Identification and characterization of class B scavenger receptor CD36 from the hard tick, Haemaphysalis longicornis

Abstract

Scavenger receptors (SRs) are cell-surface proteins and exhibit distinctive ligand-binding properties, recognizing a wide range of ligands that include microbial surface constituents and intact microbes. The class B scavenger receptor CD36 (SRB) is predominantly expressed by macrophages and is considered important in innate immunity. We here show the identification and characterization of SRB from the hard ixodid tick, Haemaphysalis longicornis (HlSRB). The full-length cDNA was 2,908 bp, including an ORF encoding of 1,518 amino acids with a pI value of 5.83. H. longicornis SRB contains a hydrophobic SRB domain and four centrally clustered cysteine residues for arrangement of disulfide bridges. Deduced amino acid sequence has an identity of 30-38% with the SRB of other organisms. RT-PCR analysis showed that mRNA transcripts were expressed in multiple organs of adult ticks but with a different transcript level in the developmental stages of H. longicornis ticks. His-tagged recombinant HlSRB was expressed in Escherichia coli with an expected molecular mass of 50 kDa. In Western blot analysis, mouse anti-rHlSRB serum recognized a strong reaction with a 50 kDa protein band in lysates prepared from egg and adult tick but showed a weak reaction with lysates of larva and nymph. In an indirect immunofluorescent antibody test, HlSRB antiserum recognized the protein located on the midgut, salivary glands, and ovary of partially fed H. longicornis females. Silencing of the HlSRB gene by RNAi led to a significant reduction in the engorged female body weight. It is noteworthy that more than a dozen SRB orthologs have been identified in the genomes of insect species with functions related to pheromone signaling, innate immunity, phagocytic clearance of apoptotic cells, and various aspects of the fatty acid metabolism. This is the first report of the identification and characterization of the SRB homologue in Chelicerata, including ticks, horseshoe crabs, scorpions, spiders, and mites.

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis

“…The Journal of Experimental Biology 216 (10) were collected as described previously (Aung et al, 2011;Fujisaki et al, 1975). RNA extraction was performed based on the manufacturer's protocol.…”

RNA Interference – A Powerful Functional Analysis Tool for Studying Tick Biology and its Control

“…However, a homologue of ENTH, Epn-I, has been identified in the hard ticks Rhiphicephalus (Boophilus) microplus and Ixodes scapularis [14]. A class B scavenger receptor identified in Haemaphysalis longicornis (HlSRB) has been demonstrated to mediate systemic RNAi in this tick [26,27]. Combined injection of dsRNA against HlSRB and other target genes, Vitellogenin-1 (HlVg-1) and Vitellogenin Receptor (HlVgR) effectively silenced these genes.…”

“…However, silencing HlSRB prior to injection of dsRNA against HlVg-1 and HlVgR inhibited the silencing of the latter two genes, suggesting that the uptake of the injected dsRNA is dependent on HlSRB in ticks. Similar to D. melanogaster scavenger receptors, HlSRB is also involved in the phagocytosis of bacteria [28], but it is expressed not only in the hemocytes but also in the other organs such as midguts, salivary glands, and ovary [26]. The presence of ENTH homologue and scavenger receptor indicates that the uptake of dsRNA in ticks is through endocytosis.…”

“…In most reports, a high concentration of at least 1 µg dsRNA per tick has been shown to be effective in inducing gene silencing [33], but in some reports, lower concentration has been found to be similarly effective [34][35][36]. Injection has been accomplished using a 33-36-gauge needle attached to a Hamilton syringe particularly in large tick species, such as Amblyomma americanum [37], Dermacentor variabilis, and D. marginatus [38], while microinjection using a microcapillary drawn to a fine point needle and inserted to a micromanipulator has been commonly employed in smaller tick species, including Ixodes [39], and Haemaphysalis [26] ticks. Different injection sites include the lower right quadrant of the ventral surface of the exoskeleton [40], the groove between the basis capituli and the scutum [37], the ventral torso of the idiosoma, away from the anal opening [39], and the coxal membrane in the fourth coxae [26,41].…”

“…Injection has been accomplished using a 33-36-gauge needle attached to a Hamilton syringe particularly in large tick species, such as Amblyomma americanum [37], Dermacentor variabilis, and D. marginatus [38], while microinjection using a microcapillary drawn to a fine point needle and inserted to a micromanipulator has been commonly employed in smaller tick species, including Ixodes [39], and Haemaphysalis [26] ticks. Different injection sites include the lower right quadrant of the ventral surface of the exoskeleton [40], the groove between the basis capituli and the scutum [37], the ventral torso of the idiosoma, away from the anal opening [39], and the coxal membrane in the fourth coxae [26,41]. In some reports, dsRNA was injected through the spiracle [29,42,43] and anal pore; the latter inducing midgut-specific silencing of the target gene [44].…”