XMU-MP-1 Blunt snout bream Megalobrama amblycephala is an he

Blunt snout bream (Megalobrama amblycephala) is an herbivorous freshwater fish native to China. Due to its fast growth, tender flesh, and high disease resistance, it has been a favorable candidate for aquaculture in China. However, it is prone to bacterial and viral diseases and is relatively sensitive to ammonia exposure [12]. Also, induction of oxidative stress and inflammation by dietary oxidized lipids has been a common issue in its culture [13]. Therefore, to overcome such issues in M. amblycephala culture research on immune-related and anti-stress genes is imperative. The goals of the present study were to: (1) achieve the molecular characterization of p38 MAPK; (2) examine p38 MAPK expression following ammonia exposure and Aeromonas hydrophila challenge; and (3) explore the role of p38 MAPK in inflammation induced by lipopolysaccharide (LPS). The outcome of this research could contribute to our understanding on fish resistance against environmental stresses and bacterial infection.
Materials and methods
Results
Discussion In the present study, p38 MAPK was cloned from liver of M. amblycephala. The ORF of p38 MAPK encodes a predicted protein of 361 XMU-MP-1 with no signal peptide. As reported for all the p38 subfamily members, the dual phosphorylation site of TGY motif and substrate binding site of ATRW are highly conserved [20,21]. The dual phosphorylation of both Thr and Tyr in TGY motif is required for all p38 MAPK activation. The ATRW domain is the kinase interaction motif (KIM) docking site binding to the linear KIM sequences and MAPK phosphatases [22]. Moreover, CD motif and ED site are important for interaction of p38 MAPK with substrate, activators and regulators [[22], [23], [24]]. In this study, p38 MAPK contained all these conversed structures as other fish species [10,25]. Phylogenetic analysis also revealed that blunt snout bream p38 MAPK is close to Sinocyclocheilus graham and Cyprinus carpio with a similarity coefficient of 99% and 98%, respectively. In mammals, p38 MAPK family includes four highly homologous isoforms: p38α, p38β, p38γ and p38; whereas to date only p38 has been identified in blunt snout bream. Generally, p38 MAPK is ubiquitously expressed in various organs including brain, muscle, kidney, gill and heart [10,25,26]. The findings in this study revealed that p38 MAPK mRNA could be found in all the tested tissues. The expression level of p38 MAPK was the highest in spleen followed by gill, head kidney, liver and intestine, respectively. This expression pattern is due to the fact that spleen and head kidney are major lymphoid organs in fish [27,28]. Spleen is the major immune organ with abundant IgM+ mature B cells [29], and head kidney has the highest concentration of developing B lymphoid cells [30] and assumes immune function [31]. Moreover, there are several reports indicating that salmon p38 MAPK is mainly expressed in the immune organs such as head kidney and spleen, and its expression could be up-regulated by immune stimuli [25]. Likewise, the expression pattern observed for p38 MAPK in the current study may indicate its role in immune function of M. amblycephala. The higher expression of p38 MAPK in gills compared to head kidney could be due to the fact that p38 MAPK is not only involved in immune function but also its distribution in ionocytes and accessory cells of teleost fish implies its ionoregulatory function [32]. Presence of p38 MAPK has been reported throughout the ionocytes specifically in areas in which sodium potassium 2 chloride cotransporter (NKCC) are absent [32]. It has been suggested that osmotic stress may activate stress-activated protein kinase (SAPK) pathway for signal transmission and in this regard p38-related MAPK is one the responsible molecules which is activated by phosphorelation through sequential actins of kinases [33,34]. Ammonia occurs in the aquatic environment resulting from agricultural run-off and decomposition of biological waste. It is toxic to all vertebrates as it may cause convulsions, coma and death [35]. Moreover, slight ammonia stress often reduces growth and induces oxidative stress in cultured fish [11,36]. There are several reports indicating that expression of p38 MAPK significantly increases under various stresses [37]. So, in the current study we examined the effects of ammonia exposure on the p38 MAPK expression, and the results showed that its expression in gill was up-regulated after 6–24 h of ammonia exposure. Gill has been recognized as the major site that is influenced by various toxicants, and ammonia exposure induces histopathologic damages to gill [38]. The up-regulation of p38 MAPK implies its role in gill damage; however, the detailed mechanism is still unknown. Expression of p38 MAPK in liver was also up-regulated between 12 and 24 h after ammonia exposure. Some studies showed that ammonia exposure induces oxidative stress and apoptosis in fish liver [39,40]. Thus, we assume that the up-regulation of p38 MAPK is associated with the oxidative stress and apoptotic events. It has been reported that thermal stress induces anti-apoptotic events via the p38 MAPK pathway [41]. Based on these results, p38 MAPK is capable of modulating stress responses, and plays key roles in protection of organisms against environmental hazards.