- Email: [email protected]
Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties
Journal of Insect Physiology 56 (2010) 157–164
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties Yunhe Li, Michael Meissle, Jo¨rg Romeis * Agroscope Reckenholz-Ta¨nikon Research Station ART, Reckenholzstr. 191, 8046 Zurich, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2009 Received in revised form 28 August 2009 Accepted 18 September 2009
We investigated the use of maize pollen as food by adult Chrysoperla carnea under laboratory and field conditions. Exposure of the insects to insecticidal Cry proteins from Bacillus thuringiensis (Bt) contained in pollen of transgenic maize was also assessed. Female C. carnea were most abundant in a maize field when the majority of plants were flowering and fresh pollen was abundant. Field-collected females contained an average of approximately 5000 maize pollen grains in their gut at the peak of pollen shedding. Comparable numbers were found in females fed ad libitum maize pollen in the laboratory. Maize pollen is readily used by C. carnea adults. When provided with a carbohydrate source, it allowed the insects to reach their full reproductive potential. Maize pollen was digested mainly in the insect’s mid- and hindgut. When Bt maize pollen passed though the gut of C. carnea, 61% of Cry1Ab (event Bt176) and 79% of Cry3Bb1 (event MON 88017) was digested. The results demonstrate that maize pollen is a suitable food source for C. carnea. Even though the pollen grains are not fully digested, the insects are exposed to transgenic insecticidal proteins that are contained in the pollen. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Bt maize Cry1Ab Cry3Bb1 Pollen digestion Environmental risk assessment
1. Introduction The common green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), is an important natural predator of insect herbivores in many different crop and non-crop habitats (New, 1975). The predatory larvae feed preferentially on aphids but can consume a wide range of soft-bodied arthropods (Principi and Canard, 1984). Adults are not predacious and live on pollen, nectar, and honeydew (Principi and Canard, 1984; Sheldon and MacLeod, 1971; Villenave et al., 2005, 2006; Li et al., 2008). The current study concerns the interactions between adult C. carnea and maize pollen. Maize produces large amounts of pollen, i.e., up to 50 million pollen grains per maize tassel over a period of 5–8 days (Goss, 1968; Treu and Emberlin, 2000). Many insects, including a number of natural enemies, consume maize pollen (Pilcher et al., 1997; Corey et al., 1998; Lundgren and Wiedenmann, 2004) because it contains organic and inorganic nutrients, such as sugars, starch, amino acids and proteins, lipids, as well as vitamins and minerals (Goss, 1968; Stanley and Linskens, 1974; Roulston and Buchmann, 2000a). While laboratory studies have shown that maize pollen can also serve as a food source for adult C. carnea (Sheldon and MacLeod, 1971; Li et al.,
* Corresponding author. Tel.: +41 44 377 72 99; fax: +41 44 377 72 01. E-mail address: [email protected] (J. Romeis). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.09.011
2008), it is still unclear to what extent adult C. carnea ingest and use maize pollen as a food source in the field. Insect-resistant genetically engineered maize varieties expressing Cry proteins from the bacterium Bacillus thuringiensis (Bt) are grown on increasing areas worldwide (Hellmich et al., 2008). Most varieties are either protected from lepidopteran pests (such as stemborers) by expressing Cry1 proteins or from corn rootworms (Diabrotica spp.; Coleoptera: Chrysomelidae) by expressing Cry3 proteins (Hellmich et al., 2008). The commercialization of Bttransgenic maize varieties has stimulated numerous studies on the potential effects on non-target organisms including those contributing to the natural regulation of herbivores (Romeis et al., 2006, 2008a; Wolfenbarger et al., 2008). Pollen-feeding species may also be exposed to Bt proteins, because Cry3Bb1-expressing varieties with the transformation events MON863 and MON88017 and Cry1Ab-expressing varieties with event Bt176 (which have been removed from the market) show pollen expression levels almost in the same order of magnitude as the green tissue (Obrist et al., 2006; Romeis et al., 2009; Meissle and Romeis, 2009a). For assessing the risks of Bt maize on natural enemies, both the level of exposure to the toxin and the hazard of being exposed must be considered (Garcia-Alonso et al., 2006; Romeis et al., 2008b). Previous hazard studies have shown that C. carnea females are not adversely affected when consuming purified Cry1Ab or Cry3Bb1 in an artificial diet or Bt maize pollen that contained the toxins in a biologically active form (Li et al., 2008; Meissle and Romeis,
158
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
2009b). This paper, however, focuses on exposure levels of adult green lacewings to Cry protein in Bt maize. A series of experiments was conducted in the laboratory and in the field to answer the following questions: (i) how abundant are adult C. carnea in maize fields during anthesis? (ii) to what extent can adult C. carnea use maize pollen as a food source? (iii) how many pollen grains are consumed and to what extent are they digested? (iv) how much Cry protein is digested by adult C. carnea when feeding on Bt maize pollen? 2. Materials and methods 2.1. Field survey Adults of C. carnea were collected in a maize field (0.5 ha) in Zurich, Switzerland. Conventional maize (DK287) was sown on 15 May 2004 and managed according to common practice. No insecticide was used. Adult C. carnea were collected by sweep-net sampling in the field center (>2 m from the field borders) and along the edge (<1 m from the field borders). Weekly samples were taken in 2004 from 20 July to 18 August, covering a period that started before anthesis, that included flowering, and that ended when pollen was no longer present. The time spent in sampling was recorded and used to calculate the number of lacewings collected per hour. Within 3 h, collected insects were individually frozen at 80 8C in 1.5 ml microreaction tubes. A binocular microscope was used to divide the lacewings into C. carnea and C. lucasina (Lacroix) (Neuroptera: Chrysopidae), following Henry et al. (2002); these two species often co-occur in Central Europe (Duelli, 2001; Villenave et al., 2006). The specimens were then divided into males and females. All studies focused on females because males were found to consume only small amounts of maize pollen in the laboratory and the field (Villenave et al., 2005; Li et al., 2008). To correlate the number of lacewings with the presence of maize pollen, the availability of pollen in the field was monitored. At each sampling date, 20 maize plants were randomly selected in the center of the maize field, and the percentage of flowering plants was recorded. In addition, the amount of pollen deposited on leaves was recorded as zero, low, or high. Aphids on these plants were also counted. Aphids may influence the abundance of adult lacewings because they serve as food for the larvae and provide honeydew to the adults (Principi and Canard, 1984). 2.2. Insect and plant material for laboratory experiments Eggs of C. carnea were collected from a permanent laboratory colony (for details on the rearing, see Romeis et al., 2004) and kept in a climatic chamber at 22 1 8C, 75 5% RH, and a 16:8 h photoperiod. After hatching, the larvae were fed with eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) provided by Biotop (Valbonne, France). After the larvae reached the second instar, they were kept individually in plastic tubes (1.5 cm diameter, 5.5 cm high) and supplied ad libitum with E. kuehniella eggs until pupation. Newly emerged (<24 h) adults were used for the experiments. Two transgenic maize varieties, Compa CB1 (Event Bt176, Syngenta, Stein am Rhein, Switzerland) and DKc5143Bt (Event MON 88017, Monsanto, St. Louis, USA) and their corresponding nontransformed near isolines Dracma1 and DKc5143, respectively, were used for the experiments. Compa CB1 plants express a modified cry1Ab gene from Bacillus thuringiensis ssp. kurstaki targeting Lepidoptera. Expression is driven by the constitutive PEPC promoter as well as a pollen-specific promoter (Koziel et al., 1993). DKc5143Bt expresses a modified cry3Bb1 gene from Bacillus thuringiensis ssp. kumamotoensis targeting corn rootworms (Vaughn et al., 2005). Expression is driven by a constitutive enhanced 35S
promoter (P-e35S) (Monsanto, 2004). Plants were grown in the glasshouse, and pollen was collected as described by Li et al. (2008). 2.3. Fitness of adult C. carnea feeding on maize pollen Newly emerged adults of C. carnea were sexed, and single pairs were confined in transparent plastic cylinders (6.0 cm diameter, 8.5 cm high). Lids with a 4 cm opening were used to allow ventilation. Between cylinder and lid, a layer of cotton gauze prevented insect escape and served as an oviposition substrate. Water was provided by a dental wick, which was positioned through a 1 cm hole at the bottom of each container. The cylinders were placed over a water reservoir so that the cotton wicks were submerged and a continuous water supply was ensured. Adults were provided with one of the following diets: (1) only water as a non-food control; (2) maize pollen (Dracma1); (3) 1 M sucrose solution (Merck, Darmstadt, Germany); (4) Maize pollen and 1 M sucrose solution; and (5) artificial diet consisting of sucrose, brewer’s yeast, and water (in proportions 7:4:4). The artificial diet served as a reference control because it is considered highly nutritious for adult C. carnea (Hagen and Tassan, 1970; Jones et al., 1977). Maize pollen and sucrose solution were offered separately in small plastic dishes (2.5 cm diameter, 0.7 cm high), which were placed on the bottom of the test containers. Artificial diet was provided on one end of a green plastic label (9.0 cm 1.6 cm). All diets were provided ad libitum and replaced every 3 days. Each diet treatment was replicated with 20 pairs of C. carnea. The experiment was conducted in a climatic chamber at 22 1 8C, 75 5% RH, and a 16:8 h photoperiod, and