Table 1.
Sequences and product length for primers used in synthesis of dsRNA.
Fig 1.
Two-step bioassay to identify the effect of the suppression of the pathway genes on WCR adults and to determine if the mortality caused by vATPase A dsRNA can be rescued by suppressing the expression of pathway genes.
Table 2.
Sequences and other relevant parameters for each primer pair used for expression analysis by qRT-PCR.
Table 3.
Sequence identity and their best sequence match from Blastx search of the NCBI database.
Fig 2.
Domain analysis of the WCR RNAi pathway genes.
(A) WCR DCR2 contain total of 6 domains, helicase ATP binding type 1 domain (helicase ATP), helicase C-terminal domain (helicase C), dicer double stranded RNA binding domain (dicer_dsrbf), PAZ domain and two ribonuclease III family domains (Rnase). (B) WCR AGO2 contains 2 domains, PAZ and PIWI. Domain’s for DCR2 and AGO2 were predicted using ScanProsite (http://prosite.expasy.org/scanprosite/).
Fig 3.
RNAi mortality suppression at day 14.
Newly emerged beetles were injected with RNAi pathway gene dsRNA on day one. On day 4 adults were exposed to vATPase A dsRNA. Mortality was collected daily. Percent mortality at day 14 represents mean adult mortality calculated from two replications each containing 30 adults for a total of 60 beetles per treatment. Different letters represent significant differences at P value < 0.05.
Fig 4.
Relative expression of RNAi pathway genes and laccasse2 in WCR adults.
Newly emerged WCR adults were injected with 600 ng of Argonaute2, Dicer2, laccase2 and GFP dsRNA or equal volume of water on day one and allowed to feed on untreated artificial diet. On day 4 adults were exposed to vATPase A dsRNA as surface treatment on artificial diet. On day 7 adults were collected and flash frozen in liquid nitrogen, and were used for gene expression analyses using qRT-PCR. (A) Relative Argonaute2 expression. (B) Relative Dicer2 expression. (C) Relative laccase2 expression. Standard errors were determined from two biological replications, each including three individual beetles (n = 6), each with two technical replications. Different letters within a figure represent significant differences at P value < 0.05.
Fig 5.
Expression analysis of vATPase A gene after both RNAi pathway gene dsRNA and vATPase A dsRNA exposures.
Newly emerged WCR adults were injected with 600 ng of dsRNA or equal volume of water and allowed to feed on untreated artificial diet. On day 4 adults were exposed to vATPase A dsRNA as surface treatment on artificial diet. Three days after vATPase A dsRNA exposure, adults were collected and flash frozen in liquid nitrogen and used for vATPase expression evaluation using qRT-PCR. Adults that were only injected with water, GFP dsRNA and laccase2 dsRNA but not exposed to vATPase A dsRNA were included as controls. Standard errors were determined from six independent biological replications (n = 6), each with two technical replications. Different letters within a figure represent significant difference at P value < 0.05.