title: Preparation of Mealybugs (Hemiptera: Pseudococcidae) for Genetic Characterization and Morphological Examination.
abstract: Mealybugs (Hemiptera: Pseudococcidae) are economically significant agricultural pests on many different crops. Because of 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 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. This study describes 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 four mealybug species. All species prepared yielded high quality slide mounts, identified as Planococcus citri Risso, Pseudococcus viburni Signoret, Rhizoecus kondonis Kuwana, or Rhizoecus californicus Ferris. DNA extracted in this manner had higher purity and yield in the final eluate than in samples extracted using standard methods. DNA extracted was successfully amplified by polymerase chain reaction using primers for the cytochrome oxidase I gene and subsequently 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.
authors.affiliation: Department of Entomology and Nematology, University of California, One Shields Ave., Davis, CA 95616 bwbahder@ucdavis.edu.
authors.forename: B W
authors.initials: BW
authors.lastname: Bahder
authors.affiliation: Department of Entomology and Nematology, University of California, One Shields Ave., Davis, CA 95616.
authors.forename: M L
authors.initials: ML
authors.lastname: Bollinger
authors.affiliation: United States Department of Agriculture, Agriculture Research Service, Department of Plant Pathology, University of California, One Shields Ave., Davis, CA 95616.
authors.forename: M R
authors.initials: MR
authors.lastname: Sudarshana
authors.affiliation: Department of Entomology and Nematology, University of California, One Shields Ave., Davis, CA 95616.
authors.forename: F G
authors.initials: FG
authors.lastname: Zalom
mesh_terms: D000818:Animals; D003576:Electron Transport Complex IV / Q000235:genetics; D005260:Female; D006430:Hemiptera / Q000033:anatomy & histology / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D003576:Electron Transport Complex IV
keywords: DNA extraction; identification; mealybug; slide-mount; taxonomy