Microarray analysis yields candidate markers for rotation resistance in the western corn rootworm beetle, Diabrotica virgifera virgifera.

As pest species may evolve resistance to chemical controls, they may also evolve resistance to cultural control methods. Yearly rotation of corn (Zea mays) with another crop interrupts the life cycle of the western corn rootworm beetle (Diabrotica virgifera virgifera, Coleoptera: Chrysomelidae), but behavioral resistance to crop rotation is now a major problem in the Midwest of the USA. Resistant adult females exhibit reduced fidelity to corn as a host and lay their eggs in the soil of both corn and soybean (Glycine max) fields. Behavioral assays suggest that the adaptation is related to increased locomotor activity, but finding molecular markers has been difficult. We used microarray analysis to search for gene expression differences between resistant and wild-type beetles. Candidates validated with real-time polymerase chain reaction exhibit predicted patterns from the microarray in independent samples across time and space. Many genes more highly expressed in the rotation-resistant females have no matches to known proteins, and most genes that were more lowly expressed are involved in antimicrobial defense.

Utility of EST-derived SSRs as population genetics markers in a beetle.

Microsatellite, or simple sequence repeat (SSR), loci can be identified by mining expressed sequence tag (EST) databases, and where these are available, marker development time and expense can be decreased considerably over conventional strategies of probing the entire genome. However, it is unclear whether they provide information on population structure similar to that generated by anonymous genomic SSRs. We performed comparative population genetic analyses between EST-derived SSRs (EST-SSRs) and anonymous SSRs developed from genomic DNA for the same set of populations of the insect Diabrotica virgifera, a beetle in the family Chrysomelidae. Compared with noncoding, nontranscribed regions, EST-SSRs were generally less polymorphic but had reduced occurrence of null alleles and greater cross-species amplification. Neutrality tests suggested the loci were not under positive selection. Across all populations and all loci, the genomic and EST-SSRs performed similarly in estimating genetic diversity, F(IS), F(ST), population assignment and exclusion tests, and detection of distinct populations. These findings, therefore, indicate that the EST-SSRs examined can be used with confidence in future genetic studies of Diabrotica populations and suggest that EST libraries can be added as a valuable source of markers for population genetics studies in insects and other animals.

Effect of four cropping systems on variant western corn rootworm (Coleoptera: Chrysomelidae) adult and egg densities and subsequent larval injury in rotated maize.

The cultural practice of rotating corn, Zea mays L., with soybean, Glycine max (L.) Merrill, to manage larval injury by the western corn rootworm, Diabrotica virgifera virgifera LeConte, was used extensively throughout east central Illinois and northern Indiana until the mid-1990s. The effectiveness of this management tactic diminished due to a shift in the ovipositional behavior of the western corn rootworm. The variant western corn rootworm has since spread as far as northwestern Illinois, southern Wisconsin, southern Michigan, and eastern Ohio. The objective of this study was to evaluate the influence of four cropping systems on adult and egg densities of the western corn rootworm and to quantify the level of root injury in rotated corn after each system. The four cropping systems used included: 1) corn; 2) soybean; 3) double-cropped winter wheat, Triticum aestivum L., followed by soybean; and 4) winter wheat. Research trials were conducted near Monmouth (northwestern), DeKalb (northern), and Urbana (east central), IL, during 2003 and 2004. Results indicated variant western corn rootworm adults can be found in all four treatments at each location and consequently no crop was immune to oviposition or root injury by corn rootworm larvae in rotated corn the following season. Adults were found primarily in corn and soybean, whereas egg densities were greatest in corn plots in all three locations in both years of the study. Root injury by larvae was most abundant in corn following corn at all three sites. Of the four systems evaluated, the use of wheat demonstrated the most potential for preventing yield reducing levels of root injury in rotated corn.

PCR-RFLP identification of Diptera (Calliphoridae, Muscidae and Sarcophagidae)--a generally applicable method.

A simple, rapid method using restriction fragment length polymorphisms (RFLPs) within the internal transcribed spacer (ITS) regions of the ribosomal DNA gene repeat allows identification of insects and other organisms. We used the method to identify the morphologically similar Diptera larvae that are important in forensic entomology for estimating the time and location of death. Polymerase chain reaction (PCR) was used to amplify a region from the 18S to the 28S rRNA genes. The ITS1 and ITS2 regions provided variation between species and homogeneity within species, with the exception of Cochliomya macellaria. Combinations of the restriction enzymes DdeI, HinfI and Sau3AI provided diagnostic bands for identification of the ten species from three families of Diptera (Calliphoridae, Muscidae and Sarcophagidae).

Assessment of parasitism of house fly and stable fly (Diptera: Muscidae) pupae by pteromalid (Hymenoptera: Pteromalidae) parasitoids using a polymerase chain reaction assay.

The internal transcribed spacer (ITS) regions of the ribosomal DNA of house flies, Musca domestica L., the stable flies, Stomoxys calcitrans (L.), and four parasitoid species in the genus Muscidifurax (Hymenoptera: Pteromalidae) were characterized to develop a method based on the polymerase chain reaction (PCR) to better define the role of pteromalid parasitism of pupae of the house fly and stable fly. Two parasitoid-specific primers were designed to anneal to the 5' end of the 5.8S rRNA gene in the parasitoid species. When paired with a universal primer at the 3' end of the 18S rRNA, the primers amplified the target ITS1 region in 10 pteromalid species. PCR allowed detection of parasitoid DNA within 24 h after females of Spalangia endius Walker oviposited into house fly puparia. PCR failed to amplify parasitoid DNA or detect parasitism in puparia that were exposed to parasitoid oviposition, allowed to develop 7 d, then killed by freezing and held at 20-24 degrees C for 4 d to allow DNA degradation. Digestion of the PCR products with restriction enzymes produced restriction fragment length polymorphisms that allowed identification of individual parasitoid species. Significantly greater levels of parasitism (P < 0.05) were detected by PCR for two of the five field collection dates in 1997. On the dates when PCR detected higher levels of parasitism than estimates provided by emergence of adult insects from samples taken at Feedlot M in 1997, more than 65% of all puparia in the emergence samples failed to produce an adult insect. Three puparia collected in 1997 produced double PCR bands that corresponded to PCR band sizes of Muscidifurax spp. and Spalangia sp., possibly indicating multiple parasitism or hyperparasitism.