Transgenic Research

, 17:345

Interactions of Bacillus thuringiensis Cry1Ac toxin in genetically engineered cotton with predatory heteropterans

Authors

    • Departamento de Agronomia-EntomologiaUniversidade Federal Rural de Pernambuco
  • John R. Ruberson
    • Department of EntomologyUniversity of Georgia
Original Paper

DOI: 10.1007/s11248-007-9109-8

Cite this article as:
Torres, J.B. & Ruberson, J.R. Transgenic Res (2008) 17: 345. doi:10.1007/s11248-007-9109-8

Abstract

A number of cotton varieties have been genetically transformed with genes from Bacillus thuringiensis (Bt) to continuously produce Bt endotoxins, offering whole plant and season-long protection against many lepidopteran larvae. Constant whole-plant toxin expression creates a significant opportunity for non-target herbivores to acquire and bio-accumulate the toxin for higher trophic levels. In the present study we investigated movement of Cry1Ac toxin from the transgenic cotton plant through specific predator-prey pairings, using omnivorous predators with common cotton pests as prey: (1) the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), with the predator Podisus maculiventris (Heteroptera: Pentatomidae); (2) the two-spotted spider mite, Tetranychus urticae (Acarina: Tetranychidae), with the predatory big-eyed bug Geocoris punctipes (Heteroptera: Geocoridae) and (3) with the predatory damsel bug Nabis roseipennis (Heteropera: Nabidae); and (4) the thrips Frankliniella occidentalis (Thysanoptera: Thripidae) with the predatory pirate bug Orius insidiosus (Heteroptera: Anthocoridae). We quantified Cry1Ac toxin in the cotton plants, and in the pests and predators, and the effects of continuous feeding on S. exigua larvae fed either Bt or non-Bt cotton on life history traits of P. maculiventris. All three herbivores were able to convey Cry1Ac toxin to their respective predators. Among the herbivores, T. urticae exhibited 16.8 times more toxin in their bodies than that expressed in Bt-cotton plant, followed by S. exigua (1.05 times), and F. occidentalis immatures and adults (0.63 and 0.73 times, respectively). Of the toxin in the respective herbivorous prey, 4, 40, 17 and 14% of that amount was measured in the predators G. punctipes, P. maculiventris, O. insidiosus, and N. roseipennis, respectively. The predator P. maculiventris exhibited similar life history characteristics (developmental time, survival, longevity, and fecundity) regardless of the prey’s food source. Thus, Cry1Ac toxin is conveyed through non-target herbivores to natural enemies at different levels depending on the herbivore species, but continuous lifetime contact with the toxin by the predator P. maculiventris through its prey had no effect on the predator’s life history. The results found here, supplemented with others already published, suggest that feeding on Cry1Ac contaminated non-target herbivores does not harm predatory heteropterans and, therefore, cultivation of Bt cotton may provide an opportunity for conservation of these predators in cotton ecosystems by reducing insecticide use.

Keywords

Transgenic cottonTritrophic interactionsBiological controlPredatory heteropteranNon-target effectsCry1AC fate

Introduction

Bt-transgenic cotton that expresses the Cry1Ac toxin from Bacillus thuringiensis is now planted on the majority of the cotton-producing land in the southeastern United States, and in large areas in Australia, Brazil, South Africa, and Argentina (Huesing and English 2004), for control of a complex of lepidopteran pests. Not all pest lepidopteran species are equally susceptible, however. Some lepidopteran species, such as armyworms (Spodoptera spp.) and loopers (several plusiine Noctuids), can survive feeding on Bt-cotton expressing Cry1Ac toxin (Adamczyk et al. 1998; Henneberry et al. 2001; Stewart et al. 2001), although they experience some deleterious effects (reduced size and survival, and longer larval development). There is also a complex of non-lepidopteran herbivores that is unaffected by the toxin. Among these unaffected taxa are stink bugs, plant bugs, aphids, whiteflies, mites, and thrips. The partially susceptible lepidopterans and non-lepidopteran herbivores can survive feeding on Bt-plants and are capable of conveying toxin to the next trophic level (Obrist et al. 2005; 2006; Harwood et al. 2005; Torres et al. 2006).

