Ecdysis triggering hormone signaling in the yellow fever mosquito Aedes aegypti☆
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.
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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.
References (24)
- et al.
Molecular identification of the first insect ecdysis triggering hormone receptors
Biochem. Biophys. Res. Commun.
(2002) - et al.
A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles
Curr. Biol.
(2006) A competitive enzyme-linked immunosorbent assay: applications in the assay of peptides, steroids, and cyclic nucleotides
Anal. Biochem.
(1989)- et al.
Two subtypes of ecdysis-triggering hormone receptor in Drosophila melanogaster
J. Biol. Chem.
(2003) - et al.
Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster
FEBS Lett.
(1999) Hormonal control of insect ecdysis: endocrine cascades for coordinating behavior with physiology
Vitam. Horm.
(2005)- et al.
Ecdysteroids regulate the release and action of eclosion hormone in the tobacco hornworm, Manduca sexta (L)
J. Insect. Physiol.
(1983) - et al.
Neuroendocrine regulation of insect ecdysis
Compr. Mol. Insect Sci.
(2005) - et al.
Steroid induction of a peptide hormone gene leads to orchestration of a defined behavioral sequence
Neuron
(1999) Aedes aegypti (L.)
Knockdown of ecdysis-triggering hormone gene with a binary UAS/GAL4 RNA interference system leads to lethal ecdysis deficiency in silkworm
Acta Biochim. Biophys. Sin. (Shanghai)
Strategic expression of ion transport peptide gene products in central and peripheral neurons of insects
J. Comp. Neurol.
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2020, General and Comparative EndocrinologyCitation Excerpt :The ETH1 elicits stronger response than ETH2 at lower concentrations (Park et al., 2003; Shi et al., 2017). However, both ETHs activated ETHR and trigger ecdysis behavior in A. aegypti at pupal stage in the absence of pre-ecdysis behavior, although the effect of both ETHs on triggering ecdysis behavior is slightly different (Dai and Adams, 2009). Both A. aegypti ETHs share the same motif FFxKxxKxxPRx, while ETH1 and ETH2 in both D. melanogaster and B. dorsalis share only the insect motif –KxxKxxPRx.
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2019, Comparative Biochemistry and Physiology -Part A : Molecular and Integrative PhysiologyCitation Excerpt :In the functional assay of PcETHR, the result demonstrated that the endogenous peptide PcETH activated the receptor in a dose-dependent manner with an EC50 of 1.41 μM. Comparatively, ligand activation potencies on ETHRs from insects D. melanogaster (Iversen et al., 2002; Park et al., 2003), M. sexta (Kim et al., 2006), A. aegypti (Dai and Adams, 2009), S. gregaria (Lenaerts et al., 2017) and B. dorsalis (Shi et al., 2017) varied diversely, with EC50s ranging from subnanomolar to micromolar level. One possible reason accounting for a low sensitivity of PcETHR is the inefficient coupling of PcETHR to the Gα16 applied in this functional CHO cell-based assay system.
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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.