Double-stranded RNA design and electroporation-mediated RNAi

Double-stranded RNAs for RNAi in P. xuthus larvae were designed using siDirect (version 2, http://sidirect2.rnai.jp/), and the target sequences were used as queries to search the P. xuthus genome to avoid off-target sites. The siRNA targeting sequences were as follows: for cll, 5′-GGGTTACCTTTAAATTTCTCTCA-3′; for abd-A, 5′-CGCAGTGAAAGAGATCAACGAGC-3′; for Abd-B, 5′-TACTATAATCTGCCAGTTGAAAG-3′; and for Lac2, 5′-ATCCTAATACCGGTTTCATGACG-3′.

For P. xuthus functional analysis, electroporation-mediated RNAi was performed, as previously described (20). Capillaries for injection were made from glass capillaries with filament (Narishige Group, Tokyo, Japan) and processed by puller-PP-830 (Narishige Group, Tokyo, Japan). A microinjector FemtoJet (Eppendorf AG, Tokyo, Japan) was used for injection. One microliter of 250 μM siRNA was injected at the intersegmental membrane between the eighth and ninth segment into the hemolymph at the third or fourth day of the third instar. Then, electroporation was performed using a NEPA21 Super Electroporator (Nepa Gene Co., Ltd., Tokyo, Japan). The parameters for electroporation were modified to avoid physical damage to larval epidermis. The poring pulse was set to 20 V lasting 5 ms, with a 50-ms interval, and was repeated twice. The transfer pulse was set to 15 V lasting 90 ms, with a 50-ms interval, and was repeated 20 times. Following electroporation, larvae were first kept in an incubator at 20°C under long-day conditions (16 hours light and 8 hours dark) for 12 hours and then subjected to normal conditions as described above. The phenotypic changes were observed and recorded during the fourth and fifth instars using a Nikon D3100. ImageJ 1.52i was used to measure the size of color marking.

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