The Mitochondrial Genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): Estimation of Divergence Dates for the Major Tick Lineages and Reconstruction of Ancestral Blood-Feeding Characters

Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae (Nuttalliella namaqua) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.     

Ixodid tick salivary gland products target host wound healing growth factors

For successful blood-feeding, ticks must confront the host immune system comprising many cells and signaling molecules, mainly cytokines and growth factors. These factors bind to specific receptors on the cell membranes, thereby initiating a signaling cascade that leads to distinct cellular activities. Ticks are able to manipulate host immune responses via molecules secreted from their salivary glands. Saliva of ixodid ticks contains factors binding important cytokines and their subgroup, chemokines. Here we demonstrate that constituents of tick salivary gland extract (SGE) also appear to bind growth factors: transforming growth factor beta (TGF-β1), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF-2), and hepatocyte growth factor (HGF), depending on tick species. SGE derived from Amblyommavariegatum reacted with TGF-β1, PDGF, FGF-2 and HGF; Dermacentorreticulatus and Rhipicephalusappendiculatus with TGF-β1, FGF-2 and HGF; and Ixodes ricinus and Ixodesscapularis with PDGF. SGE from the species targeting PDGF (A. variegatum and I. ricinus) also inhibited cell proliferation in vitro and induced a change in morphology of different cell lines. These effects correlated with disruption of the actin cytoskeleton. Such effects were not observed with SGE of the two species that did not target PDGF. Targeting of wound healing growth factors appears to be yet another strategy ixodid ticks adopt for suppression of inflammation and successful haematophagy.     

Factors influencing the toxicity of salivary gland extracts of Ixodes holocyclus Neumann

A bioassay using mice was developed to compare the toxin content of extracts of salivary glands of I. holocyclus at various stages of feeding. The quantity of toxin increased rapidly from the third day of feeding. Toxin production continued and increased in ticks removed after 3–5 days on mice and held at 30°C at 92% RH for 24 h, whereas no toxin was detected in the salivary glands of ticks fed for 3 days and treated similarly. It is suggested that major physiological changes occur in the salivary glands of I. holocyclus on the third day, which once stimulated continue independently of feeding. Toxin production in ticks was not suppressed by passively immunizing mice with anti-tick toxin but was in ticks fed upon hosts with a previous experience of tick feeding.Thus, to obtain salivary glands containing high concentrations of toxin for chemical analysis, it is necessary for salivary glands to develop 5 days from the initial attachment of the tick to a host with no previous experience of tick feeding. This can be achieved by passively immunizing mice against toxin, thus enabling the tick to feed 5 days without killing the mouse or by keeping the tick for 24 h at 30°C at 92% RH following the death of the mouse on the fourth day.     

The regulatory mechanism of Hsp90α secretion from endothelial cells and its role in angiogenesis during wound healing

Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90α can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90α from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90α in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90α but not Hsp90β is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90α localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90α neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90α localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90α can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90α as a stimulator for wound repair.     

Ixodid tick salivary gland extracts suppress human transforming growth factor-β1 triggered signalling pathways in cervical carcinoma cells

Ticks, very successful vectors and reservoirs of diverse viruses and other infectious agents, are able to counteract host defence mechanisms and wound healing processes to facilitate blood-feeding. Cutting the epidermis by the tick chelicerae and penetration of the hypostome into the dermis followed by feeding elicit a wound healing response, involving coagulation, inflammation, angiogenesis, synthesis of extracellular matrix and ground substances, as well as tissue remodelling. These repair processes of injured skin are coordinated by a wide array of cytokines, chemokines and growth factors. Tick salivary gland compounds, in addition to their vital anticoagulant and immunomodulatory functions, also possess significant anti-tumour properties and represent very promising sources of novel pharmaceuticals. Among life-threatening cancers, cervical carcinoma is still a high risk. Human transforming growth factor-beta 1 (huTGF-β1) inhibitors are significant in anti-cancer therapy. In this study, we investigated effects of salivary gland extracts (SGE) of females of two ixodid ticks, Dermacentor reticulatus (DR SGE) and Hyalomma anatolicum excavatum (HAE SGE), on signalling pathways triggered by huTGF-β1 in two cervical carcinoma cell lines, SiHa and HeLa, varying in their response to huTGF-β1. By dual luciferase reporter assay we monitored activation/inhibition of canonical SMAD or non-canonical signalling pathways in cells treated with huTGF-β1 and DR SGE/HAE SGE. Using specific monoclonal antibodies we tested effects of SGE on phosphorylation of ERK1/2 and/or AKT-1 molecules. We were the first to detect significant inhibition of huTGF-β1 induced signalling pathways, canonical as well as non-canonical, by DR and HAE SGE. Although the overall effects of both SGE on signalling pathways were similar, we revealed different patterns of effects of the two SGE on phosphorylation of the monitored signal transducers. Further investigations in this field are required.     