lasted for 28 days. Longevity, pre-oviposition period (mean number of days from emergence to the first oviposition), daily and total fecundity, and fertility (egg hatching rate) were recorded. Eggs were removed daily from the containers and counted. For the assessment of fertility, collected eggs were placed in a separate container every 3 days together with the oviposition substrate (gauze) and with ad libitum E. kuehniella eggs as food to minimize cannibalism of hatching lacewing larvae. Seven days later, hatched larvae were counted. After termination of the experiment, surviving adult C. carnea were frozen. Subsequently, the dry weights were determined on an electronic microbalance MX5 (Mettler Toledo, Greifensee, Switzerland) after drying at 50 8C for 4 days. For data analysis, three to four pairs had to be excluded from each of the pollen only, pollen and sucrose solution, and artificial diet treatments because the lacewings had escaped, were damaged, or were infertile (unmated). The water treatment was not statistically analyzed because all insects died within 7 days without laying eggs. For the sucrose solution treatment, oviposition parameters were not compared with the other treatments because females laid very few eggs. Pre-oviposition period was compared using Mann–Whitney U-tests with Bonferroni correction for three pair-wise comparisons (adjusted a = 0.017). For daily fecundity, repeated-measures ANOVA was carried out and the means were separated using the Tukey HSD test. Data on total fecundity, fertility, and dry weight (females and males separately) were analyzed using a one-way ANOVA followed byffi the Tukey HSD pffiffiffiffiffiffi test. Fertility data were transformed by arcsin ðxÞ and dry weight of males by log(x) before analysis. The survival rates of adult lacewings fed with the different diets (excluding the water only control) were compared using Chi-square (x2) tests with Bonferroni correction for six pair-wise comparisons (adjusted a = 0.0083). 2.4. Consumption of maize pollen To measure how much pollen is consumed by lacewings when maize pollen is provided ad libitum, we fed newly emerged
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
C. carnea females with artificial diet for 1 week. Then the insects were separated into two groups: one group was fed only maize pollen (DKc5143) while the other group was fed with maize pollen and 1 M sucrose solution (in separate containers). Water provision and experimental conditions were as described above. Lacewings were allowed to feed for 2 days, which was sufficient time for them to replace their gut content (unpublished observation). On the third day, 15 individuals from each group were frozen at 20 8C at 9:00, 12:00 and 18:00 h; these sampling times were used because we suspected that the quantity of pollen grains in the gut could vary with time of day. To quantify maize pollen grains in the gut of C. carnea, we thawed and excised the females, and the whole alimentary canal was pulled out. Subsequently, the gut was transferred to a 1.5 ml microreaction tube containing 100 ml fuchsin acid solution. The red color stained the pollen grains and facilitated counting. After the gut was ruptured with a thin needle, the pollen suspension was mixed using a Vortex mixer. An aliquot (5 ml) of the suspension was transferred to a glass slide, and pollen grains (including full, partially digested, and empty ones) were counted with a microscope at 50 magnification. Counting was repeated for three subsamples for each insect gut, and the mean number of pollen grains was multiplied by 20 to estimate the total number contained in the insect. Six to thirteen females were analyzed at each sampling time for both the pollen only and the pollen and sucrose solution treatments. In addition to the pollen content of laboratory-fed insects, the pollen content in the guts of six to fourteen females collected in the center of the maize field was determined for each sampling date between 20 July and 10 August 2004.
159
containers without food. After 6 h, fresh fecal pellets were collected and stored at 20 8C for digestion analysis. To observe the digestion status of the pollen grains in different gut sections of C. carnea females, we excised the alimentary canal and divided it into crop, diverticulum, midgut, and hindgut following Woolfolk and Inglis (2003). The pollen grains obtained from each gut section were transferred to a drop of 0.5 M sucrose solution on a glass slide. One drop of lactophenol cotton blue was added to stain the cytoplasm dark blue, while the cell walls were left unstained. Basic fuchsin served as a counterstain to highlight the exine in pink, which particularly helped with the counting of empty pollen shells (Human and Nicolson, 2003). After staining, the microscope slide was sealed with a cover slip and examined with a microscope at 200 magnification. For each slide, approximately 100 randomly selected pollen grains were classified into one of four groups (Fig. 1): (A) undigested grains with 90– 100% of original content; similar in shape to fresh pollen; (B) slightly digested grains with 50–90% of original content; irregular shape; (C) heavily digested grains with 10–50% of original content; shrunken; (D) fully digested grains with 0–10% of original content; heavily shrunken or burst open. Fresh pollen grains and pollen grains contained in lacewing feces were classified in the same way. Fresh pollen, gut sections, and feces were each replicated six times. In addition, females collected in the edge of the maize field were assessed for the degree of pollen digestion in the foregut (crop and diverticulum) and hindgut. Six to ten females were investigated for each of three sampling dates in 2004 (27 July, 3 August, and 10 pffiffiffiffiffiffi ffi August). After arcsin ðxÞ transformation, one-way ANOVA was conducted to compare the proportions of the undigested pollen grains in the foregut among the three sampling dates.
2.5. Digestion of maize pollen
2.6. Fate of Cry protein contained in Bt maize pollen after lacewing gut passage
For the assessment of pollen digestion, newly emerged females were fed with artificial diet for 1 week in the laboratory. Subsequently, they were provided with maize pollen (DKc5143) and sucrose solution for 2 days as described above. Half of the group was sampled and stored at 20 8C for analysis of pollen digestion. The remaining females were transferred into new
Newly emerged females of C. carnea were fed with artificial diet. After 1 week, the insects were provided with water only for at least 24 h to empty their gut. Then the insects were placed in 10 plastic containers (10 insects per container) and provided with water and Bt maize pollen of Compa CB1 or DKc5143Bt (five containers per maize treatment). After 2 days, the lacewings were transferred to
Fig. 1. Micrographs showing the different degrees of digestion of maize pollen grains in the gut of adult Chrysoperla carnea. The degree of digestion was classified as follows: (A) undigested (retaining 90–100% of content; similar in shape to fresh pollen); (B) slightly digested (retaining 50–90% of content; similar in shape to fresh pollen); (C) heavily digested (retaining 10–50% of content; shrunken); (D) fully digested (retaining 0–10% of content; shrunken or burst open).
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
160
new containers with the same food. Fresh fecal pellets were collected every 2 h. The feces were pooled for each container, and five samples were collected for each maize variety. Five subsamples of fresh pollen from each of the two Bt maize varieties were also collected. All samples were stored at 80 8C. After lyophilization, the concentrations of Cry1Ab or Cry3Bb1 were measured using Double-Antibody Sandwich Enzyme-Linked ImmunoSorbent Assay (DAS-ELISA) purchased from Agdia (Elkhard, Indiana, USA) and as described by Li et al. (2008). The measured OD values were calibrated using Cry1Ab or Cry3Bb1 standards made from purified toxins, which were provided by M. Carey (Dept. Biochemistry, Case Western Reserve University, Cleveland, OH, USA) and by Monsanto Company, respectively (Li et al., 2008). The total digestion rate of Bt toxin in pollen cannot be determined from Cry protein concentrations (Cry protein per dry weight) because the digestion process reduces both the amount of Cry protein and the weight of the pollen grains. Therefore, the mean Bt toxin content of a single pollen grain derived from feces or directly from the plant was assessed. Lyophilized fresh pollen or feces (1.0 mg) were mixed with 300 ml fuchsin acid solution. The pollen grains were counted in each of three 5 ml aliquots of the suspension with a microscope at 50 magnification. The mean number of pollen grains in the aliquots was multiplied by 60 to obtain the number in the whole sample. Finally, the weight of each sample (1.0 mg) was divided by the number of grains to obtain the mean dry weight of an individual pollen grain. For each of the two maize varieties, this procedure was repeated seven to ten times. Based on the individual dry weight of pollen grains and the Bt protein concentrations in fresh maize pollen and feces, the Bt content in single pollen grains was calculated. 3. Results 3.1. Field survey On the first sampling date (20 July), when pollen was not yet available, 1.5 C. carnea females were collected per hour of sweepnet sampling. In the early stage of pollen shedding (27 July), when about half of the maize plants were flowering and only small amounts of pollen were deposited on leaves, 5.4 females were collected per hour. Most females (17.0 per hour) were collected on 3 August when 80% of maize plants were flowering and large amounts of pollen were on the leaves. On 10 August, only 5% of maize plants were still flowering, and 4.8 females were caught per hour. On the last sampling date on 18 August, pollen was no longer available in the field and no adult C. carnea was found (Supplementary data 1). During the sampling period, a large density of aphids (21 per plant) was observed only before the start of maize flowering (20
Fig. 2. Survival (A; n = 32–40) and daily fecundity (B; mean SE, n = 16–20) of Chrysoperla carnea adults fed one of five diets.
