Hypoxia activates a variety of complex pathways at both the cellular and organism level, with the ultimate aim of reinstating oxygen homoeostasis. In the past decades, physiological and biochemical responses to hypoxia have been studied in several marine bivalve species [26,28,39,40]. Adenosine–5-triphosphate (ATP), the major energy source for cells in the body, is predominantly supplied by a series of metabolic pathways including glycolysis, the citric acid cycle, and the electron transport chain [41]. Among these, glycolysis occurs (with variations) in nearly all organisms, both aerobic and anaerobic. The wide occurrence of glycolysis indicates that it is one of the most important energy metabolic pathways [42]. It provides not only high-energy compounds like ATP, but also pyruvate, which can be used in the citric acid cycle to generate more ATP, NADH, and FADH2 [43–45]. In this study, the enzymatic assays of LDH indicated that the hepatopancreas of R. philippinarum switch to anaerobic metabolism at 8 d under hypoxia at 15 °C, while the SDH activity is likely to related to mitochondrial aerobic energy production, suggesting that aerobic and anaerobic metabolism were both existed in R. philippinarum. When Manila clams are under hypoxia, their shell valves are frequently closed, likely undergoing low oxygen consumption and reduced metabolisms [46]. The activity of LDH is closely related to cell metabolism, and its activity is often used as an indicator to evaluate the level of anaerobic metabolism [47]. As shown in Figure 8A&B, the level of dissolved oxygen in the water is insufficient with the hypoxia exposure time increased, and the clams may depend on anaerobic respiration to supply the metabolic energy, which is strongly reducing their energy requirement, so the activity of SDH and LDH decreases with prolonged hypoxia challenge. The different changes in the activities of key enzymes involved in glycolysis may indicate a diverse strategy of shellfish under hypoxia [29].
The hypoxia condition is a challenging situation because the energy production is drastically decreased in anaerobic metabolic pathways compared to aerobic energy provision. The decrease in metabolic rate at hypoxic environment is mainly caused by two mechanisms, including the depression of ATP-requiring processes and the inhibition of ATP-generating pathway. The reduction of the metabolic rate under hypoxia challenge provides benefits for facultative anaerobes. The glycogen stores of Mytilus edulis would only be enough for 3 days to provide ATP by anaerobic fermentation. However, mussels are able to sustain their life at anoxic conditions for weeks by reducing energy requirement [48]. The Manila clam exhibited strong tolerance to hypoxia as the 20-day LC50 for dissolved oxygen (DO) was estimated to be 0.57 mg L–1, and the LT50 at 0.5 mg L–1 DO was 422 hours.29 In this study, AKP activity in hepatopancreas was significantly increased at 5 d in response to hypoxia stress, indicating that the hepatopancreas is an important organ that involved in the immune response and hypoxia regulation in R. philippinarum. In the gills, however, no significant change in AKP activity observed between hypoxia challenge and control groups during 8 days, suggesting a tissue-specific response exist in R. philippinarum, which was also reported in C. gigas [1].
Previous studies explored how environmental changes are transduced into coordinated changes in physiological processes by examining gene expression cycles using microarray in intertidal bivalves [49–54]. More recently, transciptomic profiling is widely employed in elucidating the genetic basis of molecular response to different environmental factors in marine bivalve [55,56]. In this study, a transcriptomic approach was firstly employed to investigate the gene expression patterns of the Manila clam under hypoxic challenge. Hypoxia responsive genes associated with stress response, substance and energy metabolism, and immune response were corroborated by qPCR. In this study, all of the 14 DEGs detected by qPCR were consistent with the RNA-seq results. The expression profiles of these genes were characterized at different hypoxia exposure duration (0, 2, 5, and 8 d). These genes reflect different aspects of the stress adaptation and molecular mechanisms of immune response to hypoxia exposure. The KEGG analyses of DEGs show that molecular pathways related to TNF signaling, NOD-like receptor signaling, RIG-I-like receptor signaling, TGF-beta signaling AMPK signaling, NF-kappa B signaling, cAMP signaling pathway, and apoptosis are highly enriched, implicating these processes are important for physiological adaptation and immune response to hypoxia stress in R. philippinarum. Manila clam may have evolved various sophisticated signaling pathways to sense their immediate environment and orchestrate appropriate transcriptional responses that mediate adaptation in benthic environment.
