Quantitative trait locus mapping and functional genomics of an organophosphate resistance trait in the western corn rootworm, Diabrotica virgifera virgifera.

The western corn rootworm, Diabrotica virgifera virgifera, is an insect pest of corn and population suppression with chemical insecticides is an important management tool. Traits conferring organophosphate insecticide resistance have increased in frequency amongst D. v. virgifera populations, resulting in the reduced efficacy in many corn-growing regions of the USA. We used comparative functional genomic and quantitative trait locus (QTL) mapping approaches to investigate the genetic basis of D. v. virgifera resistance to the organophosphate methyl-parathion. RNA from adult methyl-parathion resistant and susceptible adults was hybridized to 8331 microarray probes. The results predicted that 11 transcripts were significantly up-regulated in resistant phenotypes, with the most significant (fold increases ≥ 2.43) being an α-esterase-like transcript. Differential expression was validated only for the α-esterase (ST020027A20C03), with 11- to 13-fold greater expression in methyl-parathion resistant adults (P < 0.05). Progeny with a segregating methyl-parathion resistance trait were obtained from a reciprocal backcross design. QTL analyses of high-throughput single nucleotide polymorphism genotype data predicted involvement of a single genome interval. These data suggest that a specific carboyxesterase may function in field-evolved corn rootworm resistance to organophosphates, even though direct linkage between the QTL and this locus could not be established.

Molecular phylogeny of Diabrotica beetles (Coleoptera: Chrysomelidae) inferred from analysis of combined mitochondrial and nuclear DNA sequences.

The phylogenetic relationships of thirteen Diabrotica (representing virgifera and fucata species groups) and two outgroup Acalymma beetle species (Coleoptera: Chrysomelidae) were inferred from the phylogenetic analysis of a combined data set of 1323 bp of mitochondrial DNA (mtDNA) cytochrome oxidase subunit 1 (COI) and the entire second internal transcribed spacer region (ITS-2) of nuclear ribosomal DNA of 362 characters. Species investigated were D. adelpha, D. balteata, D. barberi, D. cristata, D. lemniscata, D. longicornis, D. porracea, D. speciosa, D. undecimpunctata howardi, D. u. undecimpunctata, D. virgifera virgifera, D. v. zeae, D. viridula, and outgroup A. blandulum and A. vittatum. Maximum parsimony (MP), minimum evolution (ME), and maximum likelihood (ML) analyses of combined COI and ITS-2 sequences clearly place species into their traditional morphological species groups with MP and ME analyses resulting in identical topologies. Results generally confer with a prior work based on allozyme data, but within the virgifera species group, D. barberi and D. longicornis strongly resolve as sister taxa as well as monophyletic with the neotropical species, D. viridula, D. cristata and D. lemniscata also resolve as sister taxa. Both relationships are not in congruence with the prior allozyme-based hypothesis. Within the fucata species group, D. speciosa and D. balteata resolve as sister taxa. Results also strongly supported the D. virgifera and D. undecimpunctata subspecies complexes. Our proposed phylogeny provides some insight into current hypotheses regarding distribution status and evolution of various life history traits for Diabrotica.

Expression and induction of three family 4 cytochrome P450 (CYP4)* genes identified from insecticide-resistant and susceptible western corn rootworms, Diabrotica virgifera virgifera.

We have previously determined that cytochrome P450-based oxidation is involved in resistance to the insecticides methyl parathion and carbaryl in geographically distinct Nebraska western corn rootworm populations. Three new family 4 cytochrome P450 (CYP4) gene fragments (CYP4AJ1, CYP4G18 and CYP4AK1) were cloned and sequenced from insecticide-resistant and -susceptible western corn rootworms. Insecticide bioassays indicated the resistant population employed in this study was significantly resistant to the insecticides methyl parathion and carbaryl. CYP4AJ1 and CYP4G18 were cloned from both genomic PCR and RT-PCR products, although only CYP4AJ1 contains an intronic region. Alignments of inferred amino acid sequences with other homologous insect CYP4 genes indicates a high degree of similarity. Northern analysis concurrently employing mixed probes representing each of the three rootworm CYP4 fragments identified increased mRNA transcript signals (i) in resistant rootworms and (ii) following induction by the P450 inducer pentamethyl benzene. These results support our previous documentation of P450-based insecticide resistance and suggest increased CYP4 transcript abundance can serve as a molecular resistance-associated marker.

