Ecdysis triggering hormone signaling in the yellow fever mosquito Aedes aegypti

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Abstract

At the end of each developmental stage, the yellow fever mosquito Aedes aegypti performs the ecdysis behavioral sequence, a precisely timed series of behaviors that culminates in shedding of the old exoskeleton. Here we describe ecdysis triggering hormone-immunoreactive Inka cells located at branch points of major tracheal trunks and loss of staining coincident with ecdysis. Peptides (AeaETH1, AeaETH2) purified from extracts of pharate 4th instar larvae have—PRXamide C-terminal amino acid sequence motifs similar to ETHs previously identified in moths and flies. Injection of synthetic AeaETHs induced premature ecdysis behavior in pharate larvae, pupae and adults. Two functionally distinct subtypes of ETH receptors (AeaETHR-A, AeaETHR-B) of A. aegypti are identified and show high sensitivity and selectivity to ETHs. Increased ETHR transcript levels and behavioral sensitivity to AeaETHs arising in the hours preceding the 4th instar larva-to-pupa ecdysis are correlated with rising ecdysteroid levels, suggesting steroid regulation of receptor gene expression. Our description of natural and ETH-induced ecdysis in A. aegypti should facilitate future approaches directed toward hormone-based interference strategies for control of mosquitoes as human disease vectors.

Introduction

During growth and development, all insects undergo ecdysis, the periodic shedding of the exoskeleton to increase their body size and facilitate morphological changes during metamorphosis. This intricate, yet life-threatening process requires precisely-timed developmental scheduling under the control of steroid and peptide hormones. To execute the sequence successfully, a peptide signaling cascade is programmed through steroid-induced gene expression to schedule an innate, stereotypic behavior. Recent reviews summarize our understanding of this process in moths and flies (Truman, 2005, Zitnan and Adams, 2005, Zitnan et al., 2007).

Elevation of 20-hydroxyecdysone (20E) at the end of the feeding phase of each instar leads to apolysis and initiation of the molt, during which the old cuticle is broken down and recycled into new cuticle. Subsequent decline of 20E is essential for initiation of the ecdysis sequence, which terminates the molt (Kingan and Adams, 2000, Truman et al., 1983, Zitnan et al., 1999). In Manduca sexta, release of ecdysis triggering hormones (PETH and ETH) from endocrine Inka cells initiates pre-ecdysis and ecdysis behaviors (Zitnan et al., 1999). Two ETH homologs, DrmETH1 and DrmETH2, were subsequently identified in Drosophila melanogaster (Park et al., 1999). ETH null mutants showed lethal ecdysis defects, indicating that ETH peptides are both necessary and sufficient to initiate the ecdysis behavioral sequence (Park et al., 2002). ETH peptides act directly on the central nervous system (CNS) to trigger a neuropeptide signaling cascade, which includes eclosion hormone (EH), kinins, diuretic hormones, crustacean cardiactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. These peptides together with ETH recruit central pattern generators that drive pre-ecdysis, ecdysis, and post-ecdysis behaviors (Kim et al., 2006a, Kim et al., 2006b). The production and release of ETH and PETH in Inka cells are regulated by 20E levels (Zitnan et al., 1999, Zitnanova et al., 2001). In addition, central neuronal circuits are also regulated by 20E. For example, competence of the CNS to respond to ETH is under steroid control (Zitnan et al., 1999, Zitnanova et al., 2001). Further investigation of the role ecdysteroids play in CNS sensitivity to ETHs is needed.

Inka cells and ETH homologs are widely distributed in all major insect orders (Zitnan et al., 2003), including Aedes aegypti, a highly anthropophilic mosquito responsible for transmission of dengue and yellow fever around the world. Limited information is available about ecdysis in A. aegypti, especially with regard to larval and pupal ecdyses. In this work, we use A. aegypti as model disease vector to evaluate the molecular regulation of the ecdysis behavioral sequence.

Section snippets

Experimental animals and behavior observation

Mosquitoes (A. aegypti) were raised at 24 °C and fed a standard diet (Lea, 1964). For behavior observations and recordings, one to four living mosquitoes were positioned in a drop of water on a slide glass and observed with a compound microscope, which was connected to a Sony CCD camera. Behaviors recorded on videotape were analyzed and edited with Adobe Premiere. Morphological markers were used to stage insects. To elicit premature ecdysis, 10 fmol of synthetic peptide (AeaETH1 or AeaETH2) was

Inka cells in A. aegypti

Using a MasPETH antiserum, we immunolabeled Inka cells of pharate 4th instar larvae and found them to be located on the surface of lateral tracheal trunks (Fig. 1A). Inka cells exhibit strong PETH-like immunoreactivity (PETH-IR) when stained ∼3 h prior to ecdysis. However, PETH-IR had disappeared when tissues were stained shortly after ecdysis (Fig. 1B) suggesting release of Inka cell peptides during ecdysis.

Identification of two ETH peptides from Inka cells of A. aegypti

To identify ETH peptides in A. aegypti, 400 pharate 4th instar larvae were fractionated

ETHs and ETH receptors in A. aegypti are highly conserved

Inka cells and ETH-like peptides have been described in a wide range of insect orders on the basis of immunohistochemical staining and across-species bioassays (Zitnan et al., 2003). However, definitive descriptions of specific ligands and their receptors, along with regulatory elements influencing their expression have been accomplished in only a few species: The moths M. sexta and Bombyx mori (Kim et al., 2006b, Zitnan et al., 2002, Zitnan et al., 1999) and the fruit fly, D. melanogaster (

Acknowledgments

We thank Alexander Raikhel for providing mosquito eggs, Ingrid Zitnanová for help with enzyme immunoassays, Dusan Zitnan for advice on immunohistochemistry, and Timothy Kingan for helpful discussions. We are especially grateful to Yoonseong Park for help with phylogenetic analysis. This work was supported by NIH Grant GM67310.

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    The nucleotide sequences reported in this paper have been deposited in the GenBank database and have the following Accession Nos. AeaETH gene (DQ864499), AeaETHR-A (DQ864500), AeaETHR-B (DQ864501).

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    Present address: The Brain Institute, 383 Colorow Dr., Rm. 361, University of Utah, Salt Lake City, UT 84108, USA.

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