Leslie C. Lewis

Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen

Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings.

Two differentially expressed ommochrome-binding protein-like genes (obp1 and obp2) in larval fat body of the European corn borer, Ostrinia nubilalis

Abstract Ommochrome-binding proteins function in coloration and detoxification pathways by transporting tryptophan metabolites, and increase in hemolymph concentration prior to diapause. Two ommochrome-binding protein genes from the European corn borer Ostrinia nubilalis (Hübner) (Onobp1 and Onobp2; GenBank accession nos. AY819651 to AY819655 and AY862870) were isolated. Relatedness to OBP-encoding genes was suggested by peptide similarity, phylogenetic reconstruction, and expression data. 21 single nucleotide polymorphisms between obp1 and 23 polymorphisms between obp2 alleles were identified, and resultant genomic markers were inherited in a Mendelian fashion. RT-PCR showed fat body specific Onobp1 and Onobp2 transcription. The Onobp1 transcript was RT-PCR amplified from fat body of 5th instars, whereas Onobp2 was expressed in fat body of 4th and 5th instars, and peaked in 5th instar wandering and 1 week old diapausing larvae. Expression suggests gene duplicates are maintained by change in temporal expression. The significance of Onobp1 and 2 gene products to O. nubilalis diapause physiology requires additional investigation. DAP diapause-associated polypeptide OBP ommochrome binding protein Onobp1 Ostrinia nubilalis ommochrome binding protein 1 Onobp2 Ostrinia nubilalis ommochrome binding protein 2

Comparative Performance of Single Nucleotide Polymorphism and Microsatellite Markers for Population Genetic Analysis

Microsatellite loci are standard genetic markers for population genetic analysis, whereas single nucleotide polymorphisms (SNPs) are more recent tools that require assessment of neutrality and appropriate use in population genetics. Twelve SNP markers were used to describe the genetic structure of Diabrotica virgifera virgifera (LeConte; Coleoptera: Chrysomelidae) in the United States of America and revealed a high mean observed heterozygosity (0.40 +/- 0.059) and low global F(ST) (0.029). Pairwise F(ST) estimates ranged from 0.007 to 0.045, and all but 2 populations showed significant levels of genetic differentiation (P < or = 0.008). Population parameters and conclusions based on SNP markers were analogous to that obtained by use of microsatellite markers from the identical population samples. SNP-based F(ST) estimates were 3-fold higher than corresponding estimates from microsatellites, wherein lower microsatellite F(ST) estimates likely resulted from an overestimate of migration rates between subpopulations due to convergence of allele size (homoplasy). No significant difference was observed in the proportion of SNP or microsatellite markers loci that were nonneutral within populations. SNP markers provided estimates of population genetic parameters consistent with those from microsatellite data, and their low back mutation rates may result in reduced propensity for error in estimation of population parameters.

Endophytic beauveria bassiana (balsamo) vuillemin in corn: The influence of the plant growth stage and ostrinia nubilalis (hübner)

A conidial suspension of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin, was injected into corn plants at anthesis to suppress populations of the European corn borer Ostrinia nubilalis (Hubner) (Lepidoptera: Pyralidae). The fungus colonized the plants and moved, primarily upward, within the pith, possibly along with plant photosyn‐thates. Beauveria bassiana injected into the corn plants significantly reduced the amount of European corn borer tunneling. Colonization of corn plants by B. bassiana was independent of European corn borer infestation. The impact of environmental conditions on the movement and colonization of the plants by B. bassiana is discussed.

