Sample Preparation for Transcriptomics of Honeybee Larvae to Identify Varroa destructor Resistance Genes

Sammanfattning

European honeybee (Apis mellifera) populations face severe declines, primarily driven by the parasitic mite Varroa destructor and its transmission of Deformed Wing Virus (DWV). A unique honeybee population on the Swedish island of Gotland has developed natural resistance, surviving mite infestation for decades by altering chemical signals to disrupt mite reproduction. To identify the underlying genetic mechanisms via future transcriptomic sequencing, establishing a robust RNA extraction protocol from lipid-rich larvae is critical. This study aimed to optimize RNA extraction and conduct viral screening. Two commercial kits, the RNeasy Mini Kit and the GeneJET RNA Purification Kit, were compared, and a cold centrifugation step (4 °C) was evaluated to handle high lipid content. Viral prevalence was subsequently analyzed using RT-qPCR. Both kits yielded sufficient RNA, but the RNeasy Mini Kit demonstrated higher stability and consistent purity. Additionally, cold centrifugation effectively solidified lipids, substantially improving the total RNA yield. Viral screening revealed a significantly lower DWV prevalence in the resistant Gotland larvae compared to susceptible controls, despite DWV circulating as a widespread background infection. Moreover, quantitative analysis showed that Varroa infestation significantly increased the virus titer in affected larvae. In conclusion, an optimized RNA extraction protocol for lipid-rich larvae was successfully established, paving the way for downstream genomic sequencing. Furthermore, the reduced DWV prevalence in resistant larvae suggests an active antiviral immune response that synergizes with their mite-suppressing traits.