Full-length sequence, regulation and developmental studies of a second vitellogenin gene from the American dog tick, Dermacentor variabilis

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Abstract

Vitellogenin (Vg) is the precursor of vitellin (Vn) which is the major yolk protein in eggs. In a previous report, we isolated and characterized the first Vg message from the American dog tick Dermacentor variabilis. In the current study, we describe a second Vg gene from the same tick. The Vg2 cDNA is 5956 nucleotides with a 5775 nt open reading frame coding for 1925 amino acids. The conceptual amino acid translation contains a 16-residues putative signal peptide, N-terminal lipid binding domain and C-terminal von Willebrand factor type D domain present in all known Vgs. Moreover, the amino acid sequence shows a typical GLCG domain and several RXXR cleavage sites present in most isolated Vgs. Tryptic digest-mass fingerprinting of Vg and Vn recognized 11 fragments that exist in the amino acid translation of DvVg2 cDNA. Injection of virgin females with 20 hydroxyecdysone induced DvVg2 expression, vitellogenesis and oviposition. Using RT-PCR, DvVg2 expression was detected only in tick females after mating and feeding to repletion. Northern blot analysis showed that DvVg2 is expressed in fat body and gut cells of vitellogenic females but not in the ovary. DvVg2 expression was not detected in adult fed or unfed males. The characteristics that distinguish Vg from other similar tick storage proteins like the carrier protein, CP (another hemelipoglycoprotein) are discussed.

Research highlights

▶ A second vitellogenin mRNA (Vg2) is described from the American dog tick, Dermacentor variabilis. ▶ Amino acid translation contains a signal peptide, LPD_N, vWD GLCG domain and several RXXR sites. ▶ Vg2 message is present only in mated, replete female adults and found in the fat body and midgut. ▶ Vg2 is up-regulated by ecdysteroids and contributes to both hemolymph Vg and egg Vn, similar to Vg1. ▶ Methods to distinguish Vg from the other major storage protein (CP) are described.

Introduction

Oviparous animals secure resources for the completion of embryo development by depositing large amounts of nutrients into their eggs. Such resources include proteins, lipids, carbohydrates, minerals and other compounds. The major protein is vitellin (Vn). Vitellogenin (Vg), the precursor of Vn, is usually synthesized extra-ovarially, secreted into the hemolymph and taken up by the ovary to be incorporated into growing oocytes via receptor mediated endocytosis. In insects, the vitellogenesis process is well studied. Vg is synthesized in the fat body cells under the regulation of juvenile hormone (JH) and/or ecdysteroids (reviewed by Gilbert et al., 2000, Roe et al., 2008, Tufail and Takeda, 2008). In Crustacea, the mandibular organs produce methyl farnesoate which regulates vitellogenesis and egg production (Laufer and Biggers, 2001, Nagaraju, 2007).

It was originally thought that ticks produced the insect juvenile hormones and like in many insects used JH to regulate vitellogenesis (Pound and Oliver, 1979, Gaber et al., 1983, Khalil et al., 1983). However, Neese et al. (2000) was unable to detect JH in two species, the soft tick Ornithodorous parkeri and the hard tick Dermacentor variabilis. Current evidence suggests that ecdysteroids and not JH regulate the expression of tick Vg synthesis (Dees et al., 1984, Sankhon et al., 1999, Friesen and Kaufman, 2002, Friesen and Kaufman, 2004, Seixas et al., 2008, Thompson et al., 2005, Thompson et al., 2007, Mitchell et al., 2007, Ogihara et al., 2007). In ticks, the fat body was reported to be the main site of Vg synthesis (Chinzei and Yano, 1985, Diehl et al., 1982, Taylor et al., 1991). Rosell and Coons (1992) suggested both fat body and gut as sources for Vg in the American dog tick, D. variabilis. Molecular studies by Thompson et al. (2007) showed that both fat body and gut produced Vg mRNA in D. variabilis replete females exclusively. Roe et al. (2008) and Cabrera et al. (2009a) proposed a global model explaining the regulation of reproduction in female Acari (mites and ticks) where ecdysteroids and not JH initiate vitellogenesis.

Several Vg cDNAs have been sequenced and characterized from insects and Crustacea with evidences for the presence of multiple Vg genes in some species (Sappington and Raikhel, 1998, Sappington et al., 2002, Tufail and Takeda, 2008). Much less is known about multiple Vgs in ticks and their tissue source and regulation. The first full-length Vg cDNA sequence from a tick was published by Thompson et al. (2007). Donohue et al. (2009) reviewed the different aspects of storage proteins in the Chelicerata including the (common) carrier protein, CP. CP is found in both males and females throughout tick development. Although there have been reports of multiple Vgs in the same tick (Thompson et al., 2007, Boldbaatar et al., 2010), Vg and CP share many common features making their identification problematic.

In this report, we provide a description of a second, full-length Vg in the American dog tick and examine for the first time, the tissue-specific hormonal regulation of multiple Vg messages which includes Vg2 in the same tick species. Our analysis also is explicit in distinguishing Vg from CP.

Section snippets

Ticks

A pathogen-free line of the American dog tick, D. variabilis, was reared as described previously (Sonenshine, 1993). Adult ticks were confined within plastic capsules attached to New Zealand white rabbits (Oryctolagus cuniculus) and allowed to feed and mate as required. Rearing conditions were 26 ± 1 °C, 92 ± 6% relative humidity and 14:10 (L:D). All use of animals was conducted at Old Dominion University and approved by the Institutional Animal Care and Use Committee.

cDNA synthesis and 454 library preparation

The initial Vg2 cDNA fragments

Full-length sequence and structural analysis of DvVg2

The Vg2 cDNA is 5956 nt in size containing an ATG start codon (nt 77–79), a TAA stop codon (nt 5852–5854), a putative poly-A addition signal (nt 5906–5910) and a poly-A tail (nt 5873–5956). The Vg2 cDNA has an open reading frame of 5775 nt coding for 1925 amino acids with a calculated molecular weight of 217 kDa. Protein–protein BLAST (blastp) showed that DvVg2 has low similarity to another Vg isolated from the same tick species where it is 20% identical and 31% similar to DvVg1 (AAW78557) on

Structural analysis of DvVg2

The Vg2 cDNA is 5956 nt and was essentially equivalent to the ∼6 kb band shown by Northern blot analysis (Fig. 3). Searching the available public databases for tick Vgs, we found 4 full-length Vg genes isolated from ticks: D. variabilis (AY885250), Ornithodoros moubata (AB440159) and 2 putative Vgs from Ixodes scapularis (XM_002415179 and XM_002403922). Boldbaatar et al. (2010) found multiple Vgs in Haemaphysalis longicornis, but these sequences were not available in GenBank for analysis. Two

Acknowledgements

This study was supported in part by grants (IBN-0315179 and IOS-0949194) from the National Science Foundation and the NC Ag. Research Service. L.A. Jeffers was also supported by a NIH Training Grant as a Ph.D. student in Biotechnology.

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