Identification of spider-mite species and their endosymbionts using multiplex PCR
Spider mites of the genus Tetranychidae are severe crop pests. In the Mediterranean a few species coexist, but they are difficult to identify based on morphological characters. Additionally, spider mites often harbour several species of endosymbiotic bacteria, which may affect the biology of their hosts. Here, we propose novel, cost-effective, multiplex diagnostic methods allowing a quick identification of spider-mite species as well as of the endosymbionts they carry. First, we developed, and successfully multiplexed in a single PCR, primers to identify Tetranychus urticae, T. evansi and T. ludeni, some of the most common tetranychids found in southwest Europe. Moreover, we demonstrated that this method allows detecting multiple species in a single pool, even at low frequencies (up to 1/100), and can be used on entire mites without DNA extraction. Second, we developed another set of primers to detect spider-mite endosymbionts, namely Wolbachia, Cardinium and Rickettsia in a multiplex PCR, along with a generalist spider-mite primer to control for potential failure of DNA amplification in each PCR. Overall, our method represents a simple, cost-effective and reliable method to identify spider-mite species and their symbionts in natural field populations, as well as to detect contaminations in laboratory rearings. This method may easily be extended to other species.
KeywordsTetranychidae Wolbachia Cardinium Rickettsia Species identification Multiplex diagnosis tool
We are grateful to Olivier Duron for useful discussions and comments, and to Inês Santos for taking care of plants and mite populations. This work was funded by an FCT-ANR project (FCT-ANR//BIA-EVF/0013/2012) to SM and Isabelle Olivieri, and by an FCT-Tubitak project (FCT-TUBITAK/0001/2014) to SM and Ibrahim Cakmak. FZ was funded through an FCT Post-Doc fellowship (SFRH/BPD/125020/2016). Funding agencies did not participate in the design or analysis of experiments. We declare that we do not have any conflict of interest.
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