Outlines research done on quantifying the effects of various species of ladybeetles as well as the carabid beetle.

1. Uptake of Cry1Ab-Endotoxins by
Generalist Predators in Fields of
Bacillus Thringiensis Corn
Aaron Samson
Department of Entomology
University of Kentucky
CEEB Symposium
5/12/06

2. 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.

3. Outline
• Bt Overview
• Project Methodology
• Results
• Implications
• Future Research

4. 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

5. Bt Classes
Protein
Crystal Size
Gene Shape (kDa) Insect Activity
cry I [several subgroups: bipyramidal 130-138 lepidoptera larvae
A(a), A(b), A(c), B, C, D, E,
F, G]
lepidoptera and
cry II [subgroups A, B, C] cuboidal 69-71 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 various
http://helios.bto.ed.ac.uk/bto/microbes/bt.htm

6. 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

7. Generalist Predators
• Many predators
Herbivores
exposed to Bt-endotoxin
through direct
consumption on prey
• Slugs and earthworms
could provide clues to Detrivores
connection with
predators (Scarites
subterraneus)
• Anthesis may mark Arthropod
increased Bt-endotoxin Predators
in coccinellids

8. Non-target Predators
• Scarites
subterraneus,
important in
agroecosystems for
control of Deroceras
laeve
• Coccinellids
including Harmonia
axyridis, Coleomegilla
maculata, Cycloneda
munda, and
Coccinella
septempunctata

9. 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

10. Feeding Trials
D. Laeve feed
on leaves for
24 hours
Female
carabids feed
20 pairs of ad libitum on D.
carabids feed laeve for 3
ad libitum on D. hours
laeve for 4
weeks

11. ELISA (Enzyme-Linked
Immunosorbent Assay)
www.activemotif.com/ catalog/assay_kits/nr

12. Feeding Trial Results
All carabids
negative
- + Bt+ Bt- Bt+1 Bt+2 Bt-1 Bt-2
control control slug slug carabid carabid carabid carabid

13. Feeding Trial Results
All 175 field S. Subterraneus showed values lower than 0.25 ng g-1
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.
Contrast to aphid-spider-carabid linkage

14. Field-Collected Carabids
Many carabids readily consume plant material
Contrast to Araneae, Coccinellidae, Heteroptera

15. Field-Collected Coccinellids
Gut screening of 1,126 samples showed significant results:
12.8% C.
maculata
resulted in
positive levels
All species
showed
positive prior
H. Axyridis
to anthesis
and C.
maculata
screened
positive 4-5
weeks post-
anthesis

16. 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

17. 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, fitness
Occur earlier in season and before anthesis
Scarites quadriceps, Evarthus sodalis in higher
densities pre-anthesis
Harpalus pennsylvanicus, Amara cupreolata in
higher densities post-anthesis

18. Coccinellid Conclusions
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 Basidiomycota
consuming weaker coccinellids or
consumption of pollen shed earlier

19. 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 Nabid Coccinellid
Corn Orius spp. Coccinellid

20. 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

21. References
Dutton A, Klein H, Romeis J, Bigler F 2002. Uptake of Bt-toxin by herbivores feeding on transgenic maize and consequences for the predator
Chrysoperla carnea. Environmental Entomology 27: 441-447.
Harwood, J.D., Samson, R.A. & Obrycki, J.J. 2006 No evidence for the uptake of Bt Cry1Ab-endotoxins by the carabid predator Scarites
subterraneus (Coleoptera: Carabidae) in laboratory and field experiments. Biocontr. Sci. Technol. 16
Harwood JD, Wallin WG, Obrycki JJ 2005. Uptake of Bt endotoxins by nontarget herbivores and higher order arthropod predators: molecular
evidence from a transgenic corn agroecosystem. Molecular Ecology 14: 2815-2823.
James C 2003. Global Status of Commercialized transgenic Crops: 2003; ISAAA Briefs no. 30: Preview. ISAAA, Ithaca, NY.
Howald R, Zwahlen C, Nentwig W 2003. Evaluation of Bt oilseed rape on the non-target herbivore Athalia rosae. Entomologia Experimentalis
et Applicata 106: 87-93.
J Li, J Carroll and DJ Ellar, 1991.Nature 353, 815-821
Obrycki JJ, Losey JE, Taylor OR, Jesse LCH 2001. Transgenic insecticidal corn: beyond insecticidal toxicity to ecological complexity. BioScience
51: 353-361.
Obrycki JJ, Ruberson JR, Losey JE 2004. Interactions between natural enemies and transgenic insecticidal crops In: Ehler LE, Sforza R, Matielle
T, editors. Genetics, Evolution and Biological Control. Wallingford, UK: CAB International. p. 183-206.
Raps A, Kehr J, Gugerli P, Moar WJ, Bigler F, Hilbeck A 2001. Immunological analysis of phloem sap of Bacillus huringiensis corn and of the
nontarget herbivore Rhopalosiphum padi (Homoptera: Aphididae) for the presence of Cry1Ab. Molecular Ecology 10:
525-533.
Shelton AM, Zhao JZ, Roush RT 2002. Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants.
Annual Review of Entomology 47: 845-881.
Way MJ, Van Emden HF 2000. Integrated pest management in practice – pathways toward successful application. Crop Protection 19: 81-103.
Wolfenbarger LL, Phifer PR 2000. Biotechnology and ecology – the ecological risks and benefits of genetically engineered plants. Science 290:
2088-2093.