Fourteen barcoded specimens of Cornus-feeding Phyllocnistis spp. were screened for infection by Wolbachia spp. (Rickettsiaceae). The two genes, wsp and fbpA, were amplified with the three sets of primers: (1) wspecF (5’-CATACCTAT TCGAAGGGATAG-3’), wspecR (5’-AGCTTCGAGTGAAACCAATTC-3’) and (2) wsp81F (5’-TGGTCCAATAAGTGATGAAGAAAC-3’), wsp691R (5’-AAAATTAAACGCTACTCCA-3’), for the gene wsp (respectively 438 bp and ~600bp) and (3) fbpa-F1 (5’-GCTGCTCCRCTTGGYWTGAT-3’), fbpa-R1(5’-CCRCCAGARAAAAYYACTATTC-3’), for the gene fbpA (429 bp) to test for the presence of other Rickettsiaceae following the standard protocols (Baldo et al. 2006 (link), Kodandaramaiah et al. 2013 (link)).
PCR was run on a DNA-cycling machine 9800 Fast Thermal Cycler (Applied Biosystems – Foster City, CA). Gel electrophoresis was applied to visualise the amplified products in 1.5 % agarose gel using the gel electrophoresis apparatus (RunOne, EmbiTec, San Diego, CA). The gel was stained with ethidium bromide (EtBr) at a concentration of 0.5 μg/mL for 30 minutes (Lee et al. 2012 ), visualised in ultraviolet light source (wavelength 254 nm) and subsequently photographed using the image system IP-010.SD (Vilber Lourmat, France). The presence of the amplified transgene element on a gel was interpreted as evidence that the insect was infected with Wolbachia or related species parasite.
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