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United States Department of Agriculture

Agricultural Research Service

Title: Longevity of ingested mRNA transcripts in the gut of a Homopteran (Bemisia tabaci): Avoiding experimental artifacts

Authors
item Sinisterra, Xiomara - UNIV. OF FLORIDA
item Shatters, Robert
item Hunter, Wayne
item Powell, Charles - UNIV. OF FLORIDA
item McKenzie, Cindy

Submitted to: Entomologia Experimentalis et Applicata
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 15, 2006
Publication Date: November 1, 2006
Citation: Sinisterra, X.H., Shatters, R.G. Jr, Hunter, W.B., Powell, C.A., McKenzie, C.L. 2006. Longevity of ingested mRNA transcripts in the gut of a homopteran (Bemisia tabaci): avoiding experimental artifacts. Entomologia Experimentalis et Applicata. 121(3): 273-279

Interpretive Summary: Experiments examining insect vector-plant virus interactions traditionally use methods to clear vector guts of ingested pathogens or macromolecules (DNA, RNA, Proteins) by having the insect feed on either a non-host plant for the pathogen, or on an artificial diet such as a sucrose solution. We compared the efficacy of two standard methods used to clear insect alimentary tracts of ingested materials: 1) feeding on a sucrose solution and 2) feeding on a non-viral host plant. The presence of tomato transcripts (Rubisco, Chlorophyll a/b binding protein) in viruliferous and non-viruliferous whiteflies was evaluated by RT (reverse transcription)-PCR. Whiteflies reared on tomato were fed on sucrose solution for 72 hr or on cotton plants for 72 h. Plant transcripts were detected in whiteflies immediately removed from the infected tomato plants and surprisingly remained detectable after 72 h of sucrose feeding. However, plant transcripts were not detectable when the whiteflies fed on cotton for 72 h. This discovery shows that sucrose feeding is not an efficient clearing method for use in whitefly virus-vector interaction studies. Our results show the importance of using active plant feeding to degrade and 'flush' foreign molecules in whiteflies and may be applicable to homopterans as a group.

Technical Abstract: Experiments examing insect vector-plant virus interactions traditionally use methods to clear vector guts of ingested pathogens or macromolecules (DNA, RNA, Proteins) by having the insect feed on either a non-host plant for the pathogen, or on an artificial diet such as a sucrose solution. The ability to rid the insect of ingested host derived virus particles and products that do not cross the epithelium is particularly important in studies whose intent is to measure virus replication and/or virus molecular activity in the insect. We compared the efficacy of two standard methods used to clear insect alimentary tracts of ingested materials: 1) feeding on a sucrose solution and 2) feeding on a non-viral host plant. The presence of tomato transcripts (Rubisco, Chlorophyll a/b binding protein) in viruliferous and non-viruliferous whiteflies was evaluated by RT (reverse transcription)-PCR. Whiteflies reared on tomato were fed on sucrose solution for 72 h or on cotton for 72 h. Plant transcripts were detected in whiteflies immediately removed from the infected tomato and surprisingly remained detectable after 72 h of sucrose feeding. However, plant transcripts were not detectable when the whiteflies fed on cotton for 72 h. Results were the same in non-viruliferous and viruliferous whiteflies. This discovery shows that sucrose feeding is not an efficient clearing method for use in whitefly virus-vector interaction studies and additional experimental controls are necessary to ensure appropriate interpretation of the data in such studies. Our results show the importance of using active plant feeding to degrade and 'flush' foreign molecules in whiteflies and may be applicable to homopterans as a group.

Last Modified: 10/1/2014
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