Predatory heteropterans are common and important members of the natural enemy complexes of a variety of row crops worldwide (see chapters in Schaefer and Panizzi 2000). Among this group are at least six genera common to cotton fields in the southeastern United States (Torres and Ruberson 2005), and their generalized feeding habit exposes them to a range of prey. Further, they are omnivorous feeders that also feed on plants, which may expose them directly to the Cry toxins in Bt-transgenic plants. Geocoris and Orius species, two important predatory genera in cotton fields, prey on several pest species, including the two-spotted spider mite, Tetranychys urticae, and the western flower thrips, Frankliniella occidentalis (Wilson et al. 1991; Schoenig and Wilson, 1992; Ellington et al. 1997), both of which are known to acquire Cry1Ab from Bt maize (Dutton et al. 2002). Population dynamics of natural enemies in large Bt and non-Bt cotton fields are increasingly being elucidated (e.g., see papers in Environmental Entomology, v.35, n. 4, 2005), but there are still many questions about the interactions of the transgenic toxins and natural enemies. For example, the extent to which toxin moves through trophic levels, and how it might affect natural enemies is still poorly understood. We previously found that partially Bt-susceptible lepidopteran larvae from commercial Bt-cotton fields (Spodoptera exigua, Spodoptera eridania and Pseudoplusia includens) contained around 2.7-fold less toxin in their bodies than was present in the plants (Torres et al. 2006). In contrast, the relatively small predatory heteropterans Geocoris punctipes, Nabis roseipennis and Orius insidiosus did not acquire detectable levels of Cry1Ac feeding on lepidopteran larvae fed on Bt-transgenic cotton. On the other hand, Cry1Ac toxin was detected in field-collected spined soldier bugs, Podisus maculiventris, which are larger predatory bugs compared to the above assayed species, and these field results were confirmed with confined studies in the greenhouse. This predator, however, consumed considerably more prey biomass compared to the small predatory heteropterans (G. punctipes, N. roseipennis and O. insidiosus) (Torres et al. 2006). These results suggest that the amount of food ingested influences the amount toxin acquired by predators. However, acquisition of toxin can also vary significantly with prey. Obrist et al. (2006) reported that T. urticae fed Cry1Ab Bt-maize contained approximately 10-fold more toxin than the larvae of lepidopteran Spodoptera littoralis. Also, in an earlier study Obrist et al. (2005) found that the thrips, Frankliniella tenuicornis acquired Cry1Ab when feeding on Bt-maize during all activity stages.

Given the wide variability in toxin acquisition by prey and predators, we undertook a series of studies to examine the extent to which prey type and predator size/species influence toxin acquisition. To address this question, we used three prey types–two-spotted spider mites, T. urticae, western flower thrips, Frankliniella occidentalis, and the beet armyworm, Spodoptera exigua. We further examined whether exposure to the Bt toxin affects life history parameters of a predator species naturally exposed to the toxin in the field (P. maculiventris) when continuously fed prey reared on Bt-cotton. Therefore, this study had four objectives: (i) to evaluate the acquisition of Cry1Ac toxin by two-spotted spider mite and western flower thrips feeding on Bt-cotton; (ii) to quantify the levels of Cry1Ac toxin conveyed to the predators G. punctipes, N. roseipennis and O. insidiosus by these prey items; (iii) to determine whether contaminated prey fed Bt-cotton affects the development and reproductive characteristics of the predator P. maculiventris, exposed to the toxin through lepidopteran larvae in Bt-cotton fields (Torres et al. 2006); and (iv) to assess how long ingested Cry1Ac remains in the body of P. maculiventris after preying on lepidopteran larvae fed-Bt cotton.

Material and methods

Insect materials

Beet armyworm larvae

Larvae of S. exigua (BAW) used in the experiments were obtained from a culture maintained at the Biological Control Laboratory of the Coastal Plain Experiment Station, Tifton, GA. The neonate BAW larvae (<12 h) were caged on upper, fully-expanded cotton leaves using organdie fabric sleeve cages. Caging of BAW larvae on plants was done repeatedly to maintain a constant supply of insects of appropriate size for experimentation. We offered the predators somewhat older BAW larvae fed Bt-cotton than those fed non-Bt cotton in order to standardise larval size and eliminate any confounding prey-size effects (larvae grow slower on Bt cotton). Overall, BAW larvae fed Bt-cotton required an additional 4–5 days to reach a length of 1.5–2.0 cm compared to larvae fed non-Bt cotton. BAW larvae of appropriate size were harvested every morning and offered to the predator nymphs and adults. To isolate other effects than prey fed Bt-cotton, the BAW larvae were offered to the predators inside cages without host plants.

