Sequential expression of small heat shock proteins contributing to the cold response of Haemaphysalis longicornis (Acari: Ixodidae).

BACKGROUND: Haemaphysalis longicornis is an important livestock pest and a serious threat to public health. Cold is a common form of stress affecting its survival and distribution. However, H. longicornis exhibits different physiological responses to cold stress. In this study, we systematically explored the regulation and functions of small heat shock proteins (sHsps) in H. longicornis during cold stress. RESULTS: Seven sHsp genes (HlsHsp14.9, HlsHsp19.9, HlsHsp20.3, HlsHsp21.4, HlsHsp23.7, HlsHsp24.0, and HlsHsp26.1) with open reading frame lengths ranging from 408 bp (HlsHsp14.9) to 673 bp (HlsHsp26.1) were cloned from H. longicornis, and featured the typical α-crystallin domain. Phylogenetic analysis revealed high similarity with the sHsps of arachnid species. Quantitative polymerase chain reaction analysis revealed that the regulation of sHsp genes depended on the severity and duration of cold treatment. Moreover, the relative expression of each gene was largely dependent on the treatment period (P < 0.01; 3, 6, and 9 days of treatment at 8, 4, 0, and -4 °C). Among all genes, HlsHsp14.9, HlsHsp19.9, HlsHsp20.3, and HlsHsp24.0 were most sensitive to rapid cold treatment. After RNA interference, the mortality of H. longicornis was significantly increased at -14 °C (P < 0.05), suggesting that the expression of sHsp genes is closely related to cold tolerance in H. longicornis. CONCLUSION: Our results indicate that sHsps play an important role in the cold stress response of H. longicornis, which may enhance our understanding of the cold adaptation mechanisms in ticks. © 2023 Society of Chemical Industry.

Molecular characterization and modulated expression of histone acetyltransferases during cold response of the tick Dermacentor silvarum (Acari: Ixodidae).

BACKGROUND: Histone acetylation is involved in the regulation of stress responses in multiple organisms. Dermacentor silvarum is an important vector tick species widely distributed in China, and low temperature is a crucial factor restricting the development of its population. However, knowledge of the histone acetyltransferases and epigenetic mechanisms underlying cold-stress responses in this tick species is limited. METHODS: Histone acetyltransferase genes were characterized in D. silvarum, and their relative expressions were determined using qPCR during cold stress. The association and modulation of histone acetyltransferase genes were further explored using RNA interference, and both the H3K9 acetylation level and relative expression of KAT5 protein were evaluated using western blotting. RESULTS: Three histone acetyltransferase genes were identified and named as DsCREBBP, DsKAT6B, and DsKAT5. Bioinformatics analysis showed that they were unstable hydrophilic proteins, characterized by the conserved structures of CBP (ZnF_TAZ), PHA03247 super family, Creb_binding, and MYST(PLN00104) super family. Fluorescence quantitative PCR showed that the expression of DsCREBBP, DsKAT6B, and DsKAT5 increased after 3 days of cold treatment, with subsequent gradual decreases, and was lowest on day 9. Western blotting showed that both the H3K9 acetylation level and relative expression of KAT5 in D. silvarum increased after treatment at - 4, 4, and 8 °C for 3 and 6 days, whereas they decreased significantly after a 9-day treatment. RNA interference induced significant gene silencing, and the mortality rate of D. silvarum significantly increased at the respective semi-lethal temperatures. CONCLUSION: These results imply that histone acetyltransferases play an important role in tick adaptation to low temperatures and lay a foundation for further understanding of the epigenetic regulation of histone acetylation in cold-stressed ticks. Further research is needed to elucidate the mechanisms underlying histone acetylation during cold stress in ticks.

The genome-wide characterization and associated cold-tolerance function of the superoxide dismutase in the cold response of the tick Haemaphysalis longicornis.

Accumulating evidence suggests that superoxide dismutase (SOD) is the first line of antioxidant defense in organisms and plays an important role in scavenging reactive oxygen species produced during environmental stress. However, limited information is available regarding the response of SOD genes to cold stress in ticks. Therefore, in the present study, SOD genes were cloned and identified from the genome of Haemaphysalis longicornis, and the function of SOD during the cold response was further explored. Seven SOD genes were characterized: HlCCS1, HlCCS2, HlMSD, HlCSD1, HlCSD2, HlCSD3, and HlCSD4. Bioinformatics analysis showed that HlCCS1 and HlCCS2 are copper chaperones of SODs. HlCSD1-HlCSD4 belong to the Cu/Zn SOD, whereas HlMSD belongs to the Mn SOD gene family. Fluorescence quantitative PCR showed that the expression of HlCCS2, HlMSD, and HlCSD1-3 was upregulated, whereas HlCCS1 and HlCSD4 were downregulated during the cold response of H. longicornis. Western blotting confirmed changes in the relative expression of HlCSD3 and HlMSD in H. longicornis after cold treatment. Mortality of H. longicornis increased significantly after dsRNA injection of HlCCS2, HlMSD, HlCSD1, and HlCSD3. The above results show that SODs have different regulatory functions during the cold response in H. longicornis, and there might be an interaction between treatment temperature and duration. Furthermore, the results lay a foundation for subsequent research on the molecular mechanism of cold tolerance in H. longicornis and shed light on the population distribution and diffusion limit of ticks.

