Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Genetic characterisation of WCR (Western Corn Rootworm) populations in Europe

Western corn rootworm beetles collected in semiochemical-lure traps during the summer of 2000 from 23 sites in Eastern Europe were sent to the USDA-ARS laboratory in Fargo, ND (USDA-ARS; Red River Valley Agricultural Research Center, Fargo, North Dakota USA). In some cases the beetles were by trapped by aspirator and in other cases collected them by hand: Insects were shipped to Fargo, ND immediately after collection in 75% alcohol via DHL and were immediately frozen upon arrival. In Fargo the PCR analysis was done thankfully by Rich ROEHRDANZ as a substantial cooperation within this project.

Collections were concentrated in Serbia, Croatia, Bosnia-Herzegovina including the “Republic Srpska”, Hungary, and Italy. Some collection sites were so close to each other, that they were summarized under one location.

Intact DNA was recovered from individual beetles from 17 of the 23 sites. Some problems were encountered with beetles from the 6 sites with negative results (result of insects drying out in shipment), but attempts are being made to refine DNA extraction methods to successfully gather material from the beetles. The intact DNA from the 17 sites was demonstrated by successful amplification of a small PCR product using a western corn rootworm specific primer set. Results of the investigations indicate that all samples appear similar to those of laboratory reared western corn rootworms (from the Brookings, South Dakota USDA-ARS laboratory), which were collected from typical populations of insects inhabiting corn/soybean rotations. Earlier studies indicate that the Brookings lab insects are similar to most populations in the central Corn Belt. To date no samples have been shown to be related to insects ovipositing in soybean. Further studies are being conducted to refine the assay technique and to provide additional data that might be useful in pinpointing the exact origin of beetles inhabiting Europe.

DNA extracts were prepared using individual insects from the samples collected in Europe and sent to Fargo, ND. Insects were sampled from 16 of the vials or containers that were received in the USA. The remainder of the insects from all of the collections have not been examined, but remain frozen and will be used in additional studies to further clarify the genetic relationships between European and USA populations. The DNA extracts are total cellular DNA, which includes both nuclear, and mitochondrial sequences. The polymerase chain reaction (PCR) was used to amplify a portion of the cytochrome oxidase I and cytochrome oxidase II genes from the mitochondrial DNA (mtDNA). This particular PCR product is about 600 base pairs long. The region spanning cyotochrome oxidase I and II includes a leucine t-RNA gene and has been widely used for phylogenetic purposes with insects.

Amplification was successful for all of the tested DNA extracts, a total of 35 individuals. Attempts to amplify some of the samples with some other primers were not successful but most likely those failures were attributed to old primers that had not been used in over a year (i.e. the controls, WCR from laboratory colonies, did not work either).

In a paper published on mitochondrial DNA variations in western and Mexican corn rootworm populations (Szalanski et al.), good diagnostics for population identification of WCR either using mtDNA followed by cleavage with restriction endonucleases, or by obtaining nucleotide sequence data from a portion of the nuclear genome called the ITS1 spacer are described. There is so far no indication that the European WCR differs from the WCR genotype that is wide spread in North America. Nor is there any evidence, to date, of differentiation among the European collections. As a further exploration of the possible geographical distribution of genetic variation in WCR, the DNA sequence of another nuclear region will be examined. This region is another spacer in the nuclear ribosomal gene array. Three ribosomal genes, 18S, 5.8S and 28S, are present in tandem multiple copies. The coding regions for the genes are separated by noncoding regions called spacers. The spacer between the 18S and 28S genes is about 1200 base pairs in length and is called the intergenic spacer (IGS). It is often assumed that since the spacer regions do not code for important products, that they are not as tightly constrained by evolutionary forces. Therefore, the argument goes, the spacer regions should exhibit greater genetic variability.

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