Two-spotted spider mite.

Cotton plants infested with two-spotted spider mite, T. urticae, were obtained by spreading infested cucumber leaves from old plant growth in the greenhouse over the cotton plant tops. After ∼15 days, three to four upper expanded cotton leaves in the plants harbouring two-spotted spider mites were heavily infested with immatures and adults. The uppermost fully expanded infested leaves were then used in assays with caged predators, and as a source of mites to assay for toxin.

Western flower thrips

The colony of western flower thrips, F. occidentalis, was derived from old cotton plants in the greenhouse that were naturally infested with the thrips. To increase the numbers of thrips for assays, the source population was multiplied in Bt and non-Bt cotton seedlings (10–15 days-old). After 2–3 weeks, the plants became heavily infested, and could be used in the study.

Predatory bugs

Most of the predatory heteropterans used in the experiments (big-eyed bug, G. punctipes, pirate bug, O. insidiosus, and damsel bug, N. roseipennis) originated from collections in cotton fields near Tifton, GA. The two former species were reared in the laboratory using corn earworm eggs as prey for three to four generations before use in the studies (25 ± 1°C, photoperiod L:D = 14:10). Damsel bugs were collected as adults or in older nymphal stages for use in trials, and were maintained in plastic cages with pinto bean pods and medium-sized BAW larvae until experiments were initiated. The spined soldier bug, P. maculiventris, originated from a collection of females on peach trees near Plains, GA, and was cultured for five generations in the laboratory using BAW larvae as prey. All cultures were maintained at 26.1 ± 1.2°C and photoperiod L:D = 12:12 to 13:11.

Cotton plants

The various herbivore species were reared for testing on Bt-cotton plants expressing the Cry1Ac toxin (variety Deltapine 555 BB/RR, BollgardR; Delta Pine and Land, Scott, MS) and on a non-transgenic variety (Deltapine 5415 RR; Delta Pine and Land, Scott, MS), both of which were grown in the greenhouse. Seeds were sown in pots (15 x 15 cm) filled with high porosity potting soil BM6™ (Berger Peat Moss Saint-Modeste, Quebec), mixed with 14-14-14 (N-P-K) controlled-release fertilizer (Osmocote™, Scotts-Sierra Horticultural Products Company, Marysville, OH), and maintained under greenhouse conditions of 26 ± 4°C (mean ± SD) and ∼14 h of light. Plants with 5–6 fully developed leaves were infested with each of the three tested herbivores: two-spotted spider mite, T. urticae, western flower thrips, F. occidentalis, and neonate BAW. Immatures and adults of the first two species were not considered separately.

Predator exposure to the Cry1Ac toxin through prey fed Bt-cotton

Four interactions involving Bt or non-Bt cotton plants, herbivorous prey and predatory heteropterans were studied: (1) immatures and adults of western flower thrips as prey for O. insidiosus, (2) two-spotted spider mites as prey for G. punctipes, (3) two-spotted spider mites as prey for N. roseipennis, and (4) BAW larvae as prey for P. maculiventris. For each plant-herbivore-predator interaction, 10 cages were placed on the uppermost expanded cotton leaf and infested with the respective herbivores. In each cage we placed (with no regard to gender): 20 O. insidiosus (n = 200 adults/treatment), 5 G. punctipes (n = 50 adults/treatment), 3 N. roseipennis (n = 30 adults/treatment), and 3 P. maculiventris (n = 30 adults/treatment). Cages were constructed of 500-ml styrofoam cups with the bottoms removed, and the cup was enclosed in a knee-high stretch stocking, which was extended over the plant leaf and tied closed. Each predator species was starved for 24–36 h prior to being caged. Cages receiving the predator P. maculiventris enclosed eight BAW (∼1.5 cm in length) from groups of larvae reared on Bt or non-Bt cotton and moved to the tested plants the day predators were released into the cages. The numbers of thrips and mites exposed to the predators were unknown but heavily infested leaves were used so that there was always excess prey in the cage. Immatures and adults of thrips and two-spotted spider mites are readily accepted as prey by Orius, Geocoris and Nabis, regardless of prey age, while BAW larvae offered as prey to P. maculiventris were manipulated to produce similar size when reared on Bt and on non-Bt cotton (see below). Because of the logistical requirements, the predator-prey studies could not be conducted simultaneously for all species combinations, but rather were conducted one at a time. Samples of plants, prey and predators were taken 48h after caging predators on both Bt and non-Bt cotton in the greenhouse. Only living predators and prey were collected for toxin measurement. All collected materials were placed in microcentrifuge tubes and stored in a freezer at −25°C until the ELISA assays were conducted.

