In the case of rice crab symbiosis, the concentration of ammonia in the water may increase due to the application of nitrogenous fertilizers, adversely affecting the health status of E. sinensis[4]. Moreover, the gills of E. sinensis play essential role in osmoregulation, immunity, and ammonia excretion[12]. Therefore, we selected the gills as the research object of the response of E. sinensis to ammonia stress. We analyzed histopathological evaluation which revealed that tissue damage was more serious with the increase of stress time and identified DEGs related to ammonia detoxification, immune response, and apoptosis based on transcriptome data.
Histopathological evaluation
The gill filament is the basic functional unit of crab gills, responsible for excreting ammonia[28]. When the crabs were exposed to high ammonia (325.07 mg/L NH4Cl) for 12 hours, part of the gill cavity began to expand, and when the gill tissues were exposed for 24 hours, most of the gill cavity began to expand and some of the gill filaments begin to rupture. In contrast, in the control group, the gill tissue was normal and no abnormalities were observed. Therefore, we inferred that high ammonia stress causes damage to the gill tissues of E. sinensis, and the damage was more severe the longer the stress time. These histological analysis results indicated that ammonia can cause damage at the cellular level which is consistent with the molecular response of crabs.
Ammonia excretion and ammonia detoxification
In previous studies, crustaceans have reported a variety of mechanisms of ammonia excretion and ammonia detoxification in defecting high ammonia environment[11, 29–31]. Crustaceans as ammonia discharge animals and the gills of aquatic crustaceans are the first point of contact with the outside world and exchange of material[32]. Gills of crustaceans can excrete ammonia in reverse concentration into a high ammonia environment through several transporters located in the gill epithelium such as Rh, NKA, NKCC, and NHE[11, 28, 29, 33, 34]. Otherwise, some studies reported that ammonia can be loaded onto vesicles as NH4+ and transported along the microtubule network to the apical membrane[28]. Ammonia can be released in vitro by membrane fusion as well as exocytosis. This vesicle ammonia trapping mechanism has been demonstrated in Portunus trituberculatus and Carcinus maenas[28, 29]. In our study, terms which were related to vesicle cytoplasmic (GO:0031982), vesicle (GO:0031410) and intracellular vesicle (GO:0097708) among GO enrichment were significantly enriched (p < 0.05). Interestingly, most DEGs among the three terms were down-regulated. We hypothesized that the down-regulation of these unigenes was not a protective measure against a high ammonia environment, but rather was caused by ammonia toxicity. This result was consistent with results in Pacifastacus leniusculus and Metacarcinus magister, which induced that high ammonia environment destroyed ion balance and osmoregulation[35, 36]. Due to the significance of gills in ammonia extraction in E. sinensis, the capacity of ammonia excretion was reduced in our investigation, which was connected to the damage produced to gill tissues after 24 h of ammonia stress in histological experiments. Additionally, it was found in Portunus pelagicus that acute ammonia exposure can significantly alter the physiology and morphology of the anterior gills[8].
Crustaceans have an ammonia detoxification mechanism that converts excess ammonia into additional compounds including glutamine and urea, reducing the concentration of ammonia in the body[37, 38]. In other studies, urea levels increased in crustaceans including Marsupenaeus japonicus and P. trituberculatus in high-ammonia environments[29, 39]. Although urea is thought to play a vital role in the detoxification process of ammonia stress, urea is also fewer toxic and can cause adverse effects to the body[40]. Uric acid, which is the precursor to urea, can be created by the breakdown of purine nucleotides like adenine and guanine, hypoxanthine, and xanthine. This process is known as the ornithine-urea cycle. Xanthine oxidoreductase, in two forms xanthine dehydrogenase (XDH) and xanthine oxidase (XO), plays an important role in the process of degradation of purine nucleotides. XDH is a rate-limiting enzyme in purine catabolism, which catalyzes the conversion of hypoxanthine to xanthine and then xanthine converts to uric acid[40, 41]. The expression of XDH, a rate-limiting enzyme in the purine metabolism pathway, was down-regulated, which is notable. Instead, E. sinensis utilized metabolic suppression under ammonia stress, likely to lessen the physiological cost of excess urea. Penaeus monodon and P. trituberculatus both showed similar outcomes[14, 15].
In terms of ammonia detoxification, protein and amino acid breakdown results in the release of endogenous ammonia, and possibly reducing protein and amino acid hydrolysis is the most effective way to avoid ammonia accumulation in the body[42]. Our previous study found that protein degradation was inhibited in the hepatopancreas tissue of E. sinensis under the same ammonia concentration stress, reducing endogenous ammonia accumulation, which are agree with other studies[23, 42–44].
