in A. salina under various salinities is warranted.
While Artemia can maintain ion homeostasis in its cells as the external salinity increases, more energy is required for growth and reproduction [71]. Theoretically, the dissolved oxygen (DO) concentration in 35 psu media at 25 °C is 3.2 times lower than that in 220 psu media [72]. In this study, we present several pieces of evidence that suggest that low oxygen may substantially impact the growth and reproduction of A. salina when salinity increases from 35 psu to 230 psu. Additionally, marine invertebrate vision is one of the most energetically demanding functions and is highly susceptible to dissolved oxygen fluctuations [73]. In high-salinity waters, transcripts related to retinal metabolic processes were significantly up-regulated; A. salina significantly up-regulated ninaB homologous genes in high-salinity media. Many diverse metazoan species contain ninaB homolog genes (Figure S3 in Additional File 3), which are associated with the synthesis of visual pigment and oxidative stress [74–76]. Moreover, the up-regulation of genes related to oxidative stress suggests that A. salina can adapt to waters simultaneously high in salinity and low in oxygen. The activity of N-acetylgalactosamine-4-sulfatase, which was one of the down-regulated genes in A. salina, can decline when exposed to low oxygen [77]. Therefore, it is likely that the gene expression patterns of A. salina are greatly affected by low dissolved oxygen levels in its media.
Few mitochondria and a thin body surface cuticle are characteristic of Artemia in low salinity waters, whereas many mitochondria and a thick cuticle have been observed in high salinity waters [23, 27, 54]. In this study, transcripts related to mitochondrion morphogenesis and glycoprotein catabolic processes were up-regulated at high salinities, which is consistent with previous investigations. It is reasonable to infer that the rate of mitochondrion morphogenesis is inversely related to the dissolved oxygen concentration in saline waters. Meanwhile, the increased thickness of the A. salina cuticle associated with glycoprotein catabolic processes lowers the water permeability of the body surface, resulting in a relatively low rate of oxygen diffusion [23, 54]. Thus, A. salina may require more mitochondria in high salinity than low salinity environments. Most sugar (i.e., glucose and galactose) transport systems, as well as those for nucleotides and amino acids, were repressed in A. salina at high salinities. These results imply that Artemia may be unable to properly use these essential macromolecules for growth and reproduction under highly saline conditions. Even though Artemia spp. are extremely halotolerant and euryhaline, significant energy expenditures are likely required to accommodate these adaptations. However, the increased thickness of A. salina body surface layers at high salinities indicates that the reduction in permeability to water, oxygen, and essential nutrients occurs passively. Moreover, several studies have reported decreased protein, carbohydrate, and glycogen contents in some crustacean species with increasing salinity [78–80].
The cellular response of A. salina required for acclimation to intermediate salinities depends on the number and type of genes expressed. A U-shaped gene expression pattern observed along the salinity gradient in A. salina implies that it can adapt well to intermediate salinities [81, 82], whereas an inverted U-shaped pattern indicates the presence of salinity response at intermediate salinities [83]. At 150 psu, 411 genes were expressed at low points in U-shaped patterns and only 63 genes were expressed at high points in inverted U-shaped patterns, implying that A. salina is well-adapted to 150 psu; most genes were actively expressed to tolerate this salinity. Furthermore, the U-shaped expression patterns observed in A. salina represent cellular responses related to cellular signaling, catabolic processes, morphogenesis, and development. Meanwhile, the inverted U-shaped expression patterns were related to diverse environmental response factors (e.g., salt aversion, sensory perception of salty taste, cellular response to light intensity, UV-B, ozone, response to oxidative stress, and starvation). Based on enriched GO terms and KEGG metabolic pathways, the functional annotations of the U-shaped expression pattern genes were usually different from those of the inverted U-shaped expression pattern genes (Fig. 4; Table S6 in Additional File 2; Figure S4 in Additional File 3). This result indicates that the genes related to U-shaped and inverted U-shaped patterns are unequally expressed, and the expression pattern observed at a specific salinity is related to the physiological characteristics of A. salina.
Within the genus Artemia, the gene expression patterns we observed with increasing salinity were not always consistent with previously identified patterns (Fig. 5; Table S7 in Additional File 2). Intriguingly, the types of genes displaying U-shaped and inverted U-shaped expression patterns were usually differentiated at each intermediate salinity (i.e., 50, 100, and 150 psu). These results demonstrate that A. salina could be a potential model organism to study locally adapted populations called ‘local adaptation’ [19, 84, 85], and differential expression patterns of Artemia are likely to fluctuate at the population level. Campillo et al. [85] reported that rotifer populations (Brachionus plicatilis) might present different cellular responses depending on the salinity of their medium. Thus, the gene expression of A. salina could also fluctuate depending on the occurrence of ecological specialization in each population at specific salinities. Further study is needed to confirm that A. salina can provide a model for local adaptation.