I am not an expert for neither insects nor primer design, but see e.g. Toju and Baba 2018. For further correspondence please use E-mail instead of RG.

Thank you!

I have amplified ITS2 in ticks (also Acari). The region can 1-1.5 kb). You will need to amplify as two fragments. I have used the following reference primers for ticks; a full-length fragment, the forward primer 3SAF (5′-CTAAGCGGTGGATCACTCGG-3′) (Barker, 1998) and reverse primer ITS2R (5′-ATATGCTTAAATTCAGCGGG-3′) (Domanico et al. 1997). These do not give a full coverage, only the outer ends are covered, the middle region is not. So you need to design primers to cover the middle region. I designed and used the following primers for that: ITS2_Int_F1 (5′-AAGAGCCTGCAGGGAAAG-3′) and ITS2_Int_R1 (5′-CACGTTCGTAAACCCATC-3′) . They worked well for ticks. You will then need to generate a contig with the resulting 4 sequences to get the full length ITS sequence.

Dear Esther,

I will check the primers you have informed. Currently, I am trying to sequence the ITS with some primers already published for spider mites (Tetranychidae). Hopefully they may work.

Thank you so much for your detailed reply and guidance.

All the best,

Mohamed