Silencing Multiple Crustacean Hyperglycaemic Hormone-Encoding Genes in the Redclaw Crayfish Cherax quadricarinatus Induces Faster Molt Rates with Anomalies

Nickolis Black; Thomas M. Banks; Susan Glendinning; Gourab Chowdhury; Donald L. Mykles; Tomer Ventura

doi:10.3390/ijms252212314

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Silencing Multiple Crustacean Hyperglycaemic Hormone-Encoding Genes in the Redclaw Crayfish Cherax quadricarinatus Induces Faster Molt Rates with Anomalies

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Silencing Multiple Crustacean Hyperglycaemic Hormone-Encoding Genes in the Redclaw Crayfish Cherax quadricarinatus Induces Faster Molt Rates with Anomalies

Nickolis Black, Thomas M. Banks, Susan Glendinning, Gourab Chowdhury, Donald L. Mykles and Tomer Ventura

International Journal of Molecular Sciences, Vol.25(22), pp.1-18

2024

DOI: https://doi.org/10.3390/ijms252212314

PMCID: PMC11594818

PMID: 39596377

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Abstract

RNA interference

multigene silencing

chimeric dsRNA

gene blocks

crustaceans

decapods

RNA interference (RNAi)-based biotechnology has been previously implemented in decapod crustaceans. Unlike traditional RNAi methodologies that investigate single gene silencing, we employed a multigene silencing approach in decapods based on chimeric double-stranded RNA (dsRNA) molecules coined ‘gene blocks’. Two dsRNA constructs, each targeting three genes of the crustacean hyperglycaemic hormone (CHH) superfamily of neuropeptides, were produced: Type II construct targeting Cq-Molt-inhibiting hormone 1 (MIH1), Cq-MIH-like 1 (MIHL1), and Cq-MIHL2 isoforms and Type I construct targeting Cq-ion transport peptide (Cq-ITP; a putative hybrid of CHH and MIH) and Cq-CHH and Cq-CHH-like (CHHL) isoforms. Both constructs were injected into juvenile redclaw crayfish, Cherax quadricarinatus, to determine the effects of multigene knockdown on molting and developmental processes. A 20-Hydroxyecdysone (20E) enzyme-linked immunosorbent assay (ELISA) and glucose assay were used to determine the effects of RNAi on molting and hemolymph glycemic activities, respectively. Multigene silencing reduced the intermolt interval by 23%. Statistically significant elevated 20E was recorded in treated intermolt individuals, consistent with the reduced intermolt interval as well as unique and abnormal phenotypes related to the molting process, which indicates a shift in 20E-induced cascade. There was no effect of RNAi treatment on hemolymph glucose level or molt increment. Through multigene silencing and subsequent annotation of gene networks, gene blocks may provide a tailored approach to investigate complex polygenic traits with RNAi in a more efficient and scalable manner.

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Title: Silencing Multiple Crustacean Hyperglycaemic Hormone-Encoding Genes in the Redclaw Crayfish Cherax quadricarinatus Induces Faster Molt Rates with Anomalies
Authors: Nickolis Black - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Thomas M. Banks - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Susan Glendinning - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Gourab Chowdhury - University of the Sunshine Coast, Queensland, School of Science, Technology and Engineering
Donald L. Mykles - Colorado State University
Tomer Ventura (Corresponding Author) - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Publication details: International Journal of Molecular Sciences, Vol.25(22), pp.1-18
Publisher: MDPI AG
Date published: 2024
DOI: 10.3390/ijms252212314
ISSN: 1422-0067
PMID: 39596377; PMC11594818
Copyright note: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Data Availability: RNA-Seq data are available online at NCBI Sequence Read Archive with the following accession number: SAMN42566432. Additional data are contained within Supplementary Materials.
Grant note: This work was supported by the National Science Foundation (IOS-1922701).
Organisation Unit: School of Science, Technology and Engineering; Centre for Bioinnovation
Language: English
Record Identifier: 991081598702621
Output Type: Journal article

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