Abstract
Insect ecdysis behavior, shedding off the old cuticle, is under the control of specific neuropeptides with the top command by the ecdysis triggering hormone (ETH). We characterized the ETH receptor (ETHR) of the malaria mosquito, Anopheles gambiae, by manual annotation of the NCBI gene (AGAP002881) and functional analysis, using a heterologous expression system in a mammalian cell line. The two splicing variants of ETHRs, ecdysis triggering hormone receptors (AgETHR-A and AgETHR-B), a conserved feature among insects, included of four (552 aa) and five exons (635 aa), respectively. The main feature of manual annotation of the receptor was a correction of N-terminal and exon-intron boundaries of an annotated gene (AGAP002881). Interestingly, the functional expression of the receptor in Chinese hamster ovary cells required modification of the transcription initiation site for mammalian Kozak consensus. In the calcium mobilization assay using the heterologous expression of each receptor, AgETHR-B showed a higher sensitivity to AgETH-1 (28 times) and AgETH-2 (320 times) than AgETHR-A. The AgETHRs showed specificity only to the ETH group of peptides but not to other groups carrying the C-termini motifs as PRXamide, such as pyrokinin1/DH and pyrokinin2/PBAN. Ecdysis triggering hormone receptors (AgETHR-B) responded to different ETH variants of other insect species more promiscuously than AgETHR-A.
Highlights
In insect growth and development, shedding old cuticles in the ecdysis involves an innate behavioral sequence orchestrated by a set of neuropeptides
The total gene spans >34.7 kb, which consisted of two contigs AAAB01008859_59 (13,937 bp) and AAAB01008859_58 (13,289 bp in AgETHR-A and 20,779 bp in AgETHR-B; Figure 1)
While the correct N-terminal of AgETHRs was amplified by the primer (Table 1) designed from the upstream of the correct translation initiation site, it was modified by a manual annotation (Figure 1A)
Summary
In insect growth and development, shedding old cuticles in the ecdysis involves an innate behavioral sequence orchestrated by a set of neuropeptides. The top command molecule, ecdysis triggering hormone (ETH) is released from Inka cells (Zitnan et al, 1996) at the initiation of the behavior. The hormonal action of the ETH peptide exerts its function by activating ETH receptors (ETHR) in the central nervous system to coordinate preecdysis and ecdysis behaviors. The eth gene encodes two similar peptides, i.e., pre-ecdysis triggering hormone and ETH, in Manduca sexta (Zitnan et al, 1999). In addition to the role of ETH in ecdysis, it acts as an obligatory allatotropin to promote juvenile hormone production for reproduction (Areiza et al, 2014). In Aedes aegypti, ETH acts as an allatotropic regulator of juvenile hormone III. ETH is known to play a critical role in the proper functioning of octopaminergic neurons to control the reproductive tract and ovulation and local interneurons of the antennal lobe for male–male courtship inhibition in Drosophila melanogaster (Meiselman et al, 2018; Deshpande et al, 2019)
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