July). At all later dates, the aphid density was less than 1 per plant (Supplementary data 1). 3.2. Fitness of adult C. carnea feeding on maize pollen During the 4 weeks of the experiment, no mortality of adult C. carnea occurred in the artificial diet and the pollen and sucrose treatments (Fig. 2A). All adults died within 7 days in the non-food control treatment (water only) (Fig. 2A). At the end of the experiment, lacewing survival was significantly lower (x2-test; P = 0.006) in the pollen only treatment (80%) than in the artificial diet (100%) or pollen and sucrose (100%) treatments, but did not differ (x2-test; P > 0.05) from survival in the sucrose solution treatment (95%). Survival did not statistically differ (x2-test; P > 0.05) among the artificial diet, pollen and sucrose, and sucrose solution treatments. The females in the sucrose solution treatment did not produce fertile eggs (Fig. 2B, Table 1). The pre-oviposition period was similar for the artificial diet and pollen and sucrose treatments (Mann–Whitney U-test; U = 98, P = 0.11), while the pre-oviposition period was significantly longer for the pollen only treatment (artificial diet: U = 38, P < 0.001; pollen and sucrose: U = 18, P < 0.001, Table 1). Daily fecundity also differed significantly
Table 1 Life-table parameters of adult Chrysoperla carnea when fed for 4 weeks with artificial diet (sucrose: brewer’s yeast: water; 7:4:4), maize pollen, or 1 M sucrose solution either alone or in combination. Diet
Artificial diet Pollen and sucrose Pollen Sucrose
Number of pairs
17 17 16 20
Pre-oviposition period (days)a
Number of eggs laid per femaleb
Egg hatching rate (%)b,c
Adult dry weight (mg)
4.4 0.30 b 3.8 0.20 b 7.7 0.79 a –
576.3 54.97 a 640.9 49.14 a 170.5 26.07 b –
76.6 6.04 a 79.7 4.73 a 85.1 2.07 a –
9.2 0.22 9.0 0.31 6.7 0.46 6.7 0.46
Values are means SE in columns 3–5. Means in a column followed by the same letter are not significantly different (P > 0.05). a Kruskal–Wallis ANOVA, followed by pair-wise comparisons using Mann–Whitney U-test. b One-way ANOVA followed by Tukey HSD test. pffiffiffiffiffiffiffi c Percentages were transformed using arcsin ðxÞ before analysis. d Data were transformed using log(x).
Femaleb
Maleb,d a a b b
4.1 0.16 4.9 0.30 3.6 0.21 4.1 0.18
b a b ab
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
among the three treatments (repeated-measures ANOVA; F2,46 = 29.4, P < 0.0001) (Fig. 2B). While fecundity did not differ between pollen and sucrose and artificial diet treatments (Tukey HSD test; P = 0.58), females provided with pollen only laid fewer eggs per day than females provided with pollen and sucrose or artificial diet (P = 0.001). Similar results were obtained for total fecundity, for which there was a significant treatment effect (one-way ANOVA; F2,47 = 30.4, P < 0.0001), no difference between pollen and sucrose and artificial diet (Tukey HSD test; P = 0.58), and a significant reduction in the pollen only treatment (P < 0.001) (Table 1). The egg hatching rates (fertility) were similar among the three treatments (one-way ANOVA, F2,46 = 0.72, P = 0.49). At the end of the experiment, the dry weight of females (oneway ANOVA; F3,72 = 18.0, P < 0.0001) and males (F3,72 = 7.0, P = 0.0003) differed among the food treatments. Dry weights of females did not differ between artificial diet and pollen and sucrose treatments (Tukey HSD test, P = 0.99) or between pollen only and sucrose solution treatments (P = 0.99) (Table 1). However, females fed with pollen only or sucrose solution weighed significantly less than those fed on artificial diet or pollen and sucrose (P < 0.001). Males feeding on pollen and sucrose weighed significantly more than those fed on artificial diet (P = 0.035) or pollen only (P = 0.0003); they also tended to weigh more than those fed sucrose solution (P = 0.058). The weight of males did not differ
161
among the artificial diet, pollen only, and sucrose solution treatments (P > 0.2) (Table 1). 3.3. Consumption of maize pollen In the laboratory, all females provided with maize pollen for 2 days contained pollen grains in their guts (Fig. 3A). Females feeding on maize pollen and 1 M sucrose solution contained 10–30% more pollen grains than those that had access to pollen only. In both treatments, females collected in the morning contained more pollen than those collected in the evening (Fig. 3A). In the field, no pollen grains were found in the guts of females collected before maize anthesis on 20 July. Females collected in the middle of the pollen-shedding period (3 August) contained the most pollen grains, and numbers were similar to those in females with ad libitum access to pollen in the laboratory (Fig. 3A and B). Compared to females collected in the middle of pollen shedding, females collected at the beginning (27 July) or the end (10 August) of the pollen-shedding period contained less than half the number of grains (Fig. 3B). 3.4. Digestion of maize pollen Pollen collected from glasshouse grown plants and offered to females in the laboratory contained 98.5% full grains (Fig. 4A). In
Fig. 3. Number of maize pollen grains (mean SE) in the gut of female Chrysoperla carnea. (A) Insects (n = 6–13) were collected in the morning, noon, and evening after having fed on maize pollen alone or maize pollen together with a 1 M sucrose solution for more than 2 days in the laboratory. (B) Insects (n = 6–14) were collected in a maize field at different times during the pollen-shedding period: Start = 27 July, 58% of plants flowering; Middle = 3 August, 80% flowering; End = 10 August, 5% flowering.
Fig. 4. Percentage of digested maize pollen grains in different gut sections and feces of female Chrysoperla carnea. Fresh pollen was used as a reference. (A) Females collected from the laboratory after being fed with maize pollen and 1 M sucrose solution; n = 6. (B) Females collected in a maize field during anthesis; n = 6–10.
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
162
Table 2 Digestion of maize pollen grains and their Cry protein content when passing through the digestive system of female Chrysoperla carnea. Insects were fed with maize pollen and water, and the feces were collected; n = 7–10. Bt maize variety (event)
Pollen source
Weight per pollen grain (ng)
Pollen digestion rate (%) (Rp = (a1 a2)/a1 100)
Bt concentration (mg/g dry weigh)
Bt content per grain (pg) (cx = axbx 103)
Digestion rate of Cry protein (%) (RCry = (c1 c2)/c1 100)
Compa CB1 (Bt176)
Fresh pollen Feces
202.1 6.67 (a1) 98.1 2.44* (a2)
51.5
11.2 0.22 (b1) 9.1 0.27 (b2)
2.3 0.05 (c1) 0.9 0.03* (c2)
60.5
DKc5143Bt (MON88017)
Fresh pollen Feces
227.7 12.55 (a1) 87.3 2.09* (a2)
61.7
6.9 0.13 (b1) 3.8 0.32 (b2)
1.6 0.03 (c1) 0.3 0.03* (c2)
78.8
Values are means SE in columns 3, 5, and 6. * Significant differences of feces treatments when contrasted against the corresponding fresh pollen treatments (P < 0.05).