Immune response
E. sinensis belong to invertebrates and lack acquired immunity, so they can only rely on the innate immune system, which is divided into cellular immunity and humoral immunity[45]. In crustaceans, hemolymph is an important defensive line of non-specific immune defense system which is the important place of the two-immunity mentioned above[10, 46]. Previous research has shown that crustaceans like E. sinensis and Litopenaeus vannamei have a lower total hemocyte count (THC). The decrease in THC demonstrated that ammonia stress damaged crustaceans' immunocompetence[10, 46]. Phagocytosis, which may eliminate metabolic waste, apoptotic cells, and external pathogens that infiltrate the body, is the most crucial defense mechanism of hemolymph. Lysosomes, on the other hand, are active organelles that take in inputs from membrane traffic via secretion, endocytosis, autophagy, and phagocytosis[47]. In our study, GO analysis revealed that DEGs related to lysosome (GO:0005764), early phagosome (GO:0032009) and endolysosome (GO:0036019) were significantly varied and KEGG analysis revealed that DEGs related to Lysosome (ko04142), T cell receptor signaling pathway (ko04660) were significantly varied which illustrated ammonia exposure inhibited immune response.
Furthermore, we identified UCHL3 and OGT which are related to immune. Both genes were expressed differently. Ubiquitination is an important post-translational modification of eukaryotic proteins and is involved in most signaling pathways. Ubiquitination is reversible, and deubiquitination plays a crucial role in many biological functions such as apoptosis, tumor growth, cell cycle, and antioxidant immunity[48]. Ubiquitin C-terminal hydrolases (UCHs) are a subfamily of deubiquitinating enzymes (DUBs) which reverse the protein ubiquitination process[49]. It is remarkable that UCHL3 also participates in the process of immune regulation in essence. In previous study, UCHL3 in Macrobrachium nipponense may be involved in innate immunity to resist bacterial invasion through activating NF-κB signaling cascade[50]. Additionally, another key to post-translational modification of proteins is O-glycosylation. Previous studies revealed that some protein added of a single N-acetylglucosamine (GlcNAc) fragment through O-glycosylation which is related to immune response, including heat shock factor 1 and heat shock protein expression[51, 52]. The up-regulated of ogt coding more protein O-glycosylation in hepatopancreas and hemocytes of L. vannamei increased its immune capability[51]. The expression of these two genes was down-regulated in our study, indicating the immune disorder of E. sinensis under ammonia stress.
The same conclusion can also be drawn in the hepatopancreas of E. sinensis under the same stress time and concentration. The hepatopancreas is an important detoxification organ of E. sinensis, which contains a large number of immune-related genes and pathways, and most of the immune-related pathways and genes in the hepatopancreas are down-regulated[23]. The results of gills and hepatopancreas indicated us that high ammonia environment damaged the immune system of E. sinensis which were in line with L. vannamei, P. monodon and Procambarus clarkii [15, 53, 54].
Apoptosis
Apoptosis can be activated by ammonia in crustaceans[54]. Apoptosis is not considered to be an autogenous injury phenomenon under pathological conditions, but a process of death in an active struggle to better adapt to the living environment[55]. The apoptosis pathway was dramatically changed in our study (P < 0.01). Additionally, the regulation of the apoptosis-related enzymes cathepsin B (CTSB) and ubiquitin conjugating enzyme E2 W (UBE2W) was drastically changed. The cysteine family includes the gene known as CTSB, which is frequently involved in apoptosis. According previous studies, L. vannamei, Fenneropenaeus chinensis, and Palaemonetes pugio were all protected against harm from external stress by apoptosis caused by the up-regulation of CTSB [56, 57]. In mouse spermatogenic cells, the down-regulation of UBE2W increased the expression of P53, caspase 6 and caspase 9 which can induce apoptosis, and decreased the expression of Bcl-2 which Significantly inhibited cell apoptosis. The down-regulation of UBE2W activated P53 / Bcl-2 / caspase 6 / caspase 9 signaling pathway. The activation of P53 / Bcl-2 / caspase 6 / caspase 9 signaling pathway increased the cell apoptosis rate[58]. The up-regulation of CTSB and down-regulation of UBE2W further confirmed that ammonia stress can activate the apoptosis of E. sinensis and eliminated the dead cells due to immunosuppression, which can protect E. sinensis. This result explained the injury of gill under ammonia stress from the molecular level.