Influence of western corn rootworm (Coleoptera: Chrysomelidae) larval injury on yield of different types of maize.

Two field experiments were conducted in 1995-1996 to determine if there are common yield responses among maize hybrids to larval western corn rootworm, Diabrotica virgifera virgifera LeConte injury. Three yellow dent hybrids, five white food grade dent hybrids, and a popcorn hybrid were included in the study. The minimum level of rootworm injury as measured by root damage ratings (3.2-4.2) that significantly reduced yield was similar across the hybrids included in the study. However, the pattern of yield response to different rootworm injury levels varied among hybrids. This suggests that maize hybrids may inherently differ in their ability to tolerate rootworm injury and partition biomass in response to injury and other stresses. The complex interaction among hybrid, level of injury, and other stresses suggests that a common western corn rootworm injury-yield relationship may not exist within maize.

PCR-RFLP of the mitochondrial cytochrome oxidase (subunit I) gene provides diagnostic markers for selected Diabrotica species (Coleoptera: Chrysomelidae).

Adult and larval identification of Diabrotica can be difficult. Some adult identifications require considerable taxonomic experience while larvae of many Diabrotica species are morphologically indistinguishable. This study was conducted to determine whether 12 pest and non-pest Diabrotica species could be separated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A 1308 bp portion of the mitochondrial cytochrome oxidase subunit I gene (COI) was amplified using PCR and digested using several restriction endonucleases. Double digests of COI amplicons with AluI and MspI resolved on polyacrylamide gels revealed several diagnostic inter- and intraspecific polymorphisms. A key to the 12 species was constructed using the PCR-RFLP patterns.

Larval susceptibility of an insecticide-resistant western corn rootworm (Coleoptera: Chrysomelidae) population to soil insecticides: laboratory bioassays, assays of detoxification enzymes, and field performance.

Soil insecticides were evaluated in laboratory and field studies against larvae of an insecticide resistant population (Phelps County, NE) of western corn rootworm, Diabrotica virgifera virgifera LeConte. Insecticide toxicity was evaluated by topical application of technical insecticides to 3rd instars from Saunders County, NE (susceptible) and Phelps County populations. Resistance ratios (LD50 Phelps County/LD50 Saunders County) for the insecticides methyl parathion, tefluthrin, carbofuran, terbufos, and chlorpyrifos were 28.0, 9.3, 8.7, 2.6 and 1.3, respectively. Biochemical investigation of suspected enzymatic resistance mechanisms in 3rd instars identified significant elevation of esterase activity (alpha and beta naphthyl acetate hydrolysis [3.8- and 3.9-fold]). Examination of 3rd instar esterases by native PAGE identified increased intensity of several isoenzymes in the resistant population. Assays of cytochrome P450 activity (4-CNMA demethylation and aldrin epoxidation) did not identify elevated activity in resistant 3rd instars. Granular soil insecticides were applied at planting to corn, Zea mays L., in replicated field trials in 1997 and 1998 at the same Phelps County site as the source of resistant rootworms for the laboratory studies. In 1997, planting time applications of Counter 20CR, Counter 15 G (terbufos), and Lorsban 15 G (chlorpyrifos) resulted in the lowest root injury ratings (1-6 Iowa scale); 2.50, 2.55, 2.65, respectively (untreated check root rating of 4.55). In 1998, all insecticides performed similarly against a lower rootworm density (untreated check root rating of 3.72). These studies suggest that resistance previously documented in adults also is present in 3rd instars, esterases are possibly involved as resistance mechanisms, and resistance to methyl parathion in adults is also evident in larvae, but does not confer cross-resistance in larvae to all organophosphate insecticides.