the crop and diverticulum of female C. carnea, 94.1% and 95.5% pollen grains, respectively, were full (Fig. 4A). In the midgut, only 14.6% of the grains remained visibly undigested, and 50.6% and 33.4% of the pollen grains were slightly and heavily digested, respectively. The proportion of undigested pollen grains further decreased to 3.6% in the hindgut. The status of the grains in the feces was similar to that in the hindgut, and no undigested pollen grains were present in the hindgut or feces of females fed in the laboratory (Fig. 4A). In the field, the proportion of undigested pollen grains in the foregut did not differ among females collected at the start, middle, and end of maize anthesis (one-way ANOVA, F2,17 = 2.51, P = 0.11); the same was true for heavily digested pollen grains in the hindgut (F2,14 = 0.04, P = 0.96). Therefore, data from the three different sampling dates were combined. The field-collected females contained a high percentage of full pollen grains in the foregut but a high percentage of heavily digested pollen grains in the hindgut (Fig. 4B). Compared to the insects reared in the laboratory, field-collected females contained 13.8% less undigested pollen grains in the foregut, and 6.4% more fully digested grains in the hindgut (Fig. 4A and B). 3.5. Fate of Cry toxin in Bt maize pollen after C. carnea gut passage During the passage through the insect gut, the pollen grains from the two Bt maize varieties lost more than half of their initial weight, with greater digestion of DKc5143Bt pollen than Compa CB1 pollen (Table 2). In addition, the Cry protein concentration in the feces decreased more with DKc5143Bt pollen (by 44.6%) than with Compa CB1 pollen (by 18.7%). Altogether, more of the initial toxin content was digested by C. carnea per pollen grain from DKc5143Bt compared to Compa CB1 (Table 2). 4. Discussion 4.1. Attractiveness of flowering maize plants to adult C. carnea In the maize field, female C. carnea were most abundant at the peak of flowering. Similar observations were reported by Sheldon and MacLeod (1971). Interestingly, abundance declined toward the end of the flowering period even though large amounts of deposited pollen were still present on maize leaves. This suggests that the insects prefer fresh pollen as a food source. Throughout the maize flowering period, only a few male C. carnea were caught, confirming previous observations that males do not require pollen as food (Li et al., 2008). Female C. lucasina, which are known to cooccur with C. carnea in Central Europe (Duelli, 2001; Villenave et al., 2006), showed a similar abundance pattern as female C. carnea (Supplementary data 1). Aphids may also attract C. carnea adults to the field because they are consumed by the larvae and produce honeydew that is consumed by the adults (Hagen et al., 1970; Principi and Canard, 1984; Hogervorst et al., 2007). In the present study, however, aphid
densities were consistently low during the maize flowering period, when most lacewings were caught. 4.2. Fitness of adult C. carnea feeding on maize pollen Adult lacewings are unable to produce eggs without feeding on a protein-rich food source, because only a limited amount of metabolites are transferred from the larvae to the adult stage (Hagen, 1950; Hagen and Tassan, 1970; Principi and Canard, 1984; Li et al., 2008). Consequently, it is not surprising that lacewings provided with sucrose solution alone survived but did not produce fertile eggs. Maize pollen can be used by C. carnea as a protein source to provide amino acids essential for reproduction. Together with sucrose solution, pollen makes an excellent diet, comparable to the artificial diet that has been considered as highly nutritious (Hagen and Tassan, 1970; Jones et al., 1977; Li et al., 2008; Li and Romeis, 2009). Our results confirm earlier reports of an increase in lacewing reproduction when pollen was supplemented with a carbohydrate-rich food source (Elbadry and Fleschner, 1965; Sundby, 1967; Sheldon and MacLeod, 1971). Interestingly, maize pollen alone is not sufficient for C. carnea to survive and to reach their full reproductive potential. Even though maize pollen contains a substantial quantity of starch (Todd and Bretherick, 1942), C. carnea adults are apparently unable to efficiently use this carbohydrate source (Sheldon and MacLeod, 1971). 4.3. Consumption of maize pollen by female C. carnea Almost all female lacewings collected in the field during maize anthesis contained maize pollen. In addition, some insects contained small numbers of pollen grains from grasses and sunflowers and also contained fungal spores. At the peak of flowering, field-collected lacewings contained an average of approximately 5000 pollen grains, which was comparable to the amount in females fed with ad libitum maize pollen and sucrose solution in the laboratory. This number is most probably an underestimation, because we sampled the field lacewings in the afternoon, and our laboratory feeding bioassay showed that C. carnea contained more pollen in the morning than at noon or in the evening. This confirms the earlier observation that lacewings feed mainly late in the evening or early in the morning (Jones et al., 1977). Regardless, the results demonstrate that female C. carnea readily feed on maize pollen during maize anthesis in the field. The time required for female lacewings to replace their gut content was estimated with an additional laboratory study (Supplementary data 2). Females were fed maize pollen for 2 days to allow them to fill their gut. Then, they were transferred to artificial diet, and fresh feces were collected and microscopically examined every 30 min until no pollen grains were found. On average, females replaced their gut contents in 10.6 h during the day and in 12.3 h during the night. We thus expect that female C. carnea replenish their gut content approximately twice each day in a flowering maize field. Given this rate of gut passage and the
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
number of pollen grains found in the gut of field-collected insects, we estimate that on average a female lacewing can consume up to 10,000 maize pollen grains per day at the peak of flowering (5000 grains two gut passages). 4.4. Digestion of maize pollen by female C. carnea Insects have developed different ways to digest and use pollen (Roulston and Cane, 2000b; Human and Nicolson, 2003): they might (i) crack open the pollen wall mechanically; (ii) pierce the pollen wall with sharp mouthparts; (iii) dissolve the pollen wall with enzymes; (iv) induce germination or pseudo-germination; (v) burst the pollen wall through osmotic shock; and (vi) penetrate the pollen wall with digestive enzymes. Microscopic gut analyses of laboratory-fed and field-collected adult C. carnea showed that most maize pollen grains were not visibly cracked, disrupted, or germinated. This shows that lacewings, like most insects (Roulston and Cane, 2000b), are unable to break down the pollen exine. While pollen grains in the lacewing crop and diverticulum appeared undigested, almost all grains were at least partly digested in the hindgut. Apparently, most digestive activity occurs in the midgut of the lacewings. Pollen content leaking through the pores was observed with the microscope at 200 magnification. Therefore, chemical mechanisms (e.g., digestive enzymes) and diffusion processes are likely to play the major role in the digestion of pollen by lacewings.
163
In summary, flowering maize fields appear to attract females of C. carnea. These females readily consume maize pollen and are able to digest a large portion of the pollen content. In the case of transgenic varieties that express the novel insecticidal protein in the pollen, adult lacewings will be exposed to the toxin because a portion of the toxin diffuses from the grains during the gut passage. Consequently, it might be warranted to assess the risk to C. carnea resulting from such toxin exposure. Acknowledgments We thank Mario Waldburger (Agroscope Reckenholz-Ta¨nikon Research Station ART, Zurich) for providing lacewing eggs and Monsanto for providing transgenic maize seeds. We are grateful to Peter Duelli for comments on an earlier draft of this manuscript. This research was funded by the National Center of Competence in Research (NCCR) Plant Survival, a research program of the Swiss National Science Foundation.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinsphys.2009.09.011. References
4.5. Exposure of adult C. carnea to Bt protein by ingestion of transgenic maize pollen Given that maize pollen grains are not mechanically disrupted and are not completely digested during gut passage, we considered the possibility that the Cry proteins in the pollen might remain in the grains. Consequently, they would not get in contact with the insect’s gut tissue and could not bind to the gut membrane, which is a prerequisite for the toxicity of Cry proteins (De Maagd et al., 2001). Based on the number of pollen grains consumed per day by a female C. carnea in a flowering maize field (about 10,000) and the measured toxin concentration in Bt maize pollen (Table 2), a single female would ingest 22.6 ng Cry1Ab and 15.6 ng Cry3Bb1 per day in Compa CB1 and DKc5143Bt maize fields, respectively. The present study revealed that 39.5% of Cry1Ab and 21.2% of Cry3Bb1 toxin was not digested. As the toxin probably remained within the grains when Bt maize pollen passed through the lacewing gut, the absolute exposure of the insects is likely to be reduced. The higher digestion rate for Cry3Bb1 than Cry1Ab might result from differences in diffusion rates through the undamaged pollen wall or the pore. If one protein diffuses more rapidly than the other, it would experience greater exposure to digestive enzymes. Evidence for this comes from the fact that pollen from the maize variety expressing the cry3Bb1 gene loses more weight during the gut passage than pollen from the cry1Ab-expressing variety. Furthermore, a supplementary study (Supplementary data 3) in which Bt maize pollen was soaked in water for 16 h revealed that 37.9% of the Cry3Bb1 from DKc5143Bt pollen but only 20.2% of the Cry1Ab from Compa CB1 pollen entered the water. Differences in rates of digestion could also result from differences in molecule stability. This is supported by the finding that Bt protein concentrations in feces for DKc5143Bt were almost half of those in fresh pollen, while they remained almost constant for Compa CB1. Similarly, Cry3Bb1 was degraded faster than Cry1Ab when leaf material from the same Bt maize varieties was incorporated in soil (Zurbru¨gg et al., 2010), and a higher digestion rate for Cry3Bb1 than for Cry1Ab was reported when two slugs, Arion lusitanicus Mabille (Mollusca: Arionidae) and Deroceras reticulatum (Mu¨ller) (Mollusca: Agriolimacidae), were fed with Bt maize leaves (Zurbru¨gg and Nentwig, 2009).
