Isolation and expression of the retinoid X receptor from last instar nymphs and adult females of the soft tick Ornithodoros moubata (Acari: Argasidae)
Introduction
Molting and reproduction are characteristic phenomena regulated by endocrine mechanisms and these processes are regulated by ecdysteroids and juvenile hormone (JH) in numerous arthropods. Despite earlier studies indicating JH is important in the regulation of reproduction and development in ticks (reviewed by Sonenshine, 1991, Rees, 2004), more recent studies were unable to detect JH in ticks (Neese et al., 2000) or show a role for JH in the regulation of tick reproduction (reviewed by Taylor and Chinzei, 2002, Rees, 2004). To date ecdysteroids are the only hormones that have been identified in ticks. Generally, ecdysteroids are released from endocrine tissues such as the prothoracic gland or ovary and induce various events in target tissues. Hematophagous arthropods require blood feeding for growth and reproduction and these events are also regulated by ecdysteroids (Raikhel and Dhadialla, 1992). After blood feeding, ecdysteroids are released into the hemolymph and regulate gene transcription in various tissues.
Ecdysteroids directly and indirectly regulate gene expression as switches through the ecdysteroid receptor. In many insects, the functional ecdysteroid receptor is a heterodimer of the ecdysteroid receptor (EcR) and ultraspiracle (USP) (Henrich et al., 1990, Yao et al., 1992, Yao et al., 1993). The ecdysteroid/EcR/USP complex binds to ecdysteroid response elements (EcRE) in the gene transcriptional region of target genes and regulates gene transcription (Riddihough and Pelham, 1987, Yao et al., 1992, Yao et al., 1993, Thomas et al., 1993, Hall and Thummel, 1998). USP is a homologue of the vertebrate retinoid X receptor (RXR) and member of the nuclear receptor family (Oro et al., 1990, Yao et al., 1992). RXR functions with retinoic acids (RAs) in the regulation of various physiological processes such as development, differentiation and homeostasis of numerous animals (Evans, 1988). The ligand of RXR includes RAs and other members of the terpene family with 9-cis RA showing high affinity to RXR (Mangelsdorf and Evans, 1995). RXR of vertebrates is a multigene family with RXRα, β and γ having been reported. RXR contains a DNA binding domain (DBD) and ligand binding domain (LBD) as seen in all nuclear receptors. USP shows a high homology in the DBD to vertebrate RXR but low homology with the LBD of vertebrate RXR (Henrich et al., 1990). Actually, USP can not bind 9-cis RA and other RA metabolites but may bind JH another important hormone for insect molting and reproduction (Jones and Jones, 2000). In addition, USP encodes a single gene and several isoforms appear by alternative splicing in various insects (Kapitskaya et al., 1996, Jindra et al., 1997) except for Drosophila melanogaster with has only one USP isoform (Oro et al., 1990). RXR not USP has recently been identified in numerous arthropods such as crustaceans, ticks and hemimetabolic insects (Chung et al., 1998, Guo et al., 1998, Hayward et al., 1999, Durica et al., 2002, Barchuk et al., 2004, Ogura et al., 2005, Maestro et al., 2005, Kato et al., 2007, Wang et al., 2007). Tick RXRs were first reported from the hard tick Amblyomma americanum (Guo et al., 1998). Two subtypes (AamRXR1, 2) were identified from A. americanum and shown to form a heterodimer with EcR. Expression of tick RXR and EcR occurs when ecdysteroids are present. In addition, AamEcR and AamRXR can bind with Drosophila hsp27 EcRE (Palmer et al., 2002). RXR and EcR of crustaceans also form a heterodimer and work similarly (Durica et al., 2002). The above studies indicate RXR may also function as a heterodimer with EcR to regulate molting and reproduction in numerous arthropods.
Tick molting and vitellogenesis are regulated by ecdysteroids (Taylor and Chinzei, 2002, Rees, 2004). Vitellogenin (Vg) is a precursor of yolk protein and injection of ecdysteroids into unfed Ornithodoros moubata females induces Vg synthesis (Chinzei et al., 1991). Mated females of O. moubata show an increase in ecdysteroids after engorgement (Ogihara et al., 2007) and EcR of O. moubata (OmEcR) sharply increases after engorgement (Horigane et al., 2007a). In addition, engorged nymphs show an increase in ecdysteroids (Ogihara et al., 2007) and OmEcR expression before ecdysis (Horigane et al., 2007a). Therefore, molting and Vg transcription may also be induced by the ecdysteroid/EcR/RXR complex in O. moubata. However, the RXR has not been identified from O. moubata. In this study, we isolated RXR from O. moubata last instar nymphs and adult females and analyzed its expression before and after molting of last instar nymphs and throughout the reproductive cycle of adult females. This study represents the first report of RXR from a soft tick.
Section snippets
Tick rearing
Ornithodoros moubata were maintained in incubators at 30 ± 1 °C, 70%±10% RH and total darkness. Ticks were fed on rabbits (Oryctolagus cunniculus) as described by Chinzei et al. (1983). Engorged last instar nymphs were placed individually in 24-well sample plates (Falcon) (1 tick/well) and allowed to molt into virgin females. Males were added to the wells on the day of molting for mated females. Mating was confirmed by the presence of the spermatophore attached to the female gonopore. Even though
Identification of OmRXR
5′- and 3′-RACE produced the complete open reading frame (ORF) of O. moubata RXR (OmRXR) (Fig. 1). The BLAST homology search showed the sequence to be the RXR gene. OmRXR ORF is 1359 bp and encodes a 453 residue protein. In addition, OmRXR showed variations in the sequences with deletions of residues in the A/B domain (MYMK) and insertions of residues in the T-box of the hinge area (Fig. 2). The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide
Discussion
The retinoid X receptor (RXR) is the most highly conserved member of the steroid/retinoid family of nuclear receptors and plays an important role in numerous physiological processes including development and reproduction of vertebrates and invertebrates. Most studies on RXR and ultraspiracle (USP) concentrate on functions in vertebrates and higher holometabolous insects and only during the last several years have studies looked at hemimetabolous insects and other invertebrates. These studies
Acknowledgments
This research was supported in part by Grants-in-Aid for Scientific Research (16580035 and 18580050 to D.T.) from the Ministry of Education, Sciences and Culture, Japan. We thank Professor Urushibara at the University of Tsukuba for providing the real-time PCR apparatus.
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