Identification, structural modeling, gene expression analysis and RNAi effect of putative phospholipase A2 in the lone star tick Amblyomma americanum.

Amblyomma americanum, also known as the lone star tick, is a small arachnid that feeds on blood and can spread disease to humans and other animals. Despite the overlapped ecological niche, geographic distribution, and host selection, there is no proof that A. americanum transmits the pathogen Borrelia burgdorferi that causes Lyme disease. Studies have shown that phospholipase A2 (PLA2) may act as a tool to eliminate B. burgdorferi, but particular PLA2 genes in A. americanum have not been identified and functionally characterized. Using the de novo sequencing method, we identified 42 putative A. americanum PLA2 (pAaPLA2) homologs in the present study, of which three pAaPLA2 had calcium binding sites and canonical histidine catalytic sites. Then, we determined phylogenetic relationships, sequence alignments, and conserved protein motifs of these pAaPLA2s. Protein structural analysis demonstrated that pAaPLA2s primarily consisted of α-helices, ß-sheets, and random coils. These genes were predicted to be engaged in the phospholipid metabolic process, arachidonic acid secretion, and PLA2 activity by functional annotation analysis. A transcriptional factor (Bgb) was discovered that interacted with pAaPLA2 proteins that may have unrecognized roles in regulating neuronal development. Based on the RNA-seq data, we surveyed expression profiles of key pAaPLA2-related genes to reveal putative modulatory networks of these genes. RNAi knockdown of pAaPLA2_1, a dominant isoform in A. americanum, led to decreased bacterial inhibition ability, suggesting pAaPLA2 may play an important role in mediating immune responses. Collectively, this study provides essential evidence of the identification, gene structure, phylogeny, and expression analysis of pAaPLA2 genes in A. americanum, and offers a deeper understanding of the putative borreliacidal roles in the lone star tick.

Identification and characterization of ecdysis-related neuropeptides in the lone star tick Amblyomma americanum.

Introduction: The lone star tick, Amblyomma americanum, is an important ectoparasite known for transmitting diseases to humans and animals. Ecdysis-related neuropeptides (ERNs) control behaviors crucial for arthropods to shed exoskeletons. However, ERN identification and characterization in A. americanum remain incomplete. Methods: We investigated ERNs in A. americanum, assessing their evolutionary relationships, protein properties, and functions. Phylogeny, sequence alignment, and domain structures of ERNs were analyzed. ERN functionality was explored using enrichment analysis, and developmental and tissue-specific ERN expression profiles were examined using qPCR and RNAi experiments. Results and discussion: The study shows that ERN catalogs (i.e., eclosion hormone, corazonin, and bursicon) are found in most arachnids, and these ERNs in A. americanum have high evolutionary relatedness with other tick species. Protein modeling analysis indicates that ERNs primarily consist of secondary structures and protein stabilizing forces (i.e., hydrophobic clusters, hydrogen bond networks, and salt bridges). Gene functional analysis shows that ENRs are involved in many ecdysis-related functions, including ecdysis-triggering hormone activity, neuropeptide signaling pathway, and corazonin receptor binding. Bursicon proteins have functions in chitin binding and G protein-coupled receptor activity and strong interactions with leucine-rich repeat-containing G-protein coupled receptor 5. ERNs were expressed in higher levels in newly molted adults and synganglia. RNAi-mediated knockdown of burs α and burs ß expression led to a significant decrease in the expression of an antimicrobial peptide, defensin, suggesting they might act in signaling or regulatory pathways that control the expression of immune-related genes. Arthropods are vulnerable immediately after molting because new cuticles are soft and susceptible to injury and pathogen infections. Bursicon homodimers act in prophylactic immunity during this vulnerable period by increasing the synthesis of transcripts encoding antimicrobial peptides to protect them from microbial invasion. Collectively, the expression pattern and characterization of ERNs in this study contribute to a deeper understanding of the physiological processes in A. americanum.

Integrative analysis highlights molecular and immune responses of tick Amblyomma americanum to Escherichia coli challenge.

Ticks are ectoparasites that can transmit various pathogens capable of causing life-threatening illnesses in people and animals, making them a severe public health threat. Understanding how ticks respond to bacterial infection is crucial for deciphering their immune defense mechanisms and identifying potential targets for controlling tick-borne diseases. In this study, an in-depth transcriptome analysis was used to investigate the molecular and immune responses of Amblyomma americanum to infection caused by the microinjection of Escherichia coli. With an abundance of differentially expressed genes discovered at different times, the analysis demonstrated significant changes in gene expression profiles in response to E. coli challenge. Notably, we found alterations in crucial immune markers, including the antimicrobial peptides defensin and microplusin, suggesting they may play an essential role in the innate immune response. Furthermore, KEGG analysis showed that following E. coli exposure, a number of key enzymes, including lysosomal alpha-glucosidase, fibroblast growth factor, legumain, apoptotic protease-activating factor, etc., were altered, impacting the activity of the lysosome, mitogen-activated protein kinase, antigen processing and presentation, bacterial invasion, apoptosis, and the Toll and immune deficiency pathways. In addition to the transcriptome analysis, we constructed protein interaction networks to elucidate the molecular interactions underlying the tick's response to E. coli challenge. Hub genes were identified, and their functional enrichment provided insights into the regulation of cytoskeleton rearrangement, apoptotic processes, and kinase activity that may occur in infected cells. Collectively, the findings shed light on the potential immune responses in A. americanum that control E. coli infection.