Corn rootworms, Diabrotica spp., are part of the large group of chysomelid beetles, many of which attack agricultural crops (Levine & Oloumi-Sadeghi 1991). Southern corn rootworm (SCR) Diabrotica undecimpunctata howardi Barber, or as adults known as spotted cucumber beetle, is widely distributed in North America, occurring in most areas east of the Rocky Mountains, in southern Canada, and in Mexico. It is most abundant and destructive in the southern United States. This insect is multivoltine and overwinters as adults in the southern parts of its range (Branson & Krysan 1981). Southern corn rootworms infest the roots of many grass crops and weeds, as well as those of peanuts, alfalfa, and occasionally cucurbits. They are most damaging to corn and peanuts. Annually 20 to 25 million acres of corn are treated with soil insecticides to protect the crop from corn rootworm larval feeding damage (Fuller et al. 1997). Soil insecticides applied for the corn rootworm represent one of the major uses of insecticide in the United States. Costs associated with insecticides applied to control larval damage to corn roots and adult damage to corn silks, along with crop losses can approach $1 billion annually (Metcalf 1986).

Despite the possible benefits that molecular genetic analysis of SCR may provide towards diagnostics, dispersal, insecticide resistance, and the implementation of area wide control programs, very little research in this area has been conducted. Previous population genetic analysis of SCR is limited to a single study by Krafsur (1999) on allozyme diversity in leaf beetles. An assessment of 56 southern corn rootworms collected from Iowa corn fields using allozymes revealed an average heterozygosity of 11.3% among the 39 loci studied. Although this study did find genetic variation in SCR, the study was directed towards detecting genetic variation in the species and not studying the extent of variation within and among populations.

The goal of our study was to survey population diversity and to determine if molecular marker systems can be used for identifying strains and emerging phenotypes that might affect area wide control programs. We surveyed two molecular markers, the first internal transcribed spacer region (ITS1), located between the repeating array of nuclear 18S and 5.8S ribosomal genes, and portions of the mitochondrial DNA cytochrome oxidase I and II genes. These markers have been successfully used for monitoring population genetics of western corn rootworm, D. virgifera virgifera LeConte (Szalanski et al. 1999) and northern corn rootworm, D. barberi Smith and Lawrence (Roehrdanz et al. 2003) and for differentiating Diabrotica species (Szalanski & Powers 1996; Szalanski et al. 2000).

Materials and Methods

Southern corn rootworm adults were obtained from the field locations as listed in Table 1. Adult rootworms were preserved by immersion in 95% alcohol in the field or transported live to the laboratory and frozen at -80°C. Voucher specimens are maintained at the Arthropod Museum, Department of Entomology, University of Arkansas, Fayetteville, AR.

Alcohol preserved specimens were allowed to dry on filter paper, and DNA was extracted from individual thoraces using the Puregene DNA isolation kit D-5000A (Gentra, Minneapolis, MN). Extracted DNA was resuspended in 50 μl of Tris:EDTA and stored at –20°C. Polymerase chain reaction for the nuclear DNA marker was conducted using the primers rDNA2 (5’-TTGATTACGTCCCTGCCCTTT-3’) described by Vrain et al. (1992), and primer rDNA1.58s (5’-GCCACCTAGTGAGCCGAGCA-3’) by Cherry et al. (1997). These primers amplify a 3’ portion of the 18S gene, the entire ITS1 region and a 5’ section of the 5.8S gene. The 3’ portion of the mtDNA cytochrome oxidase (CO) I gene, tRNA leucine, and a 5’ portion of the CO II gene was amplified using the primers C1-J-2797 (5’-CCTCGACGTTATTCAGATTACC-3’) (Simon et al. 1994) and c2-N-3400 (5’-TCAATATCATTGATGACCAAT-3’) (Taylor et al. 1997). PCR reactions were conducted using 1 l of the extracted DNA Szalanski et al. (1997). The ITS1 PCR protocol was 40 cycles of 94°C for 45 s, 54°C for 45 s and 72°C for 60 s. The mtDNA PCR protocol consisted of 40 cycles of 94°C for 45 s, 42°C for 45 s, and 72°C for 60 s. Amplified DNA from individual beetles was purified, and concentrated using Microcon-PCR Filter Units (Millipore, Bedford, MA). Samples were sent to the University of Arkansas DNA Resource Center (Fayetteville, AR) for direct sequencing in both directions using an ABI Prism 377 DNA sequencer. Consensus sequences were obtained from the sequences using GCG Wisconsin Package software (Accelrys, San Diego, CA). GenBank accession numbers for the beetles subjected to DNA sequencing in this study are AY191606 to AY191608. The distance matrix option of PAUP* 4.0b10 (Swofford 2001) was used to calculate genetic distances according to the Kimura 2-parameter model (Kimura 1980) of sequence evolution.

