Preparation of mealybugs (Hemiptera: Pseudococcidae) for genetic characterization and morphological examination

Authors: BAHDER, BRIAN - University Of California; BOLLINGER, MICHAEL - University Of California; Sudarshana, Mysore; ZALOM, FRANK - University Of California

Submitted to: Journal of Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2015
Publication Date: 7/21/2015
Citation: Bahder, B.W., Bollinger, M., Sudarshana, M.R., Zalom, F.G. 2015. Preparation of mealybugs (Hemiptera: Pseudococcidae) for genetic characterization and morphological examination. Journal of Insect Science. doi: 10.1093/jisesa/iev086.

Interpretive Summary: Mealybugs (Hemiptera: Pseudococcidae) are economically significant agricultural pests on a wide range of crops. Due to their small size and lack of easily visible characters for identification, determination of their taxonomic status is difficult and requires technical competency to prepare a slide-mounted specimen. The standard mounting technique also does not allow for analysis of the genome of the specimen. Conversely, preparatory techniques for genetic analysis of mealybugs cause either loss of the entire individual or physical damage that can make morphology-based identification difficult. We developed a simple protocol that does not impact physical integrity of the specimen for fixation and microscopic examination yet enables simultaneous DNA extraction for DNA-based identification of three mealybug species. All species prepared yielded high quality slide-mounts that were identified as Planococcus citri Risso, Pseudococcus viburni Signoret, or Rhizoecus kondonis Kuwana. DNA extracted was successfully amplified by PCR using primers specific to cytochrome oxidase subunit I, and the amplified product was sequenced for all specimens. This protocol is likely to be applicable to other Hemiptera taxa that are preserved by slide mounting, allowing for both the preparation of a high-quality voucher specimen for morphological identification and simultaneous analysis of DNA for the same specimen. The methods used are technically less challenging than current standard procedures.

Technical Abstract: Mealybugs (Hemiptera: Pseudococcidae) are economically significant agricultural pests on a wide range of crops. Due to their small size and lack of easily visible characters for identification, determination of their taxonomic status is difficult and requires technical competency to prepare a slide-mounted specimen. The standard mounting technique also does not allow for analysis of the genome of the specimen. Conversely, preparatory techniques for genetic analysis of mealybugs cause either loss of the entire individual or physical damage that can make morphology-based identification difficult. We developed a simple protocol that does not impact physical integrity of the specimen for fixation and microscopic examination yet enables simultaneous DNA extraction for DNA-based identification of three mealybug species. All species prepared yielded high quality slide-mounts that were identified as Planococcus citri Risso, Pseudococcus viburni Signoret, or Rhizoecus kondonis Kuwana. DNA extracted was successfully amplified by PCR using primers specific to cytochrome oxidase subunit I, and the amplified product was sequenced for all specimens. This protocol is likely to be applicable to other Hemiptera taxa that are preserved by slide mounting, allowing for both the preparation of a high-quality voucher specimen for morphological identification and simultaneous analysis of DNA for the same specimen. The methods used are technically less challenging than current standard procedures.