Abstract

Amrasca biguttula biguttula (Ishida) commonly known as cotton leafhopper is a severe pest of cotton and okra. Not much is known on this insect at molecular level due to lack of genomic and transcriptomic data. To prepare for functional genomic studies in this insect, we evaluated 15 common housekeeping genes (Tub, B-Tub, EF alpha, GADPH, UbiCF, RP13, Ubiq, G3PD, VATPase, Actin, 18s, 28s, TATA, ETF, SOD and Cytolytic actin) during different developmental stages and under starvation stress. We selected early (1st and 2nd), late (3rd and 4th) stage nymphs and adults for identification of stable housekeeping genes using geNorm, NormFinder, BestKeeper and RefFinder software. Based on the different algorithms, RP13 and VATPase are identified as the most suitable reference genes for quantification of gene expression by reverse transcriptase quantitative PCR (RT-qPCR). Based on RefFinder which comprehended the results of three algorithms, RP13 in adults, Tubulin (Tub) in late nymphs, 28S in early nymph and UbiCF under starvation stress were identified as the most stable genes. We also developed methods for feeding double-stranded RNA (dsRNA) incorporated in the diet. Feeding dsRNA targeting Snf7, IAP, AQP1, and VATPase caused 56.17–77.12% knockdown of targeted genes compared to control and 16 to 48% mortality of treated insects when compared to control.

Document Type

Article

Publication Date

1-12-2018

Notes/Citation Information

Published in PLOS ONE, v. 13, no. 1, e0191116, p. 1-21.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Digital Object Identifier (DOI)

https://doi.org/10.1371/journal.pone.0191116

Funding Information

The internal funding of Punjab Agricultural University supported this research.

journal.pone.0191116.s001.doc (120 kB)
S1 Table. Descriptive analysis of all candidate reference genes by calculating standard deviation and p-values by Bestkeeper.

journal.pone.0191116.s002.docx (12 kB)
S2 Table. Mortality calculated in bioassays with dsRNA of different genes in comparison to dsGFP.

Share

COinS