We evaluated persistence of the toxin in the body of P. maculiventris after preying on BAW larvae fed Bt-cotton because we had previously found Cry1Ac in the bodies of feral P. maculiventris adults collected in Bt cotton fields (Torres et al. 2006). Adults of P. maculiventris, caged for 48 h on Bt or non-Bt cotton and provided BAW larvae as prey, were collected and the predators were separated into three groups. One group of predators was immediately frozen at −20°C (designated as time 0 h, after having been caged for 48 h in the respective prey and plant system; n = 10 adults per treatment). The other two groups were transferred to rearing cages in the laboratory where they were maintained on BAW larvae reared on non-Bt plants. The predators in these treatments were then frozen at −20°C 24 (n = 10 adults) and 48 h (n = 8 adults) after being removed from Bt-cotton plants. The bugs were subsequently analysed for presence of Cry1Ac toxin (see below).

Preimaginal development and reproduction of P. maculiventris fed Bt-cotton and non-Bt cotton reared BAW

Because we found that adult P. maculiventris collected in Bt-cotton fields contained Cry1Ac toxin (Torres et al. 2006), we investigated whether continuous feeding on Cry1Ac-contaminated prey affects the life history of P. maculiventris. Second-instar P. maculiventris nymphs from laboratory-reared predators were reared to adulthood preying on BAW larvae fed either Bt or non-Bt cotton (first instars do not feed on prey). Predator rearing was conducted in 12 × 10 × 10 cm (length, width and height) plastic cages lined with paper towels. Each of 22 cages was stocked with six 2nd-instar nymphs, and half of the cages were provisioned with ∼2.0 cm-long BAW larvae reared on Bt-cotton, and the remainder were provisioned with BAW larvae of the same size reared on non-Bt cotton. A small ball of saturated cotton was placed in each cage as a moisture source. The cages were maintained in the laboratory under 12–13 h photoperiod and at 26.1 ± 1.2°C. Each day, nymphal survival was noted and prey replaced until adult emergence.

Adults of P. maculiventris emerging from both treatments were maintained on the same diets they had experienced as nymphs, and set up in individual pairs 3 days after emergence. Adult survival and reproduction were observed for 30 days following adult emergence (n = 12 females per treatment). This 30-days period includes most of the reproductive activity of this species when fed a range of prey species (De Clercq 2000). The procedure used to provide prey and moisture to adults was identical to that used for nymphs.

