Two Splice Isoforms of Leptinotarsa Ecdysis Triggering Hormone Receptor Have Distinct Roles in Larva-Pupa Transition

Abstract

Insect ecdysis triggering hormone (ETH) receptors (ETHRs) are rhodopsin-like G protein-coupled receptors. Upon binding its ligand ETH, ETHR initiates a precisely programed ecdysis behavior series and physiological events. In Drosophila melanogaster, the ethr gene produces two functionally distinct splicing isoforms, ethra and ethrb. ETH/ETHRA activates eclosion hormone (EH), kinin, crustacean cardioactive peptide (CCAP), and bursicon (burs and pburs) neurons, among others, in a rigid order, to elicit the behavioral sequences and physiological actions for ecdysis at all developmental stages, whereas ETH/ETHRB is required at both pupal and adult ecdysis. However, the role of ETHRB in regulation of molting has not been clarified in any non-drosophila insects. In the present paper, we found that 20-hydroxyecdysone (20E) signaling triggers the expression of both ethra and ethrb in a Coleopteran insect pest, the Colorado potato beetle Leptinotarsa decemlineata. RNA interference (RNAi) was performed using double-stranded RNAs (dsRNAs) targeting the common (dsethr) or isoform-specific (dsethra, dsethrb) regions of ethr. RNAi of dsethr, dsethra, or dsethrb by the final-instar larvae arrested larva development. The arrest was not rescued by feeding 20E. All the ethra depleted larvae stopped development at prepupae stage; the body cavity was expanded by a large amount of liquid. Comparably, more than 80% of the ethrb RNAi larvae developmentally halted at the prepupae stage. The remaining Ldethrb hypomorphs became pupae, with blackened wings and highly-expressed burs, pburs and four melanin biosynthesis genes. Therefore, ETHRA and ETHRB play isoform-specific roles in regulation of ecdysis during larva-pupa transition in L. decemlineata.

RNA interference targeting Ras GTPase gene Ran causes larval and adult lethality in Leptinotarsa decemlineata

“…The same method as previously described 34,35 was used to express ds LdRan and ds egfp , derived from a 361‐bp fragment of LdRan and a 414‐bp enhanced green fluorescent protein ( egfp ) gene from Aequorea victoria , using specific primers listed in Table S1. All dsRNAs were generated by Escherichia coli HT115 (DE3) competent cells lacking RNase III following the established method 10,11 . The bacterial solutions (at a dsRNA concentration of about 0.5 μg/mL) were used for the experiment.…”

“…All dsRNAs were generated by Escherichia coli HT115 (DE3) competent cells lacking RNase III following the established method. 10,11 The bacterial solutions (at a dsRNA concentration of about 0.5 μg/mL) were used for the experiment.…”

“…The identification of candidate genes is a crucial step in the development of dsRNA insecticides 4 . In this context, ingestion of dsRNA targeting combinatorial hormone signaling networks regulating development (juvenile hormone, JH; 20‐hydroxyecdysone, 20E; insulin like peptide, ILP) can cause larval lethality in L. decemlineata 10–19 . The knockdown of genes involved in important cellular functions like vacuolar‐type adenosine triphosphatase (vATPase) 7,8 or metabolic activities such as chitin production and deposition 20–23 hampers larval growth.…”

“…4 In this context, ingestion of dsRNA targeting combinatorial hormone signaling networks regulating development (juvenile hormone, JH; 20-hydroxyecdysone, 20E; insulin like peptide, ILP) can cause larval lethality in L. decemlineata. [10][11][12][13][14][15][16][17][18][19] The knockdown of genes involved in important cellular functions like vacuolar-type adenosine triphosphatase (vATPase) 7,8 or metabolic activities such as chitin production and deposition [20][21][22][23] hampers larval growth. The beetles are multivoltine defoliators that produce multiple overlapping generations each year.…”

Stabilized Double-Stranded RNA Strategy Improves Cotton Resistance to CBW (Anthonomus grandis)

“…Since these processes are exclusive to arthropods, homologous proteins are not found in other organisms, making genes from these metabolic and signaling pathways suitable for RNAi molecule development [ 53 ]. Several studies have shown abnormal development in species in which ETH or ETHr genes were knocked down by RNAi [ 54 , 55 , 56 , 57 , 58 ]. Furthermore, our group has previously demonstrated that knockdown of ETHr in CBW larvae leads to lethality and uncompleted metamorphosis [ 59 ].…”