Toxicological and Biochemical Analyses Demonstrate the Absence of Lethal or Sublethal Effects of cry1C- or cry2A-Expressing Bt Rice on the Collembolan Folsomia candida

Abstract

Assessing the potential effects of insect-resistant genetically engineered (GE) plants on collembolans is important because these common soil arthropods may be exposed to insecticidal proteins produced in GE plants by ingestion of plant residues, crop pollen, or root exudates. Laboratory studies were conducted to evaluate the potential effects of two Bacillus thuringiensis (Bt)-rice lines expressing Cry1C and Cry2A in pollen and leaves and of their non-Bt conventional isolines on the fitness of the collembolan Folsomia candida and on the activities of its antioxidant-related enzymes, superoxide dismutase and peroxidase, and of its detoxification-related enzymes, glutathione reductase and glutathione S-transferase. Survival, development, reproduction, and the intrinsic rate of increase (r_m) were not significantly reduced when F. candida fed on the Bt rice pollen or leaf powder than on the non-Bt rice materials; these parameters, however, were significantly reduced when F. candida fed on non-Bt rice pollen or non-Bt leaf-based diets containing the protease inhibitor E-64 at 75 μg/g. The activities of the antioxidant-related and detoxification-related enzymes in F. candida were not significantly affected when F. candida fed on the Bt rice materials, but were significantly increased when F. candida fed on the non-Bt rice materials containing E-64. The results demonstrate that Cry1C and Cry2A are not toxic to F. candida, and also indicate the absence of unintended effects on the collembolan caused by any change in plant tissue nutritional composition due to foreign gene transformation.

Keywords: Bt rice leaf; Bt rice pollen; ELISA; environmental risk assessment; enzyme activity; non-target effects.

FIGURE 1

Survival of Folsomia candida on a diet consisting of rice pollen (A) or a mixture of rice leaf powder and baker’s yeast (10:1) (B). The pollen and leaf power were obtained from either of two Bt rice lines (T1C-19: Cry1C, and T2A-1: Cry2A) or their non-Bt near isoline MH63 (negative control). For the positive control, the corresponding non-Bt rice tissue was supplemented with E-64. Asterisks indicate a significant difference between the treatment and the negative control (Logrank test, P < 0.01) (n = 50).

FIGURE 2

Fecundity (numbers of eggs) produced per individual (A), hatching rate of eggs (B), and numbers of fecal pellets produced per individual (C) of F. candida fed on a diet consisting of rice pollen (left) or a mixture of rice leaf powder and baker’s yeast (10:1) (right). The rice pollen and rice leaf powder were obtained from either of two Bt rice lines (T1C-19: Cry1C and T2A-1: Cry2A) or their non-Bt near isoline MH63 (negative control). For the positive control, the corresponding non-Bt rice tissue was supplemented with E-64 (positive control) for 35 days. Values are means ± SE, n = 50. Asterisks indicate a significant difference between the treatment and the negative control (MH63) (Dunnett’s test, P < 0.05).

FIGURE 3

Enzyme activities (A: peroxidase, POD; B: superoxide dismutase, SOD; C: glutathione, GR; and D: glutathione S-transferase, GST) in F. candida that fed on a diet consisting of non-Bt rice pollen (MH63, negative control), Bt rice pollen containing Cry1C or Cry2A (T1C-19 or T2A-1), or non-Bt rice pollen containing E-64 protein (positive control) for 35 days. Values are means ± SE, n = 4. Asterisks indicate a significant difference between the treatment and the negative control (MH63) (P < 0.05).

FIGURE 4

Enzyme activities (A: POD, B: SOD, C: GR, and D: GST) in F. candida that fed on non-Bt rice leaves (MH63, negative control), Bt rice leaves containing Cry1C or Cry2A (T1C-19 or T2A-1), or non-Bt leaves containing E-64 protein (E-64, positive control) for 35 days. Rice leaf powder was mixed with baker’s yeast (10:1). Values are means ± SE, n = 4. Asterisks indicate a significant difference between the treatment and the negative control (MH63) (P < 0.05).