Results and Discussion

DNA sequencing of the ITS1 PCR-amplified product revealed a 762 bp amplicon for all of the SCR subjected to DNA sequencing (Fig. 1). The average base frequencies were A = 0.37, C = 0.14, G = 0.10, and T = 0.39. Of the 21 SCR individuals sequenced (Table 1), no ITS1 sequences were variable. For the mtDNA COI/COII amplicon, a total of 580 bp were sequenced. The average base frequencies were A = 0.30, C = 0.17, G = 0.20, and T = 0.33. One nucleotide site, 304, was variable among the 21 southern corn rootworm DNA sequences (Fig. 2). One of the SCR sampled from Brookings, SD had a C at this site, while the remaining DNA sequences had a T at this site. To verify this nucleotide polymorphism, the Brookings, SD sample was resequenced in both directions, and all four individual sequences had the polymorphism at site 304. Based on a study by Kobayashi et al. (1999) on PCR error, almost all of the Epilachna ladybird beetle mtDNA COI PCR errors, 19 out of 20, were transitions, while the substitution observed in SCR is a transition. In addition, based on the PCR error rate of 7.3 × 10^–5 substitutions per site per cycle from Kobayashi et al. (1999), we calculate that the chance of a PCR error at site 304 for two independent PCR amplications is 8.5 × 10^-6. Based on this, we believe that this nucleotide difference represents an actual polymorphism in the population.

The low level of molecular genetic differentiation within and among SCR populations has been observed in western corn rootworm, Diabrotica virgifera virgifera LeConte, and Mexican corn rootworm, D. v. zeae Krysan and Smith (Szalanski et al. 1999). No ITS or mtDNA markers were found that were diagnostic for the subspecies despite the fact that there are obvious color differences. What little genetic polymorphism that was detected had no geographical component. Low levels of genetic diversity in the ITS1 and mtDNA regions among widely dispersed populations has been reported for other insect species besides SCR, WCR, and MCR including European corn borer, Ostrinia nubialis (Hübner) (Marcon et al. 1998), stable fly, Stomoxys calcitrans L. (Szalanski et al. 1996), and alfalfa weevil, Hypera postica (Gyllenhal) (Erney et al. 1996). These species have high mobility or recent expansion from a genetic bottleneck in common. These factors may contribute to the lack of genetic variation among SCR populations.

This is not the case with northern corn rootworm, Diabrotica barberi Smith and Lawrence (Roehrdanz et al. 2003). ITS1 DNA sequencing and PCR-RFLP analysis of ITS1 and mtDNA revealed genetic variation within and genetic differentiation among northern corn rootworm populations. With both ITS1 and mtDNA markers there appeared to be a phylogeographic pattern of genotype frequencies. They observed that populations east of Illinois were homogeneous for one ITS1 genotype, two ITS1 genotypes were observed in Illinois, Wisconsin and North Dakota, while South Dakota, Nebraska, Iowa, and Kansas populations were composed of all three ITS1 genotypes. The mtDNA had 58 haplotypes that displayed a strong east-west geographical partition. The region of overlap occurred in a few counties of east-central Illinois. Possible reasons for this population structure include expansion from different glacial relicts, historic host plant differences, and Wolbachia driven reproductive incompatibilities (Roehrdanz et al. 2003). The lack of genetic distinction observed in southern corn rootworm populations suggests either high levels of dispersal or a recent geographical expansion from a relatively small base.

The lack of genetic polymorphism in the ribosomal spacer and mitochondrial COI/COII regions diminishes the prospect of using these tools to quickly find molecular markers to track insecticide resistance, or other behavioral modifications in SCR. This is especially relevant given the impending release of genetically modified corn with the Bacillus thuringiensis var. tenebrionis gene which encodes for an endotoxin specific for Coleoptera, and is targeted towards corn rootworms. Techniques detecting higher levels of polymorphism such as AFLP or microsatellites may reveal markers more suitable for population level analyses of gene flow in SCR. Assays for genes or gene products directly involved may also prove useful, especially for resistance management.