Aerobic organisms have the antioxidant mechanisms to prevent the oxidative damage and protect them against oxidant stress. This biological process involve in a number of cellular molecules and antioxidant enzymes [51]. In the last decade, however, most studies in marine bivalves investigated the activities of the antioxidant enzymes, but only a few studies reported both the biochemical responses and molecular actors governing the antioxidant following hypoxia [57,58]. In this study, we observed a rapid regulation of a set of genes involved in the molecular responses to oxidative stress. The cytochrome P450, peroxisomal membrane protein (PEX), and Glutathione peroxidase (GPx) were both upregulated under hypoxia challenge. Peroxidase, an important class of antioxidation enzymes, is responsible for the defense response to oxidative stress and degradation of ROS [59]. The expression of GPx mRNA increased under hypoxia challenge to protect cells from ROS that can be formed upon reoxygenation, which has been reported in C. gigas [1,8]. Stress-induced immune changes have been reported in many marine invertebrates, including oyster [60,61] and mussel [62,63]. The innate immunity is the mainly immunological defense mechanism in invertebrate metazoan [64]. In the present study, several immune-related genes such as the defensin, IAP and HSP70 were up-regulated under the hypoxia challenge, demonstrating that these genes are involved in defense and immune response. In addition, focal adhesion, NF-kappa B signaling pathway, apoptosis, TNF signaling pathway were enriched by KEGG analysis, suggesting that these pathways play significant roles in the immune response and defense mechanisms against hypoxia stress.
Hypoxia inducible factors (HIFs) are a family of highly conserved transcription factors that act as main regulators of oxygen homeostasis and the adaptive response to hypoxia [65]. NF-κB signaling pathway has been proved to be involved in innate immune response to bacterial infection and hypoxia stress in molluscan shellfish, including R.philippinarum [66], Meretrix meretrix [67], C. gigas [68], and H. diversicolor [17]. It is indicated that genes associated with NF-κB signaling pathway participated in the immunomodulation process to respond to hypoxia stress [17]. Therefore, we focus on the HIF–1 and NF-kappaB signaling pathways in the present study. Nuclear factor kappa B (NF-κB) is a transcription factor regulates the expression of cytokines, effector enzymes and apoptosis inhibitors in response to extracellular signals. NF-κB signaling plays a critical role in immune defense, stress responses and inflammation. NF-κB comprises RelA (p65), RelB, c-Rel, NF-κB1 (p50) and NF-κB2 (p52). NF-κB can stimulate transcription of its target genes in a very quick fashion, as it exists freely in the cytoplasm, albeit inhibited by IκB proteins. In the current study, NF-kB is upregulated, which could increase the HIF–1α mRNA level, as well as the PI3K and p70S6K are upregulated in PI3K-Akt signaling pathway.
Enolase is a hypoxic stress protein, which may contribute to hypoxic tolerance by increasing anaerobic metabolism [69]. As one the downstream genes of the HIF signaling pathway, the enolase (ENO1) is significantly upregulated in the hypoxia challenged clams, which promote anaerobic metabolism and causing reduced oxygen consumption. Concomitantly, hypoxia induced NFκB activity is preceded by temporally sequential IKK activation, IκB phosphorylation, and IκB degradation, indicating that hypoxia activates NFκB through increased IKK activity (Figure 7). The IKK-dependence of hypoxia-induced NFκB activity is consistent with most other stimuli of this pathway [70]. The role of IKK in oncogenesis and inflammation is well established, but there is emerging evidence that IKK may have antiinflammatory functions [71,72]. In this study, we also observed an increase in IKKβ in hypoxia (Figure 7). The observation that IKKβ are hypoxia-sensitive, suggests the possibility of a molecular mechanism where a p50- mediated response could be resolved over a course of hypoxia [70]. Therefore, NF-κB plays important roles in regulating genes responsible for both the immune response and hypoxia stress resistance.