Longevity of ingested mRNA transcripts in the gut of a homopteran (Bemisia tabaci): avoiding experimental artifacts

Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) biotype B, also known as Bemisia argentifolii (Bellows et al., 1994), has been characterized as the main vector of begomoviruses, a virus genus that poses a major threat to the production of many vegetable crops worldwide (Polston & Anderson, 1997; Mansoor et al., 2003). Begomoviruses are transmitted by whiteflies in a persistent circulative manner (Hunter et al., 1998; Rosell et al., 1999; Ghanim & Czosnek, 2001). However, important aspects of virus–vector interactions at the molecular level remain controversial. Replication and/or molecular activity of some begomoviruses in the whitefly have been suggested (Ghanim & Czosnek, 2000; Czosnek et al., 2001; Sinisterra et al., 2005). We have studied the genetic activity of a begomovirus, tomato yellow leaf curl virus (TYLCV), in the whitefly vector by measuring the presence of selected virus transcripts (V1, V2, and C3) using real-time reverse transcription-polymerase chain reaction (RT-PCR) methods (Sinisterra et al., 2005). In those studies, it was important to rid the insect of ingested host derived virus particles and products that do not cross the gut-epithelium. In order to conduct those quantitative studies, we needed to find a method that would guarantee the removal of any virus transcript ingested by the whiteflies and remaining in the alimentary canal, thereby allowing the quantification of virus transcripts that are either stably acquired or synthesized de novo in the insect. Laboratory manipulation of different RNA species has shown that RNA is very easily degradable due to the ubiquitous presence of RNases. These enzymes can maintain their activity even after being subjected to many forms of harsh treatments including autoclaving and boiling (Sambrook & Russel, 2001). Because high RNase activity has been identified in plant phloem sap, RNase inactivation techniques have been used in studies that intend to detect plant mRNA moieties in samples collected by severing the stylets of aphids that were allowed to feed overnight on barley plants (Doering-Saad et al., 2002). We tested the efficiency of two gut-clearing methods: (i) non-viral host feeding and (ii) artificial diet feeding by monitoring the permanence of ingested plant mRNA transcripts within non-viruliferous and viruliferous insects. The work presented is to call attention to the finding that typical RNA transcripts are not as unstable in the alimentary canal of phloem feeders such as whiteflies (and potentially other homopterans) as may have been thought. The stability of such molecules should be taken into consideration in the experimental design of studies concerning molecule retention/interaction studies in insects.