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Marker based diagnosis, molecular phylogenetics and discovery of population altering endosymbionts for selected diabroticites (Coleoptera: Chrysomelidae)

Thomas Lowell Clark, University of Nebraska - Lincoln

Abstract

Identification of Diabrotica can prove to be difficult. Larvae of many species are morphologically indistinguishable. This study was conducted to determine whether 12 Diabrotica species could be separated using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP). A 1308 by portion of the mitochondrial cytochrome oxidase subunit I gene (COI) was amplified using PCR and digested using restriction endonucleases. Digests resolved on polyacrylamide gels revealed diagnostic markers. A key to the species was constructed using PCR-RFLP patterns. ^ The phylogenetics of thirteen Diabrotica and two Acalymma beetle species were inferred from COI and the second internal transcribed spacer region (ITS-2). Parsimony and neighbor joining analysis COI and ITS-2 separated species into traditional morphological clades. Results generally conferred with prior allozyme analysis but varied within the virgifera clade, D. barberi and D. longicornis appear as sister taxa as well as D. cristata and D. lemniscata . Within the fucata clade D. speciosa and D. balteata were monophyletic. Results also strongly supported the D. virgifera and D. undecimpunctata subspecies complexes. D. porracea, D. viridula, D. adelpha formed variable topologies, however, analysis of COI data consistently placed D. viridula as monophyletic with D. barberi and D. longicornis . ^ Wolbachia has been implicated as a cause of reproductive disruption in many insects. PCR assays of the 16S rRNA gene were conducted to reveal the prevalence of Wolbachia in 12 Diabrotica and 2 Acalymma species. PCR assays revealed Wolbachia infecting 3 Diabrotica and 2 Acalymma species. Distances and neighbor joining trees of 16S rRNA and ftsZ DNA sequences on the Kimura 2-parameter measure and BlastN searches revealed that the strain of Wolbachia infecting D. lemniscata, D. v. virgifera, A. blandulum, and A. vittatum are likely the same strain of Wolbachia within the division A group. The strain infecting D. cristata while a division A strain is different from the Wolbachia infecting other Diabroticites. Implications of these infections are discussed. ^

Subject Area

Biology, Entomology

Recommended Citation

Clark, Thomas Lowell, "Marker based diagnosis, molecular phylogenetics and discovery of population altering endosymbionts for selected diabroticites (Coleoptera: Chrysomelidae)" (2000). ETD collection for University of Nebraska - Lincoln. AAI9991979.
http://digitalcommons.unl.edu/dissertations/AAI9991979

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