Cry1Ac toxin analysis

All of the organisms used in the experiments were assayed for Cry1Ac toxin using enzyme-linked immunosorbent assay (ELISA). Cotton leaves and surviving prey (of two-spotted spider mites, immature and adult western flower thrips, and BAW larvae) were collected and frozen at −25°C. Plant material of each plant-prey-predator system consisted of leaf discs collected by snapping a centrifuge tube cap down between the main veins of the each leaf (ca. ten samples/each leaf inside cages) hosting the prey and predators inside the cage for each Bt and non-Bt cotton treatment. Numbers of specimens assayed in the prey-predator systems varied as a function of insect size. We used 10 mg of immature and adult mites combined, 47 G. punctipes, 10 mg of immature thrips, 12 mg of adult thrips, 178 O. insidiosus, and 23 N. roseipennis. Specimens were frozen until extraction was conducted (5–10 days later). For the toxin retention experiment we assayed 12 S. exigua larvae (at the 0-h interval) and 28 P. maculiventris divided into three groups, corresponding to 0, 24, and 48 h after switching predators to BAW larvae reared on non-Bt cotton. Extraction of Cry1Ac toxin from plant material, S. exigua, P. maculiventris and N. roseipennis was accomplished by macerating the sample material in buffer in a 10-ml tube. Due to the small weight:volume sample ratio generated by two-spotted spider mites, western flower thrips, big-eyed bugs and insidious flower bugs, the extraction of toxin in buffer for these species was made by macerating the samples in 1.5 ml microcentrifuge tubes. Frozen materials were thawed on the day of toxin extraction, weighed, and transferred to 10-ml tubes or 1.5-ml microcentrifuge tubes, and mixed with phosphate-buffer saline solution in Tween20 (1xPBST) (Agdia® Inc., Elkhart, IN). Non-fat dried milk (0.4% w/v) and Tween20 (0.5% v/v) were added to PBST to compose the final extraction buffer, which was mixed with sample material at a rate of 1:10 (w/v). Due to the expected greater concentration of Cry1Ac toxin in the body of T. urticae (Obrist et al. 2006), the final rate dilution for this species was 1:20 (w/v). The extract supernatants were transferred to clean 1.5 ml microcentrifuge tubes and centrifuged at 5000 rpm for 1 min and loaded at a rate of 100 μl per test well. The toxin levels in the samples were assayed using antibody-coated wells in PathoScreen® plates for Bt-Cry1Ac/Cry1Ab in ELISA kits using peroxidase enzyme conjugate (Agdia® Inc., Elkhart, IN). Absorbance measurements were taken with an ELx808 microtiter plate reader (Bio-Tek Instruments Inc., Winooski, VT) reading at 450 nm. To read the results at 450 nm, 50 μl of a 3 M sulfuric acid solution was added to each well to stop further development of the reaction in the wells prior to reading. Standards of Cry1Ac at concentrations 0.625, 1.25, 2.5, 5, 10, 20 and 40 ng/ml (p.p.b.) were used to build a standard optical density curve for estimating toxin content of tested material.

Statistical analysis

Nymphal developmental time and survival, adult fresh body weight at emergence, age at first oviposition, and the number of eggs and nymphs produced per female during the first 30 days of the adult stage were measured and analysed comparing P. maculiventris provided BAW larvae fed Bt-cotton with those provided BAW larvae fed non-Bt cotton. Prior analyses, these data were tested for normality (Kolmogorov-D:Normal test) and homogeneity of variance (Bartlett’s test), and square root (x + 0.5) transformations were used with exception for nymphal survival that did not require transformation; however, untransformed means are presented in tables and figures. Further, the results were submitted to the Student’s t-test using the pooled method for equal sample variance through the Proc TTEST of SAS (SAS Institute 1999–2001) to compare predatory bugs reared BAW larvae fed Bt and those reared BAW larvae fed non-Bt cotton plants. The proportion of adult females surviving within 30 days of adult emergence was compared between treatments (females fed Bt or non-Bt cotton reared BAW larvae) by the Long-Rank test through Kaplan–Meyer method using the Proc LIFTEST of SAS (SAS Institute 1999–2001).

Results

Cry1Ac in Bt-cotton, herbivores and predators

Cry1Ac was detected in all three trophic levels of the Bt-cotton treatment (Fig. 1), whereas no toxin was detected in plants, herbivores or predators of the non-Bt cotton treatment. The average toxin titre in Bt-cotton leaves from cages confining herbivores and predators was 0.19 ± 0.01 μg Cry1Ac g−1 of fresh tissue. From this amount of Cry1Ac toxin expressed in Bt-cotton plants about 63% and 73% were detected in immature and adult thrips, respectively, at the second trophic level. Cry1Ac was detected in T. urticae at 16.8 times higher concentration (3.19 ± 0.53 μg Cry1Ac g−1 of fresh weight) than the original concentration in Bt-cotton. Toxin titres for BAW are presented below. In the third trophic level, Cry1Ac was detected in all four tested predatory bugs, regardless of prey. Although Cry1Ac was highly concentrated in T. urticae, the amount conveyed to their predators–the big-eyed bug, G. punctipes, and the damsel bug, N. roseipennis–was only 4% and 14%, respectively, of the original concentration in the prey. Also, only about 17% of Cry1Ac measured in western flower thrips’ bodies was detected in its predator O. insidiosus (Fig. 1). Data for P. maculiventris are presented below (Fig. 2).
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Fig. 1

