Uptake of Cry1Ab-Endotoxins by Generalist Predators in

Fields of Bacillus Thringiensis Corn

Aaron SamsonDepartment of Entomology

University of KentuckyCEEB Symposium

5/12/06

Acknowledgements

• Dr. James D. Harwood(post-doctoral scholar)

• Dr. John J. Obrycki (chairman)

• Casey Bayne (Agricultural Biotechnology undergraduate)Supported in part by grant from the Kentucky Science and Engineering Foundation as per Grant Agreement #KSEF-148-502-04-121 with Kentucky Science and Technology Corporation.

Outline

• Bt Overview

• Project Methodology

• Results

• Implications

• Future Research

Bacillus thuringiensis• Discovered in 1911

•Approx. 1% of agrochemical market

• Contained in bacteria plasmids

• Dried spores and toxin crystals

• Crystals a protoxin (must be activated for effect)

• Activated in increased pH conditions in mig-gut of larvae, then lyses epithelial cells to cause septicemia (safe for humans)

• Used genetically since 1996

Bt Classes

GeneCrystal

Shape

Protein Size (kDa

) Insect Activity

cry I [several subgroups: bipyramidal 130-138lepidoptera

larvae

A(a), A(b), A(c), B, C, D, E, F, G]      

cry II [subgroups A, B, C] cuboidal 69-71lepidoptera and

diptera

cry III [subgroups A, B, C]

flat/irregular 73-74 coleoptera

cry IV [subgroups A, B, C, D] bipyramidal 73-134 diptera

cry V-IX various 35-129 varioushttp://helios.bto.ed.ac.uk/bto/microbes/bt.htm

Purpose

• 67 million ha transgenic crops in 2003

• Insecticidal input decreased and crop yield increased without effects on non-target arthropods

• Concerns raised over negative effects on arthropod food chain

• Tests showed some elevated levels of Bt-endotoxin in non-target herbivores and arthropod predators

• Research used antibody-based assays to identify trophic linkages in field

Generalist Predators• Many predators exposed to Bt-endotoxin through direct consumption on prey

• Slugs and earthworms could provide clues to connection with predators (Scarites subterraneus)

• Anthesis may mark increased Bt-endotoxin in coccinellids

Herbivores

Detrivores

Arthropod Predators

Non-target Predators• Scarites subterraneus, important in agroecosystems for control of Deroceras laeve

• Coccinellids including Harmonia axyridis, Coleomegilla maculata, Cycloneda munda, and Coccinella septempunctata

Field Collection

• Adult coccinellids hand-collected from Bt-corn field at Spindletop Research Farm

• Carabids collected daily from pitfall traps

• Carabids and slugs (D. laeve) used for lab feeding collected from ceramic dishes in alfalfa

• Carabids and slugs starved 72 hours prior to feeding trials

Feeding Trials

D. Laeve feed on leaves for 24 hours

Female carabids feed ad libitum on D. laeve for 3 hours

20 pairs of carabids feed ad libitum on D. laeve for 4 weeks

ELISA (Enzyme-Linked Immunosorbent Assay)

www.activemotif.com/ catalog/assay_kits/nr

Feeding Trial Results

All carabids negative

-

control

+

control

Bt-2

carabid

Bt-1

carabid

Bt+2

carabid

Bt+1

carabid

Bt-

slug

Bt+

slug

Feeding Trial Results

Average 57.98 ng g-1 Cry1Ab Bt-endotoxin found in D. laeve slugs fed Bt-corn seedlings, but no effect on egg production rates, mean hatching success, or mean time to hatching with S. subterraneus.

All 175 field S. Subterraneus showed values lower than 0.25 ng g-1

Contrast to aphid-spider-carabid linkage

Field-Collected Carabids

Many carabids readily consume plant material

Contrast to Araneae, Coccinellidae, Heteroptera

Field-Collected CoccinellidsGut screening of 1,126 samples showed significant results:

12.8% C. maculata resulted in positive levels

All species showed positive prior to anthesis

H. Axyridis and C. maculata screened positive 4-5 weeks post-anthesis

Species Number % positive

Coleomegilla maculata 775 12.80%

Harmonia axyridis 213 3.80%

Coccinella septempunctata 82 2.40%

Cycloneda munda 56 3.60%

Proportion positive adult coccinellids

Carabid Implications

Hypothesis that Bt-endotoxin moves through corn-slug-carabid food chain is rejected

S. subterraneus avoids prey S. subterraneus employ pre-oral digestion

Exposure of Bt-endotoxin to S. subterraneus had no effect on fecundity, fitnessOccur earlier in season and before anthesis

Scarites quadriceps, Evarthus sodalis in higher densities pre-anthesis

Harpalus pennsylvanicus, Amara cupreolata in higher densities post-anthesis

Positive results for C. maculata suggests path different than corn-herbivore-coccinellid (non-pollen)

Microbial action on pollen transferred to spores and ingested

Late results could be H. axyridis consuming weaker coccinellids or consumption of pollen shed earlier

Coccinellid Conclusions

Basidiomycota

Future Research

• Observation of other species around anthesis

• Fitness and fecundity exposure lab trials for other predators

• Identification of other possible trophic linkages using field population surveys and quantitative assessments

Corn

Corn

Orius spp.

Nabid

Coccinellid

Coccinellid

Summary• Expansion of Bt-crops prompts need for research

• Some arthropods show elevated levels of Bt-endotoxin

• Hypothesized that elevated levels of Bt-endotoxin in corn-slug-carabid pathway and around anthesis

• No positive samples in field or lab carabids and no effect on fecundity, fitness

• Significant positive results in coccinellids, but no connection with anthesis

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