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LHPP (Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase) is a protein histidine phosphatase that modulates a hidden posttranslational modification called histidine phosphorylation. LHPP also acts as a tumor suppressor, which plays a pivotal role in various cellular processes. However, whether LHPP participates in the regulation of invertebrate's immunity is still unknown. Here we characterized a LHPP homolog in P. vannamei (designated PvLHPP), with a 807 bp length of open reading frame (ORF) encoding a putative protein of 268 amino acids. Sequence analysis revealed that PvLHPP contains a typical hydrolase 6 and hydrolase-like domain, which was conserved from invertebrate to vertebrate. PvLHPP was ubiquitously expressed in tissues and induced in hemocyte and hepatopancreas by Vibrio parahaemolyticus, Streptococcus iniae and white spot syndrome virus (WSSV) challenge, indicating that PvLHPP participated in the immune responses. Moreover, silencing of PvLHPP followed by V. parahaemolyticus inhibited hemocyte apoptosis. This study enriches our current insight on shrimp immunity, and provides novel perspective to understand immune-regulatory role of PvLHPP.

Anthropogenic factors and climate change have serious effects on the aquatic ecosystem and aquaculture. Among water pollutants, ammonia has the greatest impact on aquaculture organisms such as penaeid shrimp because it makes them more susceptible to infections. In this study, we explored the effects of ammonia stress (0, 50, 100, and 150 mg/L) on the molecular structure and functions of the multifunctional respiratory protein hemocyanin (HMC) in Penaeus vannamei. While the mRNA expression of Penaeus vannamei hemocyanin (PvHMC) was up-regulated after ammonia stress, both plasma hemocyanin protein and oxyhemocyanin (OxyHMC) levels decreased. Moreover, ammonia stress changed the molecular structure of hemocyanin, modulated the expression of protein phosphatase 2 A (PP2A) and casein kinase 2α (CK2α) to regulate the phosphorylation modification of hemocyanin, and enhanced its degradation into fragments by trypsin. Under moderate ammonia stress conditions, hemocyanin also undergoes glycosylation to improve its in vitro antibacterial activity and binding with Gram-negative (Vibrio parahaemolyticus) and Gram-positive (Staphylococcus aureus) bacteria, albeit differently. The current findings indicate that P. vannamei hemocyanin undergoes adaptive molecular modifications under ammonia stress enabling the shrimp to survive and counteract the consequences of the stress.

Summary: The mitogen-activated protein kinase (MAPK) intracellular signaling pathway mediates numerous biological processes, including antimicrobial immune response by inducing antimicrobial peptides (AMPs) production. Although MAPK signaling cascade proteins have been identified in penaeid shrimp, their modulation via the MKK4-p38-c-Jun cascade and effect on AMPs production is unknown. Here, we show that hemocyanin (PvHMC), antimicrobial peptides (anti-lipopolysaccharide factor, crustin, and penaeidins), and MKK4-p38-c-Jun cascade proteins are simultaneously induced by pathogens (Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus) in Penaeus vannamei. Intriguingly, knockdown of PvHMC with or without pathogen challenge attenuated the expression of MKK4-p38-c-Jun cascade proteins and their phosphorylation level, which consequently decreased AMPs expression. Further analysis revealed that PvHMC interacts via its armadillo (ARM) repeat domain with PvMKK4 to modulate the p38 MAPK signaling pathway. Thus, the ARM repeat domain enables penaeid shrimp hemocyanin to modulate AMPs expression during antimicrobial response by activating the p38 MAPK signaling pathway.