UPTAKE OF CRY1AB-ENDOTOXIN BY GENERALIST
PREDATORS IN FIELDS OF BACILLUS THURINGIENSIS CORN
R Aaron Samson, James D Harwood and John J Obrycki
Department of Entomology, University of Kentucky, Lexington KY 40546-0091.
ELISA Screening Uptake of Bt-endotoxin by Coccinellids in the Field
Each frozen sample was screened using standard ELISA protocols. Each slug, Gut screening of 1,126 coccinellids indicated small, but significant numbers of
Introduction coccinellid, and carabid was weighed and diluted based on weight of carabids and positive results:
Genetically engineered crops have been highly successful in contributing to coccinellids (guts were extracted and weighed; whole bodies of slugs were
economically valuable levels of pest control (Reed et al. 2001; Carrière et al. homogenized). Following homogenization, samples were vortex mixed, centrifuged, • While few H. axyridis, C. septempunctata, and C. munda screened positive for Bt-
2003) often with little or no consequence for non-target organisms following and plated on a standard 96-well plate. Following sample addition, Cry1Ab antibody endotoxin, 12.8% of C. maculata resulted in positive Bt-endotoxin levels.
their exposure to transgenic material in the laboratory (Hellmich et al. 2001; was aliquotted to each well, followed by enzyme conjugate and horseradish
Lundgren & Wiedenmann 2002) or field (Naranjo 2005; Pilcher et al. 2005). peroxidase. Plates were read at 450 nm after addition of dilute acid stopping solution.
However, areas planted to transgenic crops are continuing to increase
• The proportion of adults for all four species testing positive for Bt-endotoxin varied
(Lawrence 2005), giving cause for concern with regard to their impact on the throughout the enitre season, although every species showed some positive prior to
Figure 2. Diagram description of “sandwich” ELISA (Enzyme- anthesis and before access to Bt-pollen.
non-target food chain (Obrycki et al. 2004). Linked Immunosorbent Assay). The antigen screened for is
Very few studies examine the trophic movement of these transgenic plant- detected by plating on pre-coated Cry1Ab antibody wells. Cry1Ab
derived compounds in the field (Harwood et al. 2005; Harwood et al. 2006; is additionally added on top of antigen followed by secondary • Two of the most abundant species of coccinellid, H. axyridis and C. maculata,
Zwahlen & Andow 2005). Many generalist predators are exposed to Bt- antibody (conjugate) and chromophore color reaction. screened positive up to ten weeks after anthesis, reaching a peak at 4-5 weeks after
endotoxins through direction consumption of plant material and feeding on anthesis whereupon approx. 40% screened positive for Bt-endotoxin.
herbivorous/ detritivorous prey. Post-mortem gut-content analyses allow the
accurate measurement of target material within predators using antibody
technology (Sheppard & Harwood 2005). This study explored the detection of
Bt-endotoxins throughout the corn-slug-carabid chain and any effect altering
egg production in carabids. Also, we examined the significance of Bt-endotoxin Laboratory feeding trials Figure 5. Percent Bt-endotoxin positive
coccinellids for each species as
movements through the food chain of coccinellids and identified linkages within
Despite the movement of Bt-endotoxins into D. laeve, the short or long-term exposure determined by ELISA analysis at 450 nm
these food webs around anthesis. Based on previous results, it was (dotted line indicates approximate date of
of S. subterraneus to slugs showed no further movement through the food chain.
hypothesized that detectable amounts would be present in both carabids and beginning of anthesis).
These toxins were broken down very rapidly such that no detectable proteins were
coccinellids, with the highest detectable amounts in coccinellids occurring at or
present in higher order predators tested.
Figure 1. Adult Coleomegilla maculata, a
not-target predator examined for the their Conclusions
uptake of Bt-endotoxin before, during, and
after anthesis. • The hypothesis that low levels of Bt-endotoxin move through the corn-slug-carabid
food chain is rejected, suggesting that either S. subterraneus is avoiding Bt-
containing prey altogether or that pre-oral digestion by S. subterraneus is resulting in
breakdown of toxins
• Exposure of S. subterraneus to Bt-containing slugs had no effect on fecundity or
Figure 3. Absorbance levels of Bt- and nonBt-containing controls, D. laeve slugs, and
Methods Scarites carabids and their approximate percent movement through each vector. • Large positive results from C. maculata for Bt-endotoxin before and after anthesis
Field Collection on Samples suggests a pathway different from that of the corn-herbivore-coccinellid pathway
(late peak could be attributed to delayed consumption of pollen or consumption of
Four species of adult coccinellids (Harmonia axyridis, Coccinella Although an average of 57.98 ng g-1 Cry1Ab Bt-endotoxin was found to be present in pollen-feeding prey).