Occurrence of the entomopathogen Beauveria bassiana (Balsamo) Vuillemin in different tillage regimes and in Zea mays L. and virulence towards Ostrinia nubilalis (Hübner)

Abstract The entomopathogen, Beauveria bassiana (Balsamo) Vuillemin, is an important component in the corn (Zea mays L.) agroecosystem; this pathogen was found in soil, living corn plants, and field corn residue. Beauveria bassiana occurs naturally in the soil within different tillage systems (plow, chisel, no-tillage), with an average of 51–74 colony forming units g−1 of soil. The natural inoculum was present in the crop residue from the different tillage systems, killing up to 84% of European corn borer larvae (Ostrinia nubilalis (Hubner), Lepidoptera: Pyralidae) overwintering in the no-tillage regime. Of the larvae collected from the corn late in the season, 100% of those killed by Beauveria bassiana were from plants colonized by Beauveria bassiana applied foliarly at the whorl-stage (χ2 = 19.1, P

Assessing the Effects of Pest Management on Nontarget Arthropods: The Influence of Plot Size and Isolation

Abstract Evaluations of field research on the nontarget effects of pest management, particularly the production of transgenic crops with insecticidal properties, suggest the methods used are sometimes unlikely to detect real differences among treatments. Among potential problems, abundance estimates may be scale-dependent for many arthropods, which move among experimental plots and between experimental plots and the surrounding environment. Insecticide-disturbed plots of field corn in a range of sizes in 2003 (0.03–0.53 ha; 18–72 m wide) and 2004 (0.01–0.13 ha; 9–36 m wide) were used for testing effects of scale on nontarget arthropod abundance. Possible effects of artificially isolating plots by removal of vegetation around plot borders were also studied in 2003. Community and taxon-based analyses showed abundance of foliar (aboveground) arthropods depended on plot size and isolation. While abundance of foliar arthropods was generally greater in smaller plots, isolation treatments suggested some taxa may have been either repelled or attracted to isolated plots. Levels of some epigeal (ground-dwelling) taxa were also size or isolation-dependent, but community-based analysis did not indicate a strong collective response to treatments. Recommendation of a practical but rigorous minimum plot size for nontarget studies may not be appropriate because responses to plot size varied among taxa. However, because arthropod movement into and out of plots can reduce differences between treatments, results suggest the use of small plots (width <9 m) for nontarget studies on transgenic crops generally should be avoided. Similarly, the taxon-specific effects of isolating plots should be considered when planning studies or interpreting results.

Aptness of insecticides (Bacillus thuringiensis and carbofuran) with endophytic Beauveria bassiana, in suppressing larval populations of the European corn borer

Abstract Research was conducted in 1989 and 1990 to determine if Beauveria bassiana in combination with Bacillus thuringiensis or carbofuran is an effective tactic for season-long management of the European corn borer, Ostrinia nubilalis . A granular formulation of Beauveria bassiana was applied to corn at whorl stage (V6) and/or at pollen-shed stage (R1) corn. Bacillus thuringiensis or carbofuran was then applied to the same plants. Beauveria bassiana reduced larval populations of O. nubilalis when the fungus was applied to corn during both the whorl stage and the pollen-shed stage. When either Bacillus thuringiensis or carbofuran was added to plants treated with Beauveria bassiana , there was increased mortality of O. nubilalis . Although Beauveria bassiana alone reduced O. nubilalis tunneling, mostly damage was further reduced when Bacillus thuringiensis or carbofuran was applied to whorl-stage corn. When applied to pollen-shedding corn, Beauveria bassiana reduced tunneling but had no significant effect on yield, unless Bacillus thuringiensis was added. Although Beauveria bassiana, Bacillus thuringiensis , and carbofuran reduced tunneling by first generation O. nubilalis , increased yields did not always result. Tunneling by second-generation O. nubilalis was significantly reduced by Beauveria bassiana in both years of all experiments. Even so, yields were variable in all experiments which indicated that a number of abiotic and biotic factors, aside from insects, affected yields. This study is the first to document compatibility of Beauveria bassiana with a chemical insecticide. Yield data indicate that endophytic Beauveria bassiana is not a plant pathogen.