Corey, D., Kambhampati, S., Wilde, G.E., 1998. Electrophoretic analysis of Orius insidiosus (Hemiptera: Anthocoridae) feeding habits in field corn. Journal of Kansas Entomological Society 71, 11–17. De Maagd, R.A., Bravo, A., Crickmore, N., 2001. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends in Genetics 17, 193–199. Duelli, P., 2001. Lacewings in field crops. In: McEwen, P., New, T.R., Whittington, A.E. (Eds.), Lacewings in the Crop Environment. Cambridge University Press, Cambridge, pp. 158–171. Elbadry, E.A., Fleschner, C.A., 1965. The feeding habits of adults of Chrysopa californica Coq. (Neuroptera, Chrysopidae). Bulletin de la Societe entomologique d’Egypte 49, 359–366. Garcia-Alonso, M., Jacobs, E., Raybould, A., Nickson, T.E., Sowig, P., Willekens, H., van der Kouwe, P., Layton, R., Amijee, F., Fuentes, A.M., Tencalla, F., 2006. A tiered system for assessing the risk of genetically modified plants to non-target organisms. Environmental Biosafety Research 5, 57–65. Goss, J.A., 1968. Development, physiology, and biochemistry of corn and wheat pollen. The Botanical Review 34, 333–358. Hagen, K.S., 1950. Fecundity of Chrysopa californica as affected by synthetic foods. Journal of Economic Entomology 43, 101–104. Hagen, K.S., Tassan, R.L., 1970. The influence of food wheast1 and related Saccharomyces fragilis yeast products on the fecundity of Chrysopa carnea (Neuroptera: Chrysopidae). The Canadian Entomologist 120, 806–811. Hagen, K.S., Tassan, R.L., Sawall, E.F., 1970. Some ecophysiological relationships between certain Chrysopa, honeydew and yeast. Bolletino del Laboratorio di Entomologia Agraria ‘Filippo Silvestri’ 28, 113–134. Hellmich, R.L., Albajes, R., Bergvinson, D., Prasifka, J.R., Wang, Z.Y., Weiss, M.J., 2008. The present and future role of insect-resistant GM crops in maize IPM. In: Romeis, J., Shelton, A.M., Kennedy, G.G. (Eds.), Integration of Insect-resistant Genetically Modified Crops within IPM Programs. Springer Science and Business Media B. V., pp. 119–158. Henry, C.S., Brooks, S.J., Duelli, P., Johnson, J.B., 2002. Discovering the true Chrysoperla carnea (Insecta: Neuroptera: Chrysopidae) using song analysis, morphology, and ecology. Annals of the Entomological Society of America 95, 172–191. Hogervorst, P.A.M., Wa¨ckers, F.L., Romeis, J., 2007. Detecting nutritional state and food source use in field-collected insects that synthesize honeydew oligosaccharides. Functional Ecology 21, 936–946. Human, H., Nicolson, S.W., 2003. Digestion of maize and sunflower pollen by the spotted maize beetle Astylus atromaculatus (Melyridae): is there a role for osmotic shock? Journal of Insect Physiology 49, 633–643. Jones, S.L., Lingren, P.D., Bee, M.J., 1977. Diel periodicity of feeding, mating, and oviposition of adult Chrysopa carnea. Annals of the Entomological Society of America 70, 43–47. Koziel, M.G., Beland, G.L., Bowman, C., Carozzi, N.B., Crenshaw, R., Crossland, L., Dawson, J., Dasai, N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D., McPherson, K., Keghji, M.R., Merlin, E., Rhodes, R., Warren, G.W., Wright, M., Evola, S.V., 1993. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11, 194–200. Li, Y., Romeis, J., 2009. Impact of snowdrop lectin (Galanthus nivalis agglutinin; GNA) on adults of the green lacewing, Chrysoperla carnea. Journal of Insect Physiology 55, 135–142.
164
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
Li, Y., Meissle, M., Romeis, J., 2008. Consumption of Bt maize pollen expressing Cry1Ab or Cry3Bb1 does not harm adult green lacewings, Chrysoperla carnea (Neuroptera: Chrysopidae). PLoS ONE 3 (8), e2909 doi:10.1371/journal. pone.0002909. Lundgren, J.G., Wiedenmann, R.N., 2004. Nutritional suitability of corn pollen for the predator Coleomegilla maculata (Coleoptera: Coccinellidae). Journal of Insect Physiology 50, 567–575. Meissle, M., Romeis, J., 2009a. The web-building spider Theridion impressum (Araneae: Theridiidae) is not adversely affected by Bt maize resistant to corn rootworms. Plant Biotechnology Journal 7, 645–656. Meissle, M., Romeis, J., 2009b. Insecticidal activity of Cry3Bb1 expressed in Bt maize on larvae of the Colorado potato beetle, Leptinotarsa decemlineata. Entomologia Experimentalis et Applicata 131, 308–319. Monsanto, 2004. Petition for the determination of nonregulated status for Mon 88017 corn. Monsanto Petition # 04-CR-108U; OECD Unique Identifier: MON88017-3. http://www.aphis.usda.gov/brs/aphisdocs/04_12501p.pdf. New, T.R., 1975. The biology of Chrysopidae and Hemerobiidae (Neuroptera), with reference to their usage as biocontrol agents: a review. Transactions of the Royal Entomological Society of London 127, 115–140. Obrist, L.B., Dutton, A., Albajes, R., Bigler, F., 2006. Exposure of arthropod predators to Cry1Ab toxin in Bt maize fields. Ecological Entomology 31, 143–154. Pilcher, C.D., Obrycki, J.J., Rice, M.E., Lewis, L.C., 1997. Preimaginal development, survival, and field abundance of insect predators on transgenic Bacillus thuringiensis corn. Environmental Entomology 26, 446–454. Principi, M.M., Canard, M., 1984. Feeding habits. In: Canard, M., Se´me´ria, Y., New, T.R. (Eds.), Biology of Chrysopidae. Dr. W. Junk Publishers, The Hague, pp. 76–92. Romeis, J., Dutton, A., Bigler, F., 2004. Bacillus thuringiensis toxin (Cry1Ab) has no direct effect on larvae of the green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae). Journal of Insect Physiology 50, 175–183. Romeis, J., Meissle, M., Bigler, F., 2006. Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology 24, 63–71. Romeis, J., Van Driesche, R.G., Barratt, B.I.P., Bigler, F., 2008a. Insect-resistant transgenic crops and biological control. In: Romeis, J., Shelton, A.M., Kennedy, G.G. (Eds.), Integration of Insect-resistant Genetically Modified Crops within IPM Programs. Springer Science and Business Media B. V., pp. 87–117. Romeis, J., Bartsch, D., Bigler, F., Candolfi, M.P., Gielkens, M.M.C., Hartley, S.E., Hellmich, R.L., Huesing, J.E., Jepson, P.C., Layton, R., Quemada, H., Raybould, A., Rose, R.I., Schiemann, J., Sears, M.K., Shelton, A.M., Sweet, J., Vaituzis, Z., Wolt, J.D., 2008b. Assessment of risk of insect-resistant transgenic crops to nontarget arthropods. Nature Biotechnology 26, 203–208. Romeis, J., Meissle, M., Raybould, A., Hellmich, R.L., 2009. Impact of insectresistant transgenic crops on above-ground non-target arthropods. In: Ferry,
N., Gatehouse, A.M.R. (Eds.), Environmental Impact of Genetically Modified Crops. CABI Publishing, Wallingford, pp. 165–198. Roulston, T.H., Buchmann, S.L., 2000a. A phylogenetic reconsideration of the starchpollination correlation. Evolutionary Ecology Research 2, 627–643. Roulston, T.H., Cane, J.H., 2000b. Pollen nutritional content and digestibility for animals. Plant Systematics and Evolution 222, 187–209. Sheldon, J.K., MacLeod, E.G., 1971. Studies on the biology of the Chrysopidae. II. The feeding behavior of the adult of Chrysopa carnea (Neuroptera). Psyche 78, 107–121. Stanley, R.G., Linskens, H.F., 1974. Pollen: Biology, Biochemistry, Management. Springer–Verlag, New York. Sundby, R.A., 1967. Influence of food on the fecundity of Chrysopa carnea Stephens (Neuroptera, Chrysopidae). Entomophaga 12, 475–479. Todd, F.E., Bretherick, O., 1942. The composition of pollens. Journal of Economic Entomology 35, 312–317. Treu, R., Emberlin, J., 2000. Pollen dispersal in the crops maize (Zea mays) oil seed rape (Brassica napus spp. oleifera), potatoes (Solanum tuberosum), sugar beet (Beta valgaris ssp. Valgaris) and wheat (Triticum aestivum) In: Report for the Soil Association, National Pollen Research Unit, University College Worcester, UK. Vaughn, T., Cavato, T., Brar, G., Coombe, T., DeGooyer, T., Ford, S., Groth, M., Howe, A., Johnson, S., Kolacz, K., Pilcher, C., Purcell, J., Romano, C., English, L., Pershing, J., 2005. A method of controlling corn rootworm feeding using a Bacillus thuringiensis protein expressed in transgenic maize. Crop Science 45, 931–938. Villenave, J., Thierry, D., Mamun, A.A., Lode´, T., Rat-Morris, E., 2005. The pollens consumed by common green lacewings Chrysoperla spp. (Neuroptera: Chrysopidae) in cabbage crop environment in western France. European Journal of Entomology 102, 547–552. Villenave, J., Deutsch, B., Lode´, T., Rat-Morris, E., 2006. Pollen preference of the Chrysoperla species (Neuroptera: Chrysopidae) occurring in the crop environment in western France. European Journal of Entomology 103, 771–777. Wolfenbarger, L.L., Naranjo, S.E., Lundgren, J.G., Bitzer, R.J., Watrud, L.S., 2008. Bt crops effects on functional guilds of non-target arthropods: a meta-analysis. PLoS ONE 3 (5), e2118, doi:10.1371/journal.pone.0002118. Woolfolk, S.W., Inglis, G.D., 2003. Microorganisms associated with field-collected Chrysoperla rufilabris (Neuroptera: Chrysopidae) adults with emphasis on yeast symbionts. Biological Control 29, 155–168. Zurbru¨gg, C., Nentwig, W., 2009. Ingestion and excretion of two transgenic Bt corn varieties by slugs. Transgenic Research 18, 215–225. Zurbru¨gg, C., Ho¨nemann, L., Meissle, M., Romeis, J., Nentwig, W., 2010. Decomposition dynamics and structural plant components of genetically modified Bt maize leaves do not differ from leaves of conventional hybrids. Transgenic Research 19, doi:10.1007/s11248-009-9304-x.