Akt is an essential player in regulating cell/organ growth at the adult stage in the hard tick Haemaphysalis longicornis

Ticks grow rapidly during blood feeding, and their body weight may ultimately increase 100-fold more than that before feeding. The molecular mechanisms controlling growth during blood feeding in ticks remain largely unknown. The conserved insulin/PI3K/Akt signaling pathway regulates growth and metabolism in eukaryotes. Here, we show evidence for the involvement of Akt in growth during blood feeding in the parthenogenetic strain of the hard tick Haemaphysalis longicornis. We identified a homolog of the Ser/Thr kinase Akt (HlAkt) from the EST database of the H. longicornis embryo. HlAkt cDNA had a 1,590 bp ORF that encodes 529 amino acids with a predicted molecular weight of 60 kDa. HlAkt possesses a PH domain, a Ser/Thr kinase domain, a hydrophobic motif, and dual phosphorylation residues (Thr 338 and Ser 503) that are essential for kinase activation. Knockdown of HlAkt by RNA interference caused inhibition of blood feeding in female ticks. Histological observation demonstrated that HlAkt knockdown led to the arrest of growth in internal organs. HlAkt knockdown also affected the expressions of blood meal-induced genes that are essential for blood digestion, development, and reproduction in the female tick. These results strongly indicate that HlAkt is essential to complete the blood feeding process accompanied by the growth of internal organs in adult ticks. This is the first report of identification and characterization of Akt in Chelicerata, including ticks.     

Ferulic acid augments angiogenesis via VEGF,PDGF and HIF-1α

Therapeutic angiogenesis is critical to wound healing and ischemic diseases such as myocardial infarction and stroke. For development of therapeutic agents, a search for new angiogenic agents is the key. Ferulic acid, a phytochemical found in many fruits and vegetables, exhibits a broad range of therapeutic effects on human diseases, including diabetes and cancer. This study investigated the augmenting effect of ferulic acid on angiogenesis through functional modulation of endothelial cells. Through endothelial cell migration and tube formation assays, ferulic acid (10^?6–10^?4 M) was found to induce significant angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro without cytotoxicity. With chorioallantoic membrane assay, ferulic acid (10^?6–10^?5 M) was also found to promote neovascularization in vivo. Using Western blot analysis and quantitative real-time polymerase chain reaction, we found that ferulic acid increased vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression in HUVECs. Furthermore, the amounts of hypoxic-induced factor (HIF) 1α mRNA and protein, the major regulator of VEGF and PDGF, also showed up-regulation by ferulic acid. Electrophoretic migration shift assay showed that the binding activity of HIF-1α was also enhanced with ferulic acid treatment of HUVECs. Moreover, inhibitors of extracellular-signal-regulated kinase 1/2 and phosphoinositide-3 kinase (PI3K) abolished the binding activity of HIF-1α and the subsequent activation of VEGF and PDGF production by ferulic acid. Thus, both mitogen-activated protein kinase and PI3K pathways were involved in the angiogenic effects of ferulic acid. Taken together, ferulic acid serves as an angiogenic agent to augment angiogenesis both in vitro and in vivo. This effect might be observed through the modulation of VEGF, PDGF and HIF-1α.     

