Quantification of vitellogenin–mRNA during maturation and breeding of a burying beetle

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Abstract

Burying beetles (Nicrophorus orbicollis) are unusual in that to breed they require an unpredictable and valuable resource, a small carcass. Thus the timing of reproduction is unpredictable and beetles’ physiological response must be fast. We hypothesized that their pattern of vitellogenin (Vg) synthesis might reflect these requirements. We examined the expression of two Vg genes (sequenced for this study) during sexual maturation and through a reproductive bout. Vg–mRNA, juvenile hormone (JH) titers, ovarian development, and hemolymph concentrations of Vg were quantified in the same individuals. All four variables gradually increased during maturation to peak 15–20 days after eclosion. Twelve hours after the discovery of a carcass, a few hours before oviposition, mRNA was high, hemolymph Vg had decreased, JH and ovarian weight had increased. After oviposition, mRNA was low, hemolymph Vg concentrations and JH were high. This is consistent with our hypothesis that beetles produce and store Vg in the hemolymph prior to the discovery of a breeding resource and replace it quickly. Partial regression of these variables (with the effect of time removed) indicated that JH was not correlated with mRNA, hemolymph Vg, or ovarian weight at any time. Thus the role of JH as a gonadotropin remains unclear.

Introduction

Reproductive behavior of insects is extremely varied. While some emerge as adults and do not require environmental or social cues to breed, others undergo regular or opportunistic reproduction that depends on the location of specific resources. In some species, females have short or even long periods of parental care in their reproductive cycles (Trumbo, 1996, Trumbo, 2002). The hormonal control of reproductive physiology of females has been studied in dozens of species but few general rules have emerged (Nijhout, 1994; Wyatt and Davey, 1996; Bellés, 1998). In long-lived insects with multiple reproductive bouts, juvenile hormone (JH) is the common, but not universal, gonadotropin regulating the synthesis of vitellogenin (Vg), egg maturation and oviposition (Koeppe et al., 1985; Wyatt and Davey, 1996; Gilbert et al., 2000). However species differ in which aspect of reproduction is regulated. 20-hydroxyecdysone along with various neuroendocrine hormones have also been shown to play gonadotropic roles. In most species, Vg synthesis is promoted by JH. A notable exception are the Diptera, in which Vg synthesis is stimulated by ecdysteroids, although previous exposure to JH is required.

Burying beetles (Silphidae: Nicrophorus) undergo cyclical reproduction cued by a complex set of factors. To breed they require a small vertebrate carcass (typically 10–60 g), a valuable but unpredictable resource. A male and female cooperate to bury and prepare it. They are unusual in a having a hormonal surge (JH) triggered by behavioral cues associated with the discovery and assessment of the carcass (Trumbo et al., 1995). Neither a mate without a carcass, nor the nutritional content of the carcass is sufficient for ovarian development (Scott and Traniello, 1987). JH hemolymph titers drop about the time of oviposition (Trumbo, 1997) and rise again to peak when larvae hatch and parental care is most intense. Three days later JH titers fall to pre-breeding levels. Both parents feed and defend the young although typically, females remain longer with the brood than males (Scott and Traniello, 1990). In spite of these dramatic changes in JH hemolymph titers that are correlated with reproductive events, the application of JH III or its analogue, methoprene, is not sufficient to cause Vg up-take by the ovaries (Scott et al., 2001). This raises the question of the role of JH in the reproductive physiology of burying beetles.

This study is a first step in addressing this question. Here we examine the transcription of Vg–mRNA by qPCR, and look for correlations with hemolymph titers of JH, ovarian development and hemolymph concentrations of Vg which we measure in the same individuals over the 20 days after eclosion that is required for sexual maturation and during a breeding bout. To do this we first identified two vitellogenin genes in burying beetles by sequencing a portion from the conserved GL/ICG region to the C-terminus (Lee et al., 2000).

Section snippets

Animals and breeding manipulations

All beetles were laboratory-reared from a colony derived from Nicrophorus orbicollis captured in Durham, NH. Beetles were maintained in boxes containing damp paper towels with up to six like-sex individuals, fed mealworms and kidney, and maintained at 20C and 14:10 L:D. To breed, a male and female were placed in a plastic box (19×14×10 cm) of autoclaved soil with a previously frozen mouse (laboratory culls from the Dana Farber Cancer Institute and the Forsyth Institute, Boston, MA). On the day

Identification of vitellogenin genes

Based on the sequences from the conserved GL/ICG motif to the C-terminus, we identified two vitellogenin genes in N. orbicollis (AY728384, AY728385), six clones were of Vg1 and 4 clones were of Vg2. The deduced amino acid sequences were 59.3% similar. Both fragments were about 531 bp. The phylogenetic distance tree constructed by aligning Nicrophorus aa sequences with those of other insects (Fig. 2) is in general agreement with similar studies (Chen et al., 1997; Nose et al., 1997; Lee et al.,

Discussion

The eggs of most animals are provisioned by vitellogenins that are synthesized, by insects, primarily in the fat body and transported to the developing oocytes via the hemolymph (Wyatt and Davey, 1996). The primary function of Vg is to provide amino acids and nutrition (carbohydrates, lipids, etc.) for the developing embryo. Because of its nutritive function selective constraints on Vg are low and it tends to be very variable among taxa (Chen et al., 1997; Hagedorn et al., 1998; Sappington and

Acknowledgements

Special thanks to Andrew Laudano who was particularly generous with his time and equipment. Rick Cote, David Berlinsky, James Taylor, and Sue Chase provided equipment, programs and help to analyze data. David Borst generously provided the JH antibody and taught us RIA. S. Yoganathan at the Forsyth Institute supplied us with laboratory culls of mice. The project was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number

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