septempunctata, Cycloneda munda, and Coleomegilla maculata) were hand- D. laeve slugs fed Bt-corn seedlings, there was no effect on egg production rates,
collected from fields of Bt-corn during the growing season at Spindletop mean hatching success, or mean time to hatching in pairs of S. subterraneus. • Since C. maculata also feeds on spores, it is possible that microbial action on
Research Farm in Lexington,KY. Carabid beetles (Scarites subterraneus) were pollen and decaying plant material on the ground could be transferred to fungal
collected daily from pitfall traps randomly located throughout the Bt-corn field. spores and ingested.
Each sample was then transferred to individual tubes and placed in a freezer
until screening. Deroceras laeve (native marsh slug) and S. subterraneus used • Adult H. axyridis readily consume less competitive coccinellids, possibly leading to
for lab trials were collected from upturned ceramic dishes and pitfalls, Uptake of Bt-endotoxins by Scarites subterraneus in the Field a tri-trophic interaction in which C. maculata is responsible for the movement of Bt-
respectively. D. laeve was then transferred into containers and fed a diet of endotoxins through the food chain.
Overall, 175 S. subterraneus adults (158 male and 17 female) were collected from
green lettuce and Russet potatoes until a starving period of 72 hours. S.
the field and all samples showed values which were below the predetermined
subterraneus were also starved for one week prior to the feeding experiment.
threshold value of 0.25 ng g-1 (correlating to an absorbance value of 0.108 at 450 References
nm). Harwood JD, Samson RA, Obrycki JJ 2006 No evidence for the uptake of Cry1Ab Bt-endotoxins by the generalist predator
Scarites subterraneus (Coleoptera: Carabidae) in laboratory and field experiments. Biocontrol Science and Technology,
Laboratory Feeding Trials
Harwood, J.D., Wallin, W.G. & Obrycki, J.J. 2005 Uptake of Bt-endotoxins by non-target herbivores and higher order
D. Laeve were allowed to feed on fresh Bt-corn leaves for 24 hours. Thereafter, arthropod predators: molecular evidence from a transgenic corn agroecosystem. Mol. Ecol. 14, 2815-2823.
ten slugs were separated for screening with ELISA (Enzyme-Linked Hellmich, R.L., Siegfried, B.D., Sears, M.K., Stanley-Horn, D.E., Daniels, M.J., Mattila, H.R., Spencer, T., Bidne, K.G. &
Figure 4. Male Harmonia axyridis are known to readily feed Lewis, L.C. 2001 Monarch larvae sensitivity to Bacillus thuringiensis purified proteins and pollen. Proc. Nat. Acad. Sci.
Immunosorbent Assay) and female S. subterraneus were allowed to feed ad USA 98, 11925-11930.
on pollen, yet no correlation has been made between pollen
libitum on slugs for three hours. The remaining slugs were used in a four-week Lundgren, J.G. & Wiedenmann, R.N. 2002 Coleopteran-specific Cry3Bb1 toxin from transgenic corn pollen does not affect
consumption and high levels of Bt-endotoxin.
feeding trial, by which 20 pairs of male and female carabids were allowed to feed the fitness of a nontarget species, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae). Environ. Entomol. 31,
ad libitum on Bt-containing on non-Bt-containing slugs. Egg production and
Obrycki, J.J., Ruberson, J.R. & Losey, J.E. 2004 Interactions between natural enemies and transgenic insecticidal crops. In
hatching success were monitored under similar conditions. At various time Genetics, Evolution and Biological Control (eds. L.E. Ehler, R. Sforza & T. Mateille), pp. 183-206. Wallingford: CAB
periods after feeding, carabids were frozen and screened using ELISA. International.
Sheppard, S.K. & Harwood, J.D. 2005 Advances in molecular ecology: tracking trophic links through predator-prey food webs.
Funct. Ecol. 19, 751-762.