Improving RNAi in the Brown Marmorated Stink Bug: Identification of target genes and reference genes for RT-qPCR

Kanakachari Mogilicherla; Jeffrey L. Howell; Subba Reddy Palli

doi:10.1038/s41598-018-22035-z

Improving RNAi in the Brown Marmorated Stink Bug: Identification of target genes and reference genes for RT-qPCR

Kanakachari Mogilicherla, Jeffrey L. Howell, Subba Reddy Palli

Research output: Contribution to journal › Article › peer-review

42 Scopus citations

Abstract

The brown marmorated stink bug (BMSB) is native to Asia and recently invaded the USA. RNA interference (RNAi) is a gene silencing mechanism in which the introduction of double-stranded RNA (dsRNA) inhibits gene function by degrading target mRNA. In dsRNA stability assays, the dsRNases present in the hemolymph and salivary gland secretions of BMSB showed lower activity than those in the hemolymph of Heliothis virescens. We evaluated six housekeeping genes (18S rRNA, EF1-α, Actin, Ubiquitin, 60S RP and β-Tubulin) across dsRNA treatments (injection and feeding) in nymphs and adults of BMSB and identified 18S rRNA and 60S RP as the best genes to use as a reference in reverse-transcriptase quantitative PCR (RT-qPCR). Homologs of 13 genes that were shown to function as effective RNAi targets in other insects were identified and evaluated by injecting dsRNA targeting these homologs into BMSB adults. Five out of 13 dsRNAs tested caused more than 70% mortality by seven days after injection of dsRNA. Feeding dsRNA targeting five of these genes (IAP, ATPase, SNF7, GPCR, and PPI) to nymphs caused more than 70% mortality by three of the five dsRNAs tested. These data suggest that feeding dsRNA causes target gene knockdown and mortality in BMSB.

Original language	English
Article number	3720
Journal	Scientific Reports
Volume	8
Issue number	1
DOIs	https://doi.org/10.1038/s41598-018-22035-z
State	Published - Dec 1 2018

Bibliographical note

Funding Information:
This is publication number 18-08-027 from the Kentucky Agricultural Experimental Station and is published with the approval of the director. This work was supported by the USDA HATCH under 2351177000. We thank Dr. Ric Bessin for help with collection and rearing of insects.

Publisher Copyright:
© 2018 The Author(s).

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title = "Improving RNAi in the Brown Marmorated Stink Bug: Identification of target genes and reference genes for RT-qPCR",

abstract = "The brown marmorated stink bug (BMSB) is native to Asia and recently invaded the USA. RNA interference (RNAi) is a gene silencing mechanism in which the introduction of double-stranded RNA (dsRNA) inhibits gene function by degrading target mRNA. In dsRNA stability assays, the dsRNases present in the hemolymph and salivary gland secretions of BMSB showed lower activity than those in the hemolymph of Heliothis virescens. We evaluated six housekeeping genes (18S rRNA, EF1-α, Actin, Ubiquitin, 60S RP and β-Tubulin) across dsRNA treatments (injection and feeding) in nymphs and adults of BMSB and identified 18S rRNA and 60S RP as the best genes to use as a reference in reverse-transcriptase quantitative PCR (RT-qPCR). Homologs of 13 genes that were shown to function as effective RNAi targets in other insects were identified and evaluated by injecting dsRNA targeting these homologs into BMSB adults. Five out of 13 dsRNAs tested caused more than 70% mortality by seven days after injection of dsRNA. Feeding dsRNA targeting five of these genes (IAP, ATPase, SNF7, GPCR, and PPI) to nymphs caused more than 70% mortality by three of the five dsRNAs tested. These data suggest that feeding dsRNA causes target gene knockdown and mortality in BMSB.",

author = "Kanakachari Mogilicherla and Howell, {Jeffrey L.} and Palli, {Subba Reddy}",

note = "Funding Information: This is publication number 18-08-027 from the Kentucky Agricultural Experimental Station and is published with the approval of the director. This work was supported by the USDA HATCH under 2351177000. We thank Dr. Ric Bessin for help with collection and rearing of insects. Publisher Copyright: {\textcopyright} 2018 The Author(s).",

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N1 - Funding Information: This is publication number 18-08-027 from the Kentucky Agricultural Experimental Station and is published with the approval of the director. This work was supported by the USDA HATCH under 2351177000. We thank Dr. Ric Bessin for help with collection and rearing of insects. Publisher Copyright: © 2018 The Author(s).

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N2 - The brown marmorated stink bug (BMSB) is native to Asia and recently invaded the USA. RNA interference (RNAi) is a gene silencing mechanism in which the introduction of double-stranded RNA (dsRNA) inhibits gene function by degrading target mRNA. In dsRNA stability assays, the dsRNases present in the hemolymph and salivary gland secretions of BMSB showed lower activity than those in the hemolymph of Heliothis virescens. We evaluated six housekeeping genes (18S rRNA, EF1-α, Actin, Ubiquitin, 60S RP and β-Tubulin) across dsRNA treatments (injection and feeding) in nymphs and adults of BMSB and identified 18S rRNA and 60S RP as the best genes to use as a reference in reverse-transcriptase quantitative PCR (RT-qPCR). Homologs of 13 genes that were shown to function as effective RNAi targets in other insects were identified and evaluated by injecting dsRNA targeting these homologs into BMSB adults. Five out of 13 dsRNAs tested caused more than 70% mortality by seven days after injection of dsRNA. Feeding dsRNA targeting five of these genes (IAP, ATPase, SNF7, GPCR, and PPI) to nymphs caused more than 70% mortality by three of the five dsRNAs tested. These data suggest that feeding dsRNA causes target gene knockdown and mortality in BMSB.

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Improving RNAi in the Brown Marmorated Stink Bug: Identification of target genes and reference genes for RT-qPCR

Abstract

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