Engorgement of Rhipicephalus haemaphysaloides ticks blocked by silencing a protein inhibitor of apoptosis

Inhibitors of apoptosis (IAPs) are regulators of cell death and may play a role in the salivary glands of ticks during blood-feeding. We cloned the open reading frame (ORF) sequence of the IAP gene in Rhipicephalus haemaphysaloides (RhIAP). The RhIAP ORF of 1887 bp encodes a predicted protein of 607 amino acids, which contains three baculovirus IAP repeat domains and a RING finger motif. A real-time PCR assay showed that RhIAP mRNA was expressed in all the tick developmental stages (eggs, larvae, nymphs, and adults) and in all tissues examined (midgut, ovary, salivary glands, fat body, and hemolymph). Western blot showed that the protein level of RhIAP in salivary glands increased during tick blood-feeding and decreased towards the end of tick engorgement. RhIAP gene silencing in vitro experiments with salivary glands demonstrated that RhIAP could be effectively knocked down within 48 h after dsRNA treatment, and as a consequence, salivary glands displayed apoptotic morphology. RhIAP gene silencing also inhibited tick blood-feeding and decreased the engorgement rate. These data suggest that RhIAP might be a suitable RNAi target for tick control.

     

Hard Ticks as Vectors Tested Negative for Severe Fever with Thrombocytopenia Syndrome in Ganghwa-do,Korea during 2019–2020

This study aimed to characterize the seasonal abundance of hard ticks that transmit severe fever with thrombocytopenia syndrome virus from April to November 2019 and 2020 on Ganghwa-do, Incheon Metropolitan City, Korea. The ticks were collected at grassland, grave site, copse and mountain road using a collection trap method. The ixodid hard ticks comprising three species (Haemaphysalis longicornis, H. flava, and Ixodes nipponensis) collected were 6,622 in 2019 and 3,811 in 2020. H. longicornis was the most frequent (97.9% in 2019 and 96.0% in 2020), followed by H. flava (2.0% and 3.0% in 2019 and 2020, respectively) and I. nipponensis (less than 0.1%). Our study demonstrated that seasonal patterns of the tick populations examined for two years were totally unsimilar. The hard ticks tested using RT-qPCR were all negative for severe fever with thrombocytopenia syndrome virus.     

Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin’s effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin’s angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and mRNA expression in cultured HUVECs in a concentration-dependent manner. Taken together, these results suggest that scutellarin promotes angiogenesis and may form a basis for angiogenic therapy.     

The new Haemaphysalis longicornis genome provides insights into its requisite biological traits

Ticks are a large group of blood-feeding arthropods that transmit multiple human and animal pathogens and are hence of importance to public health. The tick Haemaphysalis longicornis is associated with the transmission of multiple human pathogens in Asia, and recently found invading to the United States. Here, we report the sequencing, assembly and annotation of the 3.16 gigabase genome of this species, which is larger than the previous assembled one. The present Haemaphysalis longicornis genome was characterized by 6519 scaffolds, 24,189 protein-coding genes and a high proportion of simple sequence repeats (54.72%). By genomic assembly and comparative genomic analysis, we characterized the key genes that play essential roles in iron metabolism, detoxification, and freeze tolerance of H. longicornis. Furthermore, a total of 79 endogenous viral elements were identified within the genome, which might have had a considerable impact on its evolution. Decoding the H. longicornis genome not only provides insight into the genetic underpinnings of specific biological processes but also offers the basis for the subsequent integrated control of ticks and tick-borne diseases.     

Identification of Four Novel Rhipicephalus annulatus Upregulated Salivary Gland Proteins as Candidate Vaccines

The control of Rhipicephalus annulatus ticks in Egypt and other countries relies principally on the application of acaricides which have many drawbacks. Recently, cattle vaccination against ticks showed a potential unconventional approach to control ticks. As a target, salivary glands contain various proteins that may play specific roles during attachment, feeding and may modulate the immune system of the host. We have performed immunoscreening on expression normalized cDNA library to identify unique R. annulatus proteins from salivary gland (RaSal) that are particularly expressed during engorgement. We also present the cloning and sequencing of four novel cDNAs (RaSal1–4) from salivary glands that are expressed during feeding. RaSal4 shows 13 cysteine amino acid residues forming 6 potential disulfide bonds. We detected the expression level of the four genes during embryogenesis in eggs collected at 6, 12 and 18 days after oviposition. RT-PCR analysis detected these proteins at days 12 and 18 while slight amplification was detected at day 6 for only RaSal2. The expression of these salivary genes may put forward new vaccines to control tick infestations and tick-borne diseases.