Levels of Cry1Ac toxin (mean + SE) in Bt-cotton and in predators and herbivore preys interactions: Nabis roseipennis preying on Tetranychus urticae, Geocoris punctipes preying on Tetranychus urticae and, Orius insidiosus preying on Frankliniella occidentalis immatures and adults) reared on Bt-cotton. Note that the y-axis scale differs among taxa as a result of large differences in Cry1Ac concentration. Bars in grey, dark grey and line pattern stand for first, second and third trophic levels tested, respectively

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Fig. 2

Levels of Cry1Ac toxin (mean + SE) in Bt-cotton, the herbivore (Spodoptera exigua), and in the predator (Podisus maculiventris–open circles; mean ± SE) over time fed S. exigua reared on Bt-cotton, on the day of collection (0h), and 24 h and 48 h after being switched to a diet of S. exigua reared on non-Bt cotton

Acquisition and retention of Cry1Ac toxin in the body of Podisus maculiventris

Bt toxin Cry1Ac (mean ± SE) was detected in the three trophic levels comprising Bt-cotton leaf samples, herbivorous BAW larvae, and the predator P. maculiventris (Fig. 2). Similar levels of Cry1Ac detected in Bt-cotton were acquired by the BAW (0.19 and 0.20 μg Cry1Ac g−1 fresh weight, respectively), but only about 40% of Cry1Ac from the herbivore was conveyed to P. maculiventris (0.075 ± 0.02 μg Cry1Ac g−1 predator fresh weight) frozen on the day of collection from Bt-cotton (time 0h). However, 24 h and 48 h after being switched to BAW larvae reared on non-Bt cotton as prey, spined soldier bugs had only about 13% and no detectable levels of the Cry1Ac remaining in their bodies, respectively (Fig. 2).

Preimaginal development and reproduction of P. maculiventris fed Bt-cotton and non-Bt cotton reared larvae

The predator P. maculiventris exhibited similar nymphal developmental times and reproductive output when preying on BAW larvae reared on Bt or non-Bt cotton (Table 1). Nor were nymphal survival (Table 1) or adult female survival up to 30 days after adult emergence (Fig. 3) significantly affected. The nymphal period lasted about two weeks from 2nd-instar to adult emergence, with survival from 68.2% to 75.7% for Bt and non-Bt cotton treatments, respectively. Average weight differences of newly emerged adult males and females fed BAW larvae reared on Bt or non-Bt cotton during nymphal stages were only 1.5 mg and 1.0 mg, respectively.
Table 1

Major life history characteristics (mean and 95% CI) of the spined soldier bug Podisus maculiventris nymphs fed Spodoptera exigua larvae reared on Bt- and non-Bt cotton as immatures and for the first 30 days following adult emergence

Characteristics

Cotton genotypes

Statistics

Bt

Non-Bt

Developmental time, days*

15.2 (14.7–15.7)

15.4 (15.1–15.7)

t92 = −0.71, p = 0.4782

Survival of nymphs (%)*

68.2 (48.5–87.8)

75.7 (56.1–95.4)

t20 = −0.61, p = 0.5497

Body weight, mg (♀)

76.4 (72.4–82.4)

77.47 (3.2–79.4)

t51 = −0.03, p = 0.9741

Body weight, mg (♂)

58.4 (56.3–60.5)

59.9 (58.1–6.6)

t39 = −1.14, p = 0.2632

Age of 1st oviposition

6.1 (5.2–6.9)

5.3 (4.7–5.9)

t21 = 1.49, p = 0.1508

Number of eggs/female

420.3 (299.9–540.8)

396.4 (254.8–537.9)

t21 = 0.46, p = 0.6499

Number of nymphs/female

394.1 (278.9–537.9)

364.6 (234.1–494.9)

t21 = 0.54, p = 0.5978

*Time from second instar to adult emergence including both genders

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Fig. 3

Age-specific survivorship of Podisus maculiventris females for 30 days following adult emergence, preying on Spodoptera exigua larvae reared on Bt and non-Bt cotton from the time the predators were second-instar nymphs. There is no significant difference between the survivorship curves for females through the Log-Rank test (χ2 = 0.5607, p = 0.4560) and Wilcoxon test (χ2 = 0.4558, p = 0.4996)

Within 30 days of becoming adults, P. maculiventris females reared on both prey types produced similar numbers of eggs and nymphs (Table 1), and adult survival 30 days after adult emergence did not differ significantly, whether predators were fed BAW larvae reared on Bt (66.7%) or on non-Bt cotton (58.3%) (Fig. 3).

