How the 'kitome' influences the characterization of bacterial communities in lepidopteran samples with low bacterial biomass.

Abstract

We aimed to elucidate whether the DNA extraction kit and bacteria therein affect the characterization of bacterial communities associated with butterfly samples harbouring different bacterial abundancies. We analysed bacteria associated with eggs of Pieris brassicae and with adults of this butterfly, which were either untreated or treated with antibiotics (ABs). Three DNA extraction kits were used. Regardless of the extraction kit used, PCR amplification of the bacterial 16S rRNA gene detected very low bacterial presence in eggs and AB-treated butterflies. In untreated butterflies, bacterial signal intensity varied according to the kit and primers used. Sequencing (MiSeq) of the bacterial communities in untreated and AB-treated butterflies revealed a low alpha diversity in untreated butterflies because of the dominance of few bacteria genera, which were detectable regardless of the kit. However, a significantly greater alpha diversity was found in AB-treated butterflies, evidencing a true bias of the results due to bacterial contaminants in the kit. The so-called 'kitome' can impact the profiling of Lepidoptera-associated bacteria in samples with low bacterial biomass. Our study highlights the necessity of method testing and analysis of negative controls when investigating Lepidoptera-associated bacterial communities.