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties Yunhe Li, Michael Meissle, Jo¨rg Romeis * Agroscope Reckenholz-Ta¨nikon Research Station ART, Reckenholzstr. 191, 8046 Zurich, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 June 2009 Received in revised form 28 August 2009 Accepted 18 September 2009
We investigated the use of maize pollen as food by adult Chrysoperla carnea under laboratory and field conditions. Exposure of the insects to insecticidal Cry proteins from Bacillus thuringiensis (Bt) contained in pollen of transgenic maize was also assessed. Female C. carnea were most abundant in a maize field when the majority of plants were flowering and fresh pollen was abundant. Field-collected females contained an average of approximately 5000 maize pollen grains in their gut at the peak of pollen shedding. Comparable numbers were found in females fed ad libitum maize pollen in the laboratory. Maize pollen is readily used by C. carnea adults. When provided with a carbohydrate source, it allowed the insects to reach their full reproductive potential. Maize pollen was digested mainly in the insect’s mid- and hindgut. When Bt maize pollen passed though the gut of C. carnea, 61% of Cry1Ab (event Bt176) and 79% of Cry3Bb1 (event MON 88017) was digested. The results demonstrate that maize pollen is a suitable food source for C. carnea. Even though the pollen grains are not fully digested, the insects are exposed to transgenic insecticidal proteins that are contained in the pollen. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Bt maize Cry1Ab Cry3Bb1 Pollen digestion Environmental risk assessment
1. Introduction The common green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), is an important natural predator of insect herbivores in many different crop and non-crop habitats (New, 1975). The predatory larvae feed preferentially on aphids but can consume a wide range of soft-bodied arthropods (Principi and Canard, 1984). Adults are not predacious and live on pollen, nectar, and honeydew (Principi and Canard, 1984; Sheldon and MacLeod, 1971; Villenave et al., 2005, 2006; Li et al., 2008). The current study concerns the interactions between adult C. carnea and maize pollen. Maize produces large amounts of pollen, i.e., up to 50 million pollen grains per maize tassel over a period of 5–8 days (Goss, 1968; Treu and Emberlin, 2000). Many insects, including a number of natural enemies, consume maize pollen (Pilcher et al., 1997; Corey et al., 1998; Lundgren and Wiedenmann, 2004) because it contains organic and inorganic nutrients, such as sugars, starch, amino acids and proteins, lipids, as well as vitamins and minerals (Goss, 1968; Stanley and Linskens, 1974; Roulston and Buchmann, 2000a). While laboratory studies have shown that maize pollen can also serve as a food source for adult C. carnea (Sheldon and MacLeod, 1971; Li et al.,
* Corresponding author. Tel.: +41 44 377 72 99; fax: +41 44 377 72 01. E-mail address: [email protected] (J. Romeis). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.09.011
2008), it is still unclear to what extent adult C. carnea ingest and use maize pollen as a food source in the field. Insect-resistant genetically engineered maize varieties expressing Cry proteins from the bacterium Bacillus thuringiensis (Bt) are grown on increasing areas worldwide (Hellmich et al., 2008). Most varieties are either protected from lepidopteran pests (such as stemborers) by expressing Cry1 proteins or from corn rootworms (Diabrotica spp.; Coleoptera: Chrysomelidae) by expressing Cry3 proteins (Hellmich et al., 2008). The commercialization of Bttransgenic maize varieties has stimulated numerous studies on the potential effects on non-target organisms including those contributing to the natural regulation of herbivores (Romeis et al., 2006, 2008a; Wolfenbarger et al., 2008). Pollen-feeding species may also be exposed to Bt proteins, because Cry3Bb1-expressing varieties with the transformation events MON863 and MON88017 and Cry1Ab-expressing varieties with event Bt176 (which have been removed from the market) show pollen expression levels almost in the same order of magnitude as the green tissue (Obrist et al., 2006; Romeis et al., 2009; Meissle and Romeis, 2009a). For assessing the risks of Bt maize on natural enemies, both the level of exposure to the toxin and the hazard of being exposed must be considered (Garcia-Alonso et al., 2006; Romeis et al., 2008b). Previous hazard studies have shown that C. carnea females are not adversely affected when consuming purified Cry1Ab or Cry3Bb1 in an artificial diet or Bt maize pollen that contained the toxins in a biologically active form (Li et al., 2008; Meissle and Romeis,
158
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
2009b). This paper, however, focuses on exposure levels of adult green lacewings to Cry protein in Bt maize. A series of experiments was conducted in the laboratory and in the field to answer the following questions: (i) how abundant are adult C. carnea in maize fields during anthesis? (ii) to what extent can adult C. carnea use maize pollen as a food source? (iii) how many pollen grains are consumed and to what extent are they digested? (iv) how much Cry protein is digested by adult C. carnea when feeding on Bt maize pollen? 2. Materials and methods 2.1. Field survey Adults of C. carnea were collected in a maize field (0.5 ha) in Zurich, Switzerland. Conventional maize (DK287) was sown on 15 May 2004 and managed according to common practice. No insecticide was used. Adult C. carnea were collected by sweep-net sampling in the field center (>2 m from the field borders) and along the edge (<1 m from the field borders). Weekly samples were taken in 2004 from 20 July to 18 August, covering a period that started before anthesis, that included flowering, and that ended when pollen was no longer present. The time spent in sampling was recorded and used to calculate the number of lacewings collected per hour. Within 3 h, collected insects were individually frozen at 80 8C in 1.5 ml microreaction tubes. A binocular microscope was used to divide the lacewings into C. carnea and C. lucasina (Lacroix) (Neuroptera: Chrysopidae), following Henry et al. (2002); these two species often co-occur in Central Europe (Duelli, 2001; Villenave et al., 2006). The specimens were then divided into males and females. All studies focused on females because males were found to consume only small amounts of maize pollen in the laboratory and the field (Villenave et al., 2005; Li et al., 2008). To correlate the number of lacewings with the presence of maize pollen, the availability of pollen in the field was monitored. At each sampling date, 20 maize plants were randomly selected in the center of the maize field, and the percentage of flowering plants was recorded. In addition, the amount of pollen deposited on leaves was recorded as zero, low, or high. Aphids on these plants were also counted. Aphids may influence the abundance of adult lacewings because they serve as food for the larvae and provide honeydew to the adults (Principi and Canard, 1984). 2.2. Insect and plant material for laboratory experiments Eggs of C. carnea were collected from a permanent laboratory colony (for details on the rearing, see Romeis et al., 2004) and kept in a climatic chamber at 22 1 8C, 75 5% RH, and a 16:8 h photoperiod. After hatching, the larvae were fed with eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) provided by Biotop (Valbonne, France). After the larvae reached the second instar, they were kept individually in plastic tubes (1.5 cm diameter, 5.5 cm high) and supplied ad libitum with E. kuehniella eggs until pupation. Newly emerged (<24 h) adults were used for the experiments. Two transgenic maize varieties, Compa CB1 (Event Bt176, Syngenta, Stein am Rhein, Switzerland) and DKc5143Bt (Event MON 88017, Monsanto, St. Louis, USA) and their corresponding nontransformed near isolines Dracma1 and DKc5143, respectively, were used for the experiments. Compa CB1 plants express a modified cry1Ab gene from Bacillus thuringiensis ssp. kurstaki targeting Lepidoptera. Expression is driven by the constitutive PEPC promoter as well as a pollen-specific promoter (Koziel et al., 1993). DKc5143Bt expresses a modified cry3Bb1 gene from Bacillus thuringiensis ssp. kumamotoensis targeting corn rootworms (Vaughn et al., 2005). Expression is driven by a constitutive enhanced 35S