Discussion

It is apparent that Cry1Ac toxin is able to move across trophic levels in the cotton system, as the toxin was found in all three trophic levels (Fig. 1). Cry1Ac or Cry1Ab toxins expressed in Bt-cotton and Bt-maize, the two most widely cultivated Bt transgenic crops worldwide, have been detected in herbivores (Harwood et al. 2005; Dutton et al. 2002; Obrist et al. 2005; Torres et al. 2006), but to date few significant effects of the toxins on predator life history characteristics have been observed in the limited tritrophic number of studies conducted. Zhang et al. (2006) observed that a Bt-cotton variety expressing both Cry1Ab and Cry1Ac toxins and a variety expressing only Cry1Ac toxin reduced weight gain, developmental rate and survival for second-instar Spodoptera litura, as expected for a partially susceptible lepidopteran species. These contaminated larvae offered as prey to first instars of the predatory coccinellid Propylea japonica reduced weight gain (18%) and survival of predators relative to those reared on prey from non-Bt cotton. On the other hand, when comparing the effects of prey reared on a single or dual Bt toxins transformed variety (Cry1Ab and Cry1Ac), the predators fed prey reared on the dual-gene plants exhibited greater weight gain (14%) and larval survival (20%) than those given prey from the single-gene plant, although S. litura larvae reared on the dual-gene plants contained ∼26% more toxin than larvae reared in the single-gene plants. Therefore, the predator larvae grew and survived better on prey larvae conveying greater amounts of toxin to the third trophic level. This result suggests other factors besides the toxins themselves and contaminated prey were at play in the outcomes of these experiments during their short evaluation period. Further, this same predator, P. japonica, when fed either cotton aphid, Aphis gossypii (Homoptera: Aphididae) reared on Bt-cotton varieties or on planthopper, Nilaparvata lugens (Homoptera: Delphacidae), reared on Cry1Ab-transgenic rice, exhibited similar development, survival and fecundity to predators reared on prey from non-Bt plants (Zhang et al. 2006b; Bai et al. 2006).

Retention of ingested Bt-toxins in non-targeted herbivores and predators, whether acquired through prey or directly from purified toxins, appears to be relatively short. In the present study, Cry1Ac levels in the predator P. maculiventris, fed BAW larvae reared on Bt-cotton decreased to 13% and 0% within 24 h and 48 h of the predators being switched from Bt-intoxicated prey to BAW larvae reared on non-Bt cotton (Fig. 2). Short toxin retention was also found in G. punctipes drinking 16 and 32 ppm purified Cry1Ac toxin water concentrations (Torres et al. 2006). These authors found detectable levels at lower and higher Cry1Ac concentrations up to 24 and 48 h, respectively, and significant decreases 1, 12, 24h, 48 and 72 h after ingestion were observed. In addition, high levels of toxin were found in G. punctipes faeces in the intervals 0–12, 12–24 and 24–48 h. Obrist et al. (2005) quantified time of retention and excretion of Cry1Ab toxin by the thrips F. tenuicornis reared on Bt-maize and found that only about 3% of the original toxin levels present on day 0 remained in the thrips’ bodies 24 h after being switched to non-Bt maize, and only a trace of toxin was detected at day 4. In addition, the author reported high levels of Cry1Ab toxin in dry faeces of the thrips. These results and those obtained for P. maculiventris and G. punctipes suggest that Bt-toxins acquired by herbivores and conveyed to predators have low persistence in the predators’ bodies. Presently no data exist about the fate of ingested Bt-toxins in the digestive system of heteropterans. Regarding the high levels detected in their faeces, we can hypothesize that it is excreted as undigested food, or at least a portion of the toxin is sufficiently intact to produce a reaction with ELISA. Whether or not the toxin is broken down, the short persistence of the toxin in the body of predatory heteropterans is probably related to the pre-oral digestion and liquid feeding behaviour of these predators, which allow much shorter food retention times in the gut for digestion and absorption compared to solid feeders.

