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Ampicilin-resistant bacteria in excrement of Oulema melanopus larvae
data: L. Meile and G. Hugenschmidt; Text: T. Schlaich and C. Sautter
Larvae of the cereal leaf beetle (Oulema melanopus) feed on single wheat plants, provided the plants are not too densely planted. The larvae do not loose theirexcrements, but carry them on their back as a protectant shield. At about mid of May adult beetles lay eggs on wheat leaves. Four weeks later thelarvae were developed and had accumulated a significant amount of their excrements. The 11th of June 2004, two larvae were sampled from each plot. The larvae collectedfrom plots bearing the variety Greina were submersed in 1 ml of physiological saline. The larvae feeding from Golin plants were stored without liquid. The samples werekept for 3 days at 4°C. We ignored the variety Frisal. The samples were subsequentlyanalysed by PD Dr. Leo Meile (Inst.f. Lebensmittel-u. Ernährungswissensschaften, ETH Zürich).
Four samples, each in duplicate, i.e. 2 animals from each repetetive plot (e.g. C4, F1, J2, L3) were diluted in 1 ml of a sterile medium containing 0.85 % NaCl and 0.1 % peptone. The mixture was vortexed for 20 seconds. From a total of 8 samples aliquotsof 0.1ml were pooled and from this pool a dilution series was prepared. From eachdilution step, an aliquot of 0.1 ml was plated in duplicate, either on plate-count-agar(PCA) (Oxoid) or PCA supplemented with 50 µg/g ampicillin. The plates were incubated for 2 days at 30°C. The relation between number of colonies growing on PCA and number of colonies growing on PCA + ampicillin was calculated (see Fig. 1). For the treatment ”Golin transgenic control” (i.e. not inoculated with stinkingssmut), the sample was lost. esistan
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Figure 1: Percentage of Ampicilin-resistant bacteria (colonies growing on plates)
We pooled the numbers by wheat lines (Figure 2). The standarddeviations have been calculated from the replication and are are shownwith the exception of the line Golin transgenic, since one sample was lost (see above).
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Figure 2: Percentage of ampicillin-resistant bacteria pooled per wheat line.
Discussion
Expectedly, the number of colonies growing on PC should be higher than thenumber of colonies growing in the presence of the antibiotic, on PC + ampicillin. The percentage of ampicillin-resistant bacteria was in general quite high. Methodical variation can then lead to the seeming paradox of 104 % of ampicillin-resistant bacteria (e.g. treatment Greina transgene control in figure 1). Thedifferent varieties, Golin and Greina cannot be compared directly, since thesampling was different. This could have had a selective effect.
A significant difference between the samples from animals feeding on transgenicor on wildtype plants was not detectable. The in general very high percentage of ampicillin resistant bacteria in thisexperiment does not allow for the conclusion of such a high percentage of ampicillin-resistant bacteria in the intestinal flora of Oulema. Only a small numberof the bacterial species of the Oulema intestinal flora might grow under the appliedmethodical conditions. Therefore, the numbers might not be representative for theactual biodiversity in Oulema intestinal flora, although the relative numbers allowfor a comparison between wildtype fed and GMO fed animals.

Source: http://www.feldversuch.ethz.ch/docs/abschluss/Ampicillin.pdf