promoter (P-e35S) (Monsanto, 2004). Plants were grown in the glasshouse, and pollen was collected as described by Li et al. (2008). 2.3. Fitness of adult C. carnea feeding on maize pollen Newly emerged adults of C. carnea were sexed, and single pairs were confined in transparent plastic cylinders (6.0 cm diameter, 8.5 cm high). Lids with a 4 cm opening were used to allow ventilation. Between cylinder and lid, a layer of cotton gauze prevented insect escape and served as an oviposition substrate. Water was provided by a dental wick, which was positioned through a 1 cm hole at the bottom of each container. The cylinders were placed over a water reservoir so that the cotton wicks were submerged and a continuous water supply was ensured. Adults were provided with one of the following diets: (1) only water as a non-food control; (2) maize pollen (Dracma1); (3) 1 M sucrose solution (Merck, Darmstadt, Germany); (4) Maize pollen and 1 M sucrose solution; and (5) artificial diet consisting of sucrose, brewer’s yeast, and water (in proportions 7:4:4). The artificial diet served as a reference control because it is considered highly nutritious for adult C. carnea (Hagen and Tassan, 1970; Jones et al., 1977). Maize pollen and sucrose solution were offered separately in small plastic dishes (2.5 cm diameter, 0.7 cm high), which were placed on the bottom of the test containers. Artificial diet was provided on one end of a green plastic label (9.0 cm 1.6 cm). All diets were provided ad libitum and replaced every 3 days. Each diet treatment was replicated with 20 pairs of C. carnea. The experiment was conducted in a climatic chamber at 22 1 8C, 75 5% RH, and a 16:8 h photoperiod, and lasted for 28 days. Longevity, pre-oviposition period (mean number of days from emergence to the first oviposition), daily and total fecundity, and fertility (egg hatching rate) were recorded. Eggs were removed daily from the containers and counted. For the assessment of fertility, collected eggs were placed in a separate container every 3 days together with the oviposition substrate (gauze) and with ad libitum E. kuehniella eggs as food to minimize cannibalism of hatching lacewing larvae. Seven days later, hatched larvae were counted. After termination of the experiment, surviving adult C. carnea were frozen. Subsequently, the dry weights were determined on an electronic microbalance MX5 (Mettler Toledo, Greifensee, Switzerland) after drying at 50 8C for 4 days. For data analysis, three to four pairs had to be excluded from each of the pollen only, pollen and sucrose solution, and artificial diet treatments because the lacewings had escaped, were damaged, or were infertile (unmated). The water treatment was not statistically analyzed because all insects died within 7 days without laying eggs. For the sucrose solution treatment, oviposition parameters were not compared with the other treatments because females laid very few eggs. Pre-oviposition period was compared using Mann–Whitney U-tests with Bonferroni correction for three pair-wise comparisons (adjusted a = 0.017). For daily fecundity, repeated-measures ANOVA was carried out and the means were separated using the Tukey HSD test. Data on total fecundity, fertility, and dry weight (females and males separately) were analyzed using a one-way ANOVA followed byffi the Tukey HSD pffiffiffiffiffiffi test. Fertility data were transformed by arcsin ðxÞ and dry weight of males by log(x) before analysis. The survival rates of adult lacewings fed with the different diets (excluding the water only control) were compared using Chi-square (x2) tests with Bonferroni correction for six pair-wise comparisons (adjusted a = 0.0083). 2.4. Consumption of maize pollen To measure how much pollen is consumed by lacewings when maize pollen is provided ad libitum, we fed newly emerged
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
C. carnea females with artificial diet for 1 week. Then the insects were separated into two groups: one group was fed only maize pollen (DKc5143) while the other group was fed with maize pollen and 1 M sucrose solution (in separate containers). Water provision and experimental conditions were as described above. Lacewings were allowed to feed for 2 days, which was sufficient time for them to replace their gut content (unpublished observation). On the third day, 15 individuals from each group were frozen at 20 8C at 9:00, 12:00 and 18:00 h; these sampling times were used because we suspected that the quantity of pollen grains in the gut could vary with time of day. To quantify maize pollen grains in the gut of C. carnea, we thawed and excised the females, and the whole alimentary canal was pulled out. Subsequently, the gut was transferred to a 1.5 ml microreaction tube containing 100 ml fuchsin acid solution. The red color stained the pollen grains and facilitated counting. After the gut was ruptured with a thin needle, the pollen suspension was mixed using a Vortex mixer. An aliquot (5 ml) of the suspension was transferred to a glass slide, and pollen grains (including full, partially digested, and empty ones) were counted with a microscope at 50 magnification. Counting was repeated for three subsamples for each insect gut, and the mean number of pollen grains was multiplied by 20 to estimate the total number contained in the insect. Six to thirteen females were analyzed at each sampling time for both the pollen only and the pollen and sucrose solution treatments. In addition to the pollen content of laboratory-fed insects, the pollen content in the guts of six to fourteen females collected in the center of the maize field was determined for each sampling date between 20 July and 10 August 2004.
159
containers without food. After 6 h, fresh fecal pellets were collected and stored at 20 8C for digestion analysis. To observe the digestion status of the pollen grains in different gut sections of C. carnea females, we excised the alimentary canal and divided it into crop, diverticulum, midgut, and hindgut following Woolfolk and Inglis (2003). The pollen grains obtained from each gut section were transferred to a drop of 0.5 M sucrose solution on a glass slide. One drop of lactophenol cotton blue was added to stain the cytoplasm dark blue, while the cell walls were left unstained. Basic fuchsin served as a counterstain to highlight the exine in pink, which particularly helped with the counting of empty pollen shells (Human and Nicolson, 2003). After staining, the microscope slide was sealed with a cover slip and examined with a microscope at 200 magnification. For each slide, approximately 100 randomly selected pollen grains were classified into one of four groups (Fig. 1): (A) undigested grains with 90– 100% of original content; similar in shape to fresh pollen; (B) slightly digested grains with 50–90% of original content; irregular shape; (C) heavily digested grains with 10–50% of original content; shrunken; (D) fully digested grains with 0–10% of original content; heavily shrunken or burst open. Fresh pollen grains and pollen grains contained in lacewing feces were classified in the same way. Fresh pollen, gut sections, and feces were each replicated six times. In addition, females collected in the edge of the maize field were assessed for the degree of pollen digestion in the foregut (crop and diverticulum) and hindgut. Six to ten females were investigated for each of three sampling dates in 2004 (27 July, 3 August, and 10 pffiffiffiffiffiffi ffi August). After arcsin ðxÞ transformation, one-way ANOVA was conducted to compare the proportions of the undigested pollen grains in the foregut among the three sampling dates.
2.5. Digestion of maize pollen
2.6. Fate of Cry protein contained in Bt maize pollen after lacewing gut passage
For the assessment of pollen digestion, newly emerged females were fed with artificial diet for 1 week in the laboratory. Subsequently, they were provided with maize pollen (DKc5143) and sucrose solution for 2 days as described above. Half of the group was sampled and stored at 20 8C for analysis of pollen digestion. The remaining females were transferred into new
Newly emerged females of C. carnea were fed with artificial diet. After 1 week, the insects were provided with water only for at least 24 h to empty their gut. Then the insects were placed in 10 plastic containers (10 insects per container) and provided with water and Bt maize pollen of Compa CB1 or DKc5143Bt (five containers per maize treatment). After 2 days, the lacewings were transferred to
Fig. 1. Micrographs showing the different degrees of digestion of maize pollen grains in the gut of adult Chrysoperla carnea. The degree of digestion was classified as follows: (A) undigested (retaining 90–100% of content; similar in shape to fresh pollen); (B) slightly digested (retaining 50–90% of content; similar in shape to fresh pollen); (C) heavily digested (retaining 10–50% of content; shrunken); (D) fully digested (retaining 0–10% of content; shrunken or burst open).
