Differentiation of Diaprepes abbreviatus and Pachnaeus litus (Coleoptera: Curculionidae)egg masses: PCR-Restriction Fragment-Length Polymorphism and species-specific PCR amplification of 18S rDNA products

Authors: Weathersbee Iii, Albert; Bullock, Robert; Panchal, Tanvi; Dang, Phat

Submitted to: Annals of the Entomological Society of America
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/2003
Publication Date: 9/1/2003
Citation: Weathersbee III, A.A., Bullock, R.C., Panchal, T.D., Dang, P.M. 2003. Differentiation of Diaprepes abbreviatus and Pachnaeus litus (Coleoptera: Curculionidae) egg masses: PCR-Restriction Fragment-Length Polymorphism and species-specific PCR amplification of 18S rDNA products. Ann. Entomol. Soc. Am. 96(5):637-642

Interpretive Summary: The root weevils Diaprepes abbreviatus (L.) and Pachnaeus litus (Germar) are both pests of Florida horticulture but D. abbreviatus is a regulated exotic species that causes severe plant damage while P. litus is a native species considered to be a minor pest. Egg masses of these two weevils cannot be distinguished when they are found on infested plants. We have developed and compared two molecular approaches to differentiating the egg masses. One approach uses a restriction fragment length polymorphism marker and the other uses species-specific, polymerase chain reaction primers to target a single nucleotide polymorphism present in the 18S rDNAs of these two weevil species. Both of the molecular approaches are effective, specific, and yield consistent results but the species-specific primer approach is quicker and more economical.

Technical Abstract: The root weevils Diaprepes abbreviatus (L.) and Pachnaeus litus (Germar) are both pests of Florida horticulture but D. abbreviatus is a regulated exotic species that causes severe damage whereas P. litus is considered a minor pest. Egg masses of these two weevil species are indistinguishable when they are detected on host plants. Two approaches to differentiating the egg masses are described here. Total genomic DNAs extracted from D. abbreviatus and P. litus were used for polymerase chain reaction (PCR) amplification and the PCR products were sequenced to obtain the 2014 base pair (bp) sequence of the complete 18S rRNA gene for each species. A 446 bp region amplified from the 5' end of the 18S rDNA of P. litus contained a restriction fragment length polymorphism (RFLP) marker, an extra BstU I recognition site that was not present in D. abbreviatus. Agarose gel electrophoresis of the restriction enzyme digested PCR products produced a restriction pattern that enabled differentiation of the egg masses. Additionally, two species-specific reverse primers were designed to exploit a single nucleotide polymorphism (SNP) at the RFLP marker site. These reverse primers differed only by a single nucleotide at the 3' end. When used in concert with a standard 18S rDNA forward primer, each species-specific reverse primer distinctly amplified a 256 base pair product only when the 3' terminal nucleotide of the primer complimented the template nucleotide present at the position of the SNP. The PCR amplification and restriction digestion pattern were specific and consistent.