DNA Methylation Variation Is a Possible Mechanism in the Response of Haemaphysalis longicornis to Low-Temperature Stress.

Ticks are notorious ectoparasites and transmit the greatest variety of pathogens than any other arthropods. Cold tolerance is a key determinant of tick abundance and distribution. While studies have shown that DNA methylation is one of the important epigenetic regulations found across many species and plays a significant role in their response to low-temperature stress, its role in the response of ticks to low-temperature stress remains unexplored. Herein, we explored the DNA methylation profile of the tick, Haemaphysalis longicornis, exposed to low-temperature stress (4 °C) using whole-genome bisulfite sequencing (WGBS). We found that approximately 0.95% and 0.94% of the genomic C sites were methylated in the control and low-temperature groups, respectively. Moreover, the methylation level under the CG context was about 3.86% and 3.85% in the control and low-temperature groups, respectively. In addition, a total of 6087 differentially methylated regions (DMRs) were identified between the low-temperature and control groups, including 3288 hypermethylated and 2799 hypomethylated DMRs. Further, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially methylated genes revealed that most of the DMGs were significantly enriched in binding and RNA transport pathways. Taken together, this research confirmed, for the first time, the whole genome DNA methylation profile of H. longicornis and provided new insights into the DNA methylation changes relating to low-temperature stress in H. longicornis, as well as provided a foundation for future studies on the epigenetic mechanism underlying the responses of ticks to abiotic stress.

The toxicity of the monoterpenes from lemongrass is mitigated by the detoxifying symbiosis of bacteria and fungi in the tick Haemaphysalis longicornis.

The entry mode of terpenes into the atmosphere is via volatilization of hydrocarbons from foliage over heavily forested areas besides entering the environment through surface water runoff. Some monoterpenes in essential oils are phytotoxins, acting as plant chemical defenses against bacteria or fungi infections and plant-eating insects. For organisms to survive, their enzymatic systems are activated in response to an assault by potentially harmful compounds. Certain bacterial and fungal genera have developed special abilities to transform toxic terpenes into less toxic derivatives. Here, we investigated the response of the bacterial and fungal community in Haemaphysalis longicornis exposed to Cymbopogon citratus (lemongrass) essential oil (EO) and citronellal. Sequencing of bacterial 16S rRNA and fungal ITS1 regions on an Illumina NovaSeq PE250 sequencing platform was performed for H. longicornis tick samples treated with 15 and 20 mg/mL of lemongrass essential oil and citronellal. The diversity recorded in samples treated with C. citratus EO was higher in comparison to those treated with citronellal but significantly lower in the control samples as reflected by the Shannon diversity index. All major H. longicornis bacterial phyla, including Proteobacteria (93.81 %), Firmicutes (2.58 %), and Bacteroidota (0.99 %) were detected. A switch of dominance from Coxiella to Pseudomonas, which has high biotransformation capacity, was observed in the bacterial community, whereas the phylum Ascomycota (Genera: Aspergillus, Archaeorhizomyces, Alternaria, and Candida) dominated in the fungal community indicating detoxifying symbiosis. Other significantly abundant bacterial genera include Ralstonia, Acinetobacter, Vibrio, and Pseudoalteromonas, while Ganoderma and Trichosporon (yeasts) spp. represented the fungi Basidiomycota. This study expanded the understanding of enzymatic modification of phytotoxic substances by microorganisms, which could provide deeper insights into the mitigation of harmful phytotoxins and the synthesis of eco-friendly derivatives for the control of ticks.

miR-2a and miR-279 are functionally associated with cold tolerance in Dermacentor silvarum (Acari: Ixodidae).

Ticks are obligate blood-sucking ectoparasites that can attack mammals, birds, reptiles as well as amphibians. Dermacentor silvarum, an important vector of various pathogenic bacteria, viruses, and protozoans, is widely distributed in China. MicroRNAs (miRNAs) are ~22 nucleotide non-coding small RNA molecules, involved in the regulation of various physiological and cellular processes. Previous studies demonstrated the vital roles of miRNAs during the reproduction and development of ticks, whereas, the regulatory/functional roles of microRNAs during the cold response of ticks remain unexplored. Here, we identified and functionally explored D. silvarum miRNAs involved in cold response to gain further understanding of the molecular regulatory mechanisms underlying cold stress in ticks. The microRNA libraries of D. silvarum were established via high-throughput sequencing after exposure to different cold treatments. A total of 147 miRNAs, including 44 known miRNAs and 103 new miRNAs, were identified. The verification of six highly differentially expressed miRNAs (miR-2a, miR-5305, miR-7, miR-279, miR-993, and novel-3) via RT-qPCR were consistent with the high-throughput sequence results. miR-2a peaked by day 6 and miR-279 expression was lowest by day 3 after cold treatment. The potential target genes of miR-2a and miR-279 were the glycogen phosphorylase (GPase) gene and serine gene, respectively. After injecting D. silvarum ticks with miR-2a and miR-279 antagonists, their respective target genes were up-regulated and vice-versa after injection with the agonists. These results indicated that these two miRNAs and their target genes may be involved in the cold response of D. silvarum ticks.