In light of the results of Dutton et al. (2002), Obrist et al. (2005, 2006) and those reported here, there is some probability that predatory heteropterans in the field will contain detectable amounts of Bt-toxin, but this probability will depend on the amount and types of prey consumed, and the capacity of the prey to acquire and concentrate the toxin. In this case, the higher Bt-toxin concentration in particular herbivorous prey, such as two-spotted spider mites and western flower thrips, are likely to be conveyed at detectable levels to the small predatory heteropterans (Geocoris, Orius and Nabis), while prey with lower toxin concentrations, such as BAW larvae, are less likely to do so. On the other hand, larger predators, such as P. maculiventris, that consume large amounts of contaminated prey are able to acquire enough toxin from these low-concentration prey to reach detectable levels.

The absence or minimising of negative impacts on natural enemies is important because the final output may result in an additive control considering the multipest cotton ecosystem–Bt against lepidopteran larvae plus natural enemies against non-targeted Bt pests–or possibly in a synergistic outcome when predators consume greater numbers of prey that are partially affected by the toxin expressed in the plant, and without effect on predator performance. In the Bt-cotton ecosystem, BAW larvae and other partially susceptible lepidopteran species exhibit slowed development rates that extend the window of larval susceptibility to these predators, generating a positive interaction between host plant resistance and natural enemies (Bottrell et al. 1998). Torres et al. (2006) found that P. maculiventris consumed nearly twice as many BAW larvae of the same age reared on Bt-cotton (10.1 larvae) compared to larvae reared on non-Bt cotton (5.7 larvae). However, it is clear from the results of the present work that increased consumption of intoxicated caterpillars does not adversely affect the life history of this predator. Likewise, the big-eyed bug, G. punctipes, exhibited similar life history characteristics fed BAW larvae caged either on Bt- or on non-Bt cotton plants in the field (Torres and Ruberson 2006).

Although all the three predatory bugs investigated in this study are important in cotton fields, and the results found in this study showed their exposure to Cry1Ac toxin through prey fed Bt-cotton, we focused on the life history characteristics of only P. maculiventris for two major reasons. First, like the other heteropterans studied here, it is a common predator in cotton fields; however it is the only heteropteran predator that was found to have detectable levels of Cry1Ac toxin in feral field populations (Torres et al. 2006). Second, previous studies already investigated the development and reproduction of G. punctipes feeding on BAW larvae reared on Bt-cotton (Torres and Ruberson 2006). In a long-term field study, Torres and Ruberson (2006) evaluated development and reproduction of G. punctipes during two cotton seasons when fed young BAW larvae confined on Bt-cotton (Cry1Ac toxin; variety DPL 458RR) and non-Bt cotton (variety DPL 5690RR). As was the case in the present study, the life history of the predator was unaffected by Bt and non-Bt cotton plants, or by prey acquiring the toxin. Likewise, the predator Orius majusculus feeding on the thrips Anaphothrips obscurus reared on Bt-maize (Zwahlen et al. 2000) and O. insidiosus feeding on larvae of the lepidopteran Ostrinia nubilalis reared on diet mixed with Bt-toxins (Al-Deeb et al. 2001). Nabids have not yet been studied. Nevertheless, when these results are all considered, there is little evidence for adverse effects of Bt toxins (at least Cry1Ac) on predatory bugs, which are important predators in many agroecosystems.

Our data add more specific information on the tritrophic interactions of Bt-cotton, herbivores and predatory heteropterans to the growing body of data on population dynamics of naturally occurring predatory taxa in Bt-cotton fields. Genetically modified cotton expressing Cry1Ac or combined Cry1Ac/2Ab is highly effective against many lepidopteran larvae, but management of herbivorous pests not targeted by the Bt toxin still benefits from suppression provided by predators and parasitoids occurring in the crop agroecosystem. The results of other experiments cited above, and those of the present study reach the same conclusion–predatory bugs are not affected when feeding on prey conveying Bt-toxins, even though levels of toxin in the prey can be much higher than that expressed by the genetically modified plants.

Acknowledgments

We are greatly indebted to Evelyn Perry and Stephen Mullis (Horticulture/UGA, Tifton) for allowing us to use their lab facilities to run ELISA assays. This research was supported in part by Georgia Cotton Commission and Cotton Incorporated and “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES Foundation Brazil) with grant to J.B.T.

Copyright information

© Springer Science+Business Media B.V. 2007