Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta

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Abstract

The juvenile hormones (JHs) regulate a diverse array of insect developmental and reproductive processes. One molecular target of JH action is its transporter, hemolymph JH binding protein (hJHBP); in the larva of the tobacco hornworm, Manduca sexta, low doses of JH can immediately increase hJHBP gene expression. Less explored are the effects of JH on embryological development, where early hormonal treatment has been shown to affect embryonic development and pupation. This study examines the egg form of JHBP and its gene expression during embryogenesis of M. sexta, as well as the phenotypic effect JH treatment has on embryos and on JHBP gene expression. We here demonstrate that the preponderance of JHBP found in the egg is maternally derived and that the embryonic gene and protein appear identical to those found in the larva. Expression of the JHBP gene begins in both the embryo itself and extra-embryonic tissues 15 h after fertilization, long before emergence of a functional fat body and circulatory system. Topical application of low JH doses to early embryos resulted in larval abnormalities while high doses of the hormone induced embryonic mortality. These effects are not mediated through regulation of the JHBP gene, since embryonic expression appears invariant in response to JH challenge. The toxicity of JH is tightly correlated with the concentration of unbound hormone.

Introduction

The development of the insect embryo requires appropriate spatial and temporal expression of a developmental program encoded by both the maternal and zygotic genomes (St Johnston and Nusslein-Volhard, 1992). Maternally derived mRNAs guide early body plan formation when translated in the zygote, activating specific gene-expression cascades leading to specification and differentiation of embryonic tissues. Disruption of these pathways results in a variety of immediate or latent morphogenetic defects (St Johnston and Nusslein-Volhard, 1992).

The juvenile hormones (JHs) and their analogs (JHAs) can profoundly alter the course of embryonic development depending upon their time of application. When unfertilized eggs of hemipterans and lepidopterans are subjected to JHs and/or JHAs, the embryos do not develop beyond the blastoderm stage (Riddiford and Williams, 1967, Enslee and Riddiford, 1977, Dorn, 1982, Kelly and Huebner, 1987). Yet, when JHs and JHAs are applied after fertilization but prior to blastoderm formation, the developing embryos appear normal but give rise to larvae that either fail to hatch or die before pupation (Riddiford and Williams, 1967, Riddiford, 1970). During the later stages of embryogenesis, JH appears to be required for production of the first larval cuticle (Dorn, 1982), as well as the gut and fat body (Brüning and Lanzrein, 1987).

In Manduca sexta larvae, the hemolymph JH binding protein (hJHBP) plays an important role in protecting JH from enzymatic degradation (Hammock et al., 1975) and in maintaining its bioavailability at peripheral sites (Goodman, 1990, Touhara and Prestwich, 1994). The tissue responsible for hJHBP synthesis in both the larva and adult is the fat body (Nowock et al., 1975, Orth et al., 2003). While the roles of hJHBP in the larval stage are well defined, the role of embryonic JHBP is less clear. JHBP or a hJHBP-like protein is present at fertilization and remains at detectable levels throughout embryogenesis (Touhara et al., 1994); yet levels of JH do not rise until the insect is midway through embryonic development. In addition, the circulatory system is not fully functional until late in embryogenesis, suggesting that JHBP plays roles that have yet to be determined. A clue to the resolution of this paradox may come from Locusta migratoria. In this species, JHBP similar to that found in the hemolymph is present in both the embryo and extra-embryonic serosa, prior to the development of the corpora allata and a functional circulatory system. Hartmann et al. (1987) demonstrated that the serosa has methyl transferase activity and is capable of converting JH acid to JH. Thus, JHBP synthesized by the serosa and secreted into the amniotic cavity may serve either to distribute the hormone or serve as a buffer to protect the embryo from excess JH.

Following dorsal closure in M. sexta, the corpora allata become active (Dorn et al., 1987) and JH titers rise (Bergot et al., 1981). Previous studies have demonstrated that JH regulates hJHBP gene expression in larvae of the black larval strain of M. sexta (Orth and Goodman, 1995, Orth et al., 1999), raising the possibility that JH fluctuations may regulate JHBP expression in the egg. Thus, two hypotheses were tested here. First, given our observation that exogenous JH in the larval stage modifies hJHBP expression, could such a response be observed in the embryo? Second, if JHBP gene expression is modified, is this the source of embryonic toxicity associated with over- or under-expression of JHBP induced by excess JH?

Section snippets

Egg collection

Insects were reared under a 16:8 L:D photoregime as described by Goodman et al. (1985a). Eggs were collected from tobacco leaves within 20 min of oviposition and incubated at 25±1 °C. Embryonic development was monitored using a dissecting microscope to record the onset of germ band formation, appendage formation and pigmentation of various structures (Dow et al., 1988). The eggs used to determine JHBP and total protein titers were collected and frozen at −80 °C. Each sample contained 10 eggs,

Homology of the embryonic protein and gene with the larval form

Previous studies by Touhara et al. (1994) demonstrated that a protein with molecular mass and binding characteristics similar to hJHBP was present in the egg of M. sexta; however, no developmental information about the putative embryonic JHBP or expression of its gene was presented. To avoid confusion, the term embryonic, unless otherwise stated, includes both the embryo and serosa. The western blot analysis in this study supports the hypothesis that embryonic JHBP and hJHBP are identical (Fig.

Discussion

In this tribute to the accomplishments of Larry Gilbert, it is fitting that we recall it was in his lab that Elaine Whitmore (Whitmore and Gilbert, 1972) first demonstrated the presence of hemolymph proteins capable of binding the naturally occurring homologs of JH. That pioneering work stimulated the subsequent identification and purification of high affinity JH binding proteins from the hemolymph of M. sexta and a number of other species (Goodman, 1990). During the past 30 years, this work

Acknowledgements

This work is supported by grants from the National Institutes of Health and the College of Agricultural and Life Sciences of the University of Wisconsin—Madison. APO was supported in part by the National Science Foundation Graduate Research Fellowship Program. We thank Ms. Hedda Goodman for her editorial assistance.

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    Present address: Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, Suite F117, La Jolla, CA 92121, USA

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