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
160
new containers with the same food. Fresh fecal pellets were collected every 2 h. The feces were pooled for each container, and five samples were collected for each maize variety. Five subsamples of fresh pollen from each of the two Bt maize varieties were also collected. All samples were stored at 80 8C. After lyophilization, the concentrations of Cry1Ab or Cry3Bb1 were measured using Double-Antibody Sandwich Enzyme-Linked ImmunoSorbent Assay (DAS-ELISA) purchased from Agdia (Elkhard, Indiana, USA) and as described by Li et al. (2008). The measured OD values were calibrated using Cry1Ab or Cry3Bb1 standards made from purified toxins, which were provided by M. Carey (Dept. Biochemistry, Case Western Reserve University, Cleveland, OH, USA) and by Monsanto Company, respectively (Li et al., 2008). The total digestion rate of Bt toxin in pollen cannot be determined from Cry protein concentrations (Cry protein per dry weight) because the digestion process reduces both the amount of Cry protein and the weight of the pollen grains. Therefore, the mean Bt toxin content of a single pollen grain derived from feces or directly from the plant was assessed. Lyophilized fresh pollen or feces (1.0 mg) were mixed with 300 ml fuchsin acid solution. The pollen grains were counted in each of three 5 ml aliquots of the suspension with a microscope at 50 magnification. The mean number of pollen grains in the aliquots was multiplied by 60 to obtain the number in the whole sample. Finally, the weight of each sample (1.0 mg) was divided by the number of grains to obtain the mean dry weight of an individual pollen grain. For each of the two maize varieties, this procedure was repeated seven to ten times. Based on the individual dry weight of pollen grains and the Bt protein concentrations in fresh maize pollen and feces, the Bt content in single pollen grains was calculated. 3. Results 3.1. Field survey On the first sampling date (20 July), when pollen was not yet available, 1.5 C. carnea females were collected per hour of sweepnet sampling. In the early stage of pollen shedding (27 July), when about half of the maize plants were flowering and only small amounts of pollen were deposited on leaves, 5.4 females were collected per hour. Most females (17.0 per hour) were collected on 3 August when 80% of maize plants were flowering and large amounts of pollen were on the leaves. On 10 August, only 5% of maize plants were still flowering, and 4.8 females were caught per hour. On the last sampling date on 18 August, pollen was no longer available in the field and no adult C. carnea was found (Supplementary data 1). During the sampling period, a large density of aphids (21 per plant) was observed only before the start of maize flowering (20
Fig. 2. Survival (A; n = 32–40) and daily fecundity (B; mean SE, n = 16–20) of Chrysoperla carnea adults fed one of five diets.
July). At all later dates, the aphid density was less than 1 per plant (Supplementary data 1). 3.2. Fitness of adult C. carnea feeding on maize pollen During the 4 weeks of the experiment, no mortality of adult C. carnea occurred in the artificial diet and the pollen and sucrose treatments (Fig. 2A). All adults died within 7 days in the non-food control treatment (water only) (Fig. 2A). At the end of the experiment, lacewing survival was significantly lower (x2-test; P = 0.006) in the pollen only treatment (80%) than in the artificial diet (100%) or pollen and sucrose (100%) treatments, but did not differ (x2-test; P > 0.05) from survival in the sucrose solution treatment (95%). Survival did not statistically differ (x2-test; P > 0.05) among the artificial diet, pollen and sucrose, and sucrose solution treatments. The females in the sucrose solution treatment did not produce fertile eggs (Fig. 2B, Table 1). The pre-oviposition period was similar for the artificial diet and pollen and sucrose treatments (Mann–Whitney U-test; U = 98, P = 0.11), while the pre-oviposition period was significantly longer for the pollen only treatment (artificial diet: U = 38, P < 0.001; pollen and sucrose: U = 18, P < 0.001, Table 1). Daily fecundity also differed significantly
Table 1 Life-table parameters of adult Chrysoperla carnea when fed for 4 weeks with artificial diet (sucrose: brewer’s yeast: water; 7:4:4), maize pollen, or 1 M sucrose solution either alone or in combination. Diet
Artificial diet Pollen and sucrose Pollen Sucrose
Number of pairs
17 17 16 20
Pre-oviposition period (days)a
Number of eggs laid per femaleb
Egg hatching rate (%)b,c
Adult dry weight (mg)
4.4 0.30 b 3.8 0.20 b 7.7 0.79 a –
576.3 54.97 a 640.9 49.14 a 170.5 26.07 b –
76.6 6.04 a 79.7 4.73 a 85.1 2.07 a –
9.2 0.22 9.0 0.31 6.7 0.46 6.7 0.46
Values are means SE in columns 3–5. Means in a column followed by the same letter are not significantly different (P > 0.05). a Kruskal–Wallis ANOVA, followed by pair-wise comparisons using Mann–Whitney U-test. b One-way ANOVA followed by Tukey HSD test. pffiffiffiffiffiffiffi c Percentages were transformed using arcsin ðxÞ before analysis. d Data were transformed using log(x).
Femaleb
Maleb,d a a b b
4.1 0.16 4.9 0.30 3.6 0.21 4.1 0.18
b a b ab
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
among the three treatments (repeated-measures ANOVA; F2,46 = 29.4, P < 0.0001) (Fig. 2B). While fecundity did not differ between pollen and sucrose and artificial diet treatments (Tukey HSD test; P = 0.58), females provided with pollen only laid fewer eggs per day than females provided with pollen and sucrose or artificial diet (P = 0.001). Similar results were obtained for total fecundity, for which there was a significant treatment effect (one-way ANOVA; F2,47 = 30.4, P < 0.0001), no difference between pollen and sucrose and artificial diet (Tukey HSD test; P = 0.58), and a significant reduction in the pollen only treatment (P < 0.001) (Table 1). The egg hatching rates (fertility) were similar among the three treatments (one-way ANOVA, F2,46 = 0.72, P = 0.49). At the end of the experiment, the dry weight of females (oneway ANOVA; F3,72 = 18.0, P < 0.0001) and males (F3,72 = 7.0, P = 0.0003) differed among the food treatments. Dry weights of females did not differ between artificial diet and pollen and sucrose treatments (Tukey HSD test, P = 0.99) or between pollen only and sucrose solution treatments (P = 0.99) (Table 1). However, females fed with pollen only or sucrose solution weighed significantly less than those fed on artificial diet or pollen and sucrose (P < 0.001). Males feeding on pollen and sucrose weighed significantly more than those fed on artificial diet (P = 0.035) or pollen only (P = 0.0003); they also tended to weigh more than those fed sucrose solution (P = 0.058). The weight of males did not differ
161
among the artificial diet, pollen only, and sucrose solution treatments (P > 0.2) (Table 1). 3.3. Consumption of maize pollen In the laboratory, all females provided with maize pollen for 2 days contained pollen grains in their guts (Fig. 3A). Females feeding on maize pollen and 1 M sucrose solution contained 10–30% more pollen grains than those that had access to pollen only. In both treatments, females collected in the morning contained more pollen than those collected in the evening (Fig. 3A). In the field, no pollen grains were found in the guts of females collected before maize anthesis on 20 July. Females collected in the middle of the pollen-shedding period (3 August) contained the most pollen grains, and numbers were similar to those in females with ad libitum access to pollen in the laboratory (Fig. 3A and B). Compared to females collected in the middle of pollen shedding, females collected at the beginning (27 July) or the end (10 August) of the pollen-shedding period contained less than half the number of grains (Fig. 3B). 3.4. Digestion of maize pollen Pollen collected from glasshouse grown plants and offered to females in the laboratory contained 98.5% full grains (Fig. 4A). In
Fig. 3. Number of maize pollen grains (mean SE) in the gut of female Chrysoperla carnea. (A) Insects (n = 6–13) were collected in the morning, noon, and evening after having fed on maize pollen alone or maize pollen together with a 1 M sucrose solution for more than 2 days in the laboratory. (B) Insects (n = 6–14) were collected in a maize field at different times during the pollen-shedding period: Start = 27 July, 58% of plants flowering; Middle = 3 August, 80% flowering; End = 10 August, 5% flowering.
Fig. 4. Percentage of digested maize pollen grains in different gut sections and feces of female Chrysoperla carnea. Fresh pollen was used as a reference. (A) Females collected from the laboratory after being fed with maize pollen and 1 M sucrose solution; n = 6. (B) Females collected in a maize field during anthesis; n = 6–10.
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
162
Table 2 Digestion of maize pollen grains and their Cry protein content when passing through the digestive system of female Chrysoperla carnea. Insects were fed with maize pollen and water, and the feces were collected; n = 7–10. Bt maize variety (event)
Pollen source
Weight per pollen grain (ng)
Pollen digestion rate (%) (Rp = (a1 a2)/a1 100)
Bt concentration (mg/g dry weigh)
Bt content per grain (pg) (cx = axbx 103)
Digestion rate of Cry protein (%) (RCry = (c1 c2)/c1 100)
Compa CB1 (Bt176)
Fresh pollen Feces
202.1 6.67 (a1) 98.1 2.44* (a2)
51.5
11.2 0.22 (b1) 9.1 0.27 (b2)
2.3 0.05 (c1) 0.9 0.03* (c2)
60.5
DKc5143Bt (MON88017)
Fresh pollen Feces
227.7 12.55 (a1) 87.3 2.09* (a2)
61.7
6.9 0.13 (b1) 3.8 0.32 (b2)
1.6 0.03 (c1) 0.3 0.03* (c2)
78.8
Values are means SE in columns 3, 5, and 6. * Significant differences of feces treatments when contrasted against the corresponding fresh pollen treatments (P < 0.05).
the crop and diