Immune Responses to Gram-Negative Bacteria in Hemolymph of the Chinese Horseshoe Crab, Tachypleus tridentatus

Abstract

Chinese horseshoe crab, Tachypleus tridentatus, is an ancient marine arthropod with a long evolutionary history. As a kind of living fossil species, the pathogen defenses of horseshoe crabs entirely depend on the innate immune system. Although, there are abundant immune molecules found in the horseshoe crab hemolymph, the biological mechanisms underlying their abilities of distinguishing and defending against invading microbes are still unclear. In this study, we used high-throughput sequencing at mRNA and protein levels and bioinformatics analysis methods to systematically analyze the innate immune response to Gram-negative bacteria in hemolymph of Chinese horseshoe crab. These results showed that many genes in the complement and coagulation cascades, Toll, NF-κB, C-type lectin receptor, JAK-STAT, and MAPK signaling pathways, and antimicrobial substances were activated at 12 and 24 h post-infection, suggesting that Gram-negative bacteria could activate the hemolymph coagulation cascade and antibacterial substances release via the above pathways. In addition, we conjectured that Toll and NF-κB signaling pathway were most likely to participate in the immune response to Gram-negative bacteria in hemolymph of horseshoe crab through an integral signal cascade. These findings will provide a useful reference for exploring the ancient original innate immune mechanism.

Identification of a Novel Antimicrobial Peptide From the Ancient Marine Arthropod Chinese Horseshoe Crab, Tachypleus tridentatus

“…In the previous study, we performed a multi-omics (transcriptome and peptidome) analysis of Chinese horseshoe crab hemolymph to identify genes induced by bacterial infection and found that the gene, termed Tatritin in this study, was significantly ( p < 0.05) induced by pathogenic V. parahaemolyticus ( 29 ). The open reading frame (ORF) and 3′ UTR sequences of Tatritin (accession number OM128176) were identified via RT-PCR.…”

“…The antimicrobial peptide named Tatritin was newly identified from the transcriptome of Chinese horseshoe crab ( 29 ) and showed a significantly upregulated expression ( p < 0.05) in hemolymph of Chinese horseshoe crab after infection with Gram-negative bacteria. The Tatritin gene consists of three exons, coding 68 amino acid residues.…”

“…According to our previous study ( 29 ), a putative antimicrobial peptide gene (termed Tatritin in this study) was identified in RNA-seq datasets (NCBI accession number SRP267502). The open reading frame (ORF) of Tatritin was amplified by PCR based on the transcript sequence.…”

“…Meanwhile, the relative expression levels of Tatritin in Chinese horseshoe crabs infected with Vibrio parahaemolyticus in different time points (0, 12, 24, 36, and 72 h) postinfection were determined by qRT-PCR. The challenge process was described in the previous study ( 29 ). qRT-PCR was conducted using a CFX Connect™ Real-Time PCR detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA) as previously described ( 29 ).…”

“…In the present study, to our knowledge, a new potential AMP gene, termed Tatritin , was identified and characterized in Chinese horseshoe crab hemolymph via transcriptome analysis postinfection with Gram-negative bacteria ( 29 ). Although Tatritin did not show similarity to reported sequences, it was characterized as a new AMP that is important for antibacterial immunity of Chinese horseshoe crab.…”