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CHARACTERIZATION OF GYPSY MOTH POPULATIONS AND RELATED SPECIES USING A NUCLEAR DNA MARKER

Published online by Cambridge University Press:  31 May 2012

Tom A. Pfeifer ,
Leland M. Humble ,
Mark Ring  and
Tom A. Grigliatti
Tom A. Pfeifer
Affiliation:
Zoology Department, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4
Leland M. Humble
Affiliation:
Forestry Canada, Pacific Forestry Centre, 506 West Burnside Road, Victoria, British Columbia, Canada V8Z 1M5
Mark Ring
Affiliation:
Zoology Department, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4
Tom A. Grigliatti*
Affiliation:
Zoology Department, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z4
*
1Author to whom correspondence should be addressed.
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Abstract

The discovery of egg masses of Asian gypsy moth onboard Russian freighters in Pacific ports of North America and the capture of adult male gypsy moths in pheromone traps underscored the need for a positive identification of Asian and European gypsy moth males. We have devised a method for differentiation of these two populations based on restriction endonuclease cleavage of a polymerase chain reaction (PCR) generated product. DNA primers for the 5S and 28S rDNA were employed to amplify the ITS2 region using PCR. The ends of the amplified DNA products were sequenced and gypsy moth specific oligonucleotide primers were designed to amplify the gypsy moth ITS2 region. Eleven of 28 restriction enzymes tested cleaved the amplified product. The restriction enzymes ClaI, PvuI, and TaqI generated distinct restriction fragment patterns for the Asian and European gypsy moth PCR product. This diagnostic was applied to samples of field-collected gypsy moths in a double blind test and correctly identified them as Asian or European. Additional studies demonstrated that these primers and restriction enzymes could be used to differentiate among Lymantria dispar, L. monacha, and L. mathura.

Résumé

La découverte de masses d’oeufs de spongieuses asiatiques à bord de cargos russes dans des ports nord-américains de la côte du Pacifique et la capture de mâles adultes dans des pièges à phéromone ont démontré la nécessité de pouvoir distinguer avec certitude les mâles de la population asiatique de ceux de la population européenne de la Spongieuse. Nous avons mis au point une méthode de différenciation de ces deux populations basée sur le clivage par des endonucléases de restriction d’un fragment obtenu par l’amplification en chaîne par polymérase (PCR). Les amorces d’ADN capables de reconnaître les fragments 5S et 28S d’ADN ribosomique ont été utilisées pour amplifier la région ITS2 par la réaction PCR. Les extrémités des fragments d’ADN amplifiés ont été soumises à un séquençage et des amorces d’oligonucléotides spécifiques aux spongieuses ont été choisies pour amplifier la région ITS2 de spongieuse. Onze des 28 enzymes de restriction essayées ont opéré le clivage du fragment amplifié. Les enzymes de restriction ClaI, PvuI, et TaqI ont donné lieu à des séquences distinctes des fragments obtenus par PCR chez les spongieuses européennes et asiatiques. Ce diagnostic a été confirmé en nature au cours de tests aveugles répétés et il permet d’identifier correctement les spongieuses asiatiques et européennes. Des travaux additionnels ont démontré que ces amorces et enzymes de restriction peuvent également servir à distinguer Lymantria dispar, L. monacha et L. mathura.

[Traduit par la Rédaction]

Type
Articles
Information
The Canadian Entomologist , Volume 127 , Issue 1 , February 1995 , pp. 49 - 58
Copyright
Copyright © Entomological Society of Canada 1995

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