Effects of Cry1Ab-Expressing Corn Anthers on Monarch Butterfly Larvae

Abstract Previous studies suggest that exposure to corn, Zea mays L., anthers expressing Bacillus thuringiensis (Bt)-derived protein may have adverse effects on the larvae of monarch butterfly, Danaus plexippus (L.). To examine the potential effects of Bt anthers on monarch butterflies, studies were designed to test toxicity in the laboratory; examine anther distribution in space and time; compare distributions of anthers, pollen, and larval feeding; and measure effects of long-term exposure in the field. In the laboratory, monarch butterfly larvae fed on whole corn anthers, but anther feeding was sporadic. Larvae exposed to 0.3 anther/cm2 fed and weighed less after 4 d compared with larvae exposed to non-Bt anthers. Adverse effects increased with increasing anther density. Monarch butterfly larvae exposed to 0.9 anther/cm2 had reduced feeding, weight, and survival and increased developmental time compared with larvae exposed to non-Bt anthers. Later instars were more tolerant of Bt toxin. For all studies, laboratory testing probably magnified effects because larvae were confined to petri dishes. Field studies showed toxic anther densities are uncommon on milkweed (Asclepias) leaves in and near cornfields during anthesis. Mean anther densities on milkweed leaves in cornfields during peak anthesis were between 0.06 and 0.1 anther/cm2 (≈ 3–5 anthers per leaf). When exposure to a density of five anthers per leaf was tested in field-cage studies, no effects on growth, development, or survival were detected. Based on probability of exposure to toxic densities, Bt anthers alone are not likely to pose a significant risk to monarch butterflies in Iowa.

Allelic variation of a Beauveria bassiana (Ascomycota: Hypocreales) minisatellite is independent of host range and geographic origin

The minisatellite locus, BbMin1, was isolated from a partial Beauveria bassiana genomic library that consisted of poly(GA) flanked inserts. Polymerase chain reaction (PCR) of the BbMin1 repeat demonstrated allele size variation among 95 B. bassiana isolates. Amplification was also observed from single isolates of Beauveria amorpha, Beauveria brongniartii, and Beauveria caledonica. Eight alleles were identified at the haploid locus, where repeat number fluctuated between one and fourteen. AMOVA and theta (Fst) indicated that fixation of repeat number has not occurred within pathogenic ecotypes or geographically isolated samples of B. bassiana. Selective neutrality of allele size, the rate of BbMin1 mutation, and the age of the species may contribute to host and geographic independence of the marker. Presence of alleles with a large number of repeat units may be attributed to the rare occurrence of somatic recombination or DNA replication error. The molecular genetic marker was useful for the identification of genetic types of B. bassiana and related species.

Mining an Ostrinia nubilalis midgut expressed sequence tag (EST) library for candidate genes and single nucleotide polymorphisms (SNPs)

Genes expressed in lepidopteran midgut tissues are involved in digestion and Bacillus thuringiensis (Bt) toxin resistance traits. Five hundred and thirty five unique transcripts were annotated from 1745 high quality O. nubilalis larval midgut expressed sequence tags (ESTs). Full‐length cDNA sequence of 12 putative serine proteinase genes and 3 partial O. nubilalis aminopeptidase N protein genes, apn1, apn3, and apn4, were obtained, and genes may have roles in plant feeding and Bt toxin resistance traits of Ostrinia larvae. The EST library was not normalized and insert frequencies reflect transcript levels under the initial treatment conditions and redundancy of inserts from highly expressed transcripts allowed prediction of putative single nucleotide polymorphisms (SNPs). Ten di‐, tri‐ or tetranucleotide repeat unit microsatellite loci were identified, and minisatellite repeats were observed within the C‐termini of two encoded serine proteinases. Molecular markers showed polymorphism at 28 SNP loci and one microsatellite locus, and Mendelian inheritance indicated that markers were applicable to genome mapping applications. This O. nubilalis larval midgut EST collection is a resource for gene discovery, expression information, and allelic variation for use in genetic marker development.