Transcriptomic Analysis of Gill and Hepatopancreas in Razor Clam (Sinonovacula constricta) Exposed to Acute Ammonia

Abstract

Accumulation of excessive ammonia is a big threat to aquatic animals, which causes adverse effects on the health, production reduction, and even high mortality. The razor clam Sinonovacula constricta, a bivalve living in intertidal mudflat with a deep-burrowing lifestyle, often faces a high concentration of ambient ammonia. However, there is less available information concerning the toxic effects of ammonia on razor clam and its molecular mechanisms of adaptation to ammonia stress. The aim of this study was to investigate the effects of ammonia exposure on the gill and hepatopancreas of razor clam by transcriptome sequencing. The results showed that the median lethal concentration of ammonia was 244.55 mg/L for 96 h. A total of 1,415 and 306 differentially expressed genes (DEGs) were identified in the gill and hepatopancreas, respectively. The functional annotation showed that DEGs of the gill were mainly involved in the regulation of nitrogen compound metabolic process, nitrogen compound transport, and amide transport. The DEGs of the hepatopancreas were mostly enriched in oxidation-reduction process, response to stress, and amine metabolic process. The expression levels of NH3/NH4+ transporting channels and H+ excreting-related genes, including Rhesus glycoproteins (Rh), Na+/K+-ATPase (NKA), Na+/H+ exchanger, V-ATPase (VHA), and carbonic anhydrase (CA), were upregulated significantly in the gill (p < 0.05). In addition, the expression levels of glutamine and urea synthesis-related genes that played vital roles in ammonia detoxification, such as glutamine synthetase (GS), arginase (ARG), and argininosuccinate synthetase (ASS), were also increased obviously in the hepatopancreas (p < 0.05). Taken together, our results indicate that the synergistic action of ammonia excretion in the gill and ammonia metabolism in the hepatopancreas might be the mechanism through which the clams tolerate to environmental ammonia. This study provides a molecular basis for the better evaluation of the responding mechanism of ammonia tolerance.

Expression and Functional Analysis of AMT1 Gene Responding to High Ammonia Stress in Razor Clam (Sinonovacula constricta)

“…+ or NH 3 [11]. Here, the hemolymph ammonia concentration in the clams injected with NC or DEPC-W under 12.85 mmol/L ammonia stress showed similar values to that determined in our previous study (nearly 2000 µmol/L) [22]. However, there was a onefold increase in the hemolymph ammonia concentration after RNAi of Sc-AMT1, supporting the importance of Sc-AMT1 in ammonia transport.…”

“…Remarkably, as benthic animals are often faced with higher ammonia concentrations compared with pelagic animals, they have evolved several mechanisms to adapt to high ambient ammonia [19]. In fact, excellent ammonia tolerance has been found in many marine bivalves, especially S. constricta, which lives under 30-40 cm of mud in ponds with an extreme high ammonia concentration [22,29]. A majority of aquatic invertebrates are ammonotelic and excrete ammonia primarily in the form of NH 3 or NH 4 + , rather than relying on ammonia metabolism [19,30].…”

“…Before the challenge treatment, six healthy clams were dissected to investigate the distribution of Sc-AMT1 in different tissues including gill, hepatopancreas, intestine, siphon, mantle, foot, and adductor muscle (posterior and anterior adductor muscles). Based on the median lethal concentration (LC 50 -96 h) of ammonia-N (17.46 mmol/L) according to our previous toxicity tests [22], the clams were randomly divided into an experimental group (12.85 mmol/L NH 4 Cl) and control group (<0.02 mmol/L ammonia-N). Each group contained a total of 120 clams in three replicate tanks (40 clams in each tank).…”

“…For example, several investigations of seawater composition in intensive aquaculture ponds showed considerable concentrations of ammonia in a range between 100 and 500 µmol/L, but also up to 2.5 mmol/L in a sediment depth of 4-9 cm [19,20]. Therefore, the razor clam (Sinonovacula constricta), as a burrowing bivalve, is a suitable model species to explore the molecular mechanism of tolerance to ammonia in mollusks [20][21][22]. Genes such as glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamic oxaloacetic transaminase (GOT), glutathione S-transferase (GST), heat shock protein 90 (HSP90), and Rh showed distinct expressional patterns during continuous exposure to high ammonia in razor clams [22][23][24][25].…”

“…Therefore, the razor clam (Sinonovacula constricta), as a burrowing bivalve, is a suitable model species to explore the molecular mechanism of tolerance to ammonia in mollusks [20][21][22]. Genes such as glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamic oxaloacetic transaminase (GOT), glutathione S-transferase (GST), heat shock protein 90 (HSP90), and Rh showed distinct expressional patterns during continuous exposure to high ammonia in razor clams [22][23][24][25]. Furthermore, abundant expression of transport-, immune-response-, and apoptosis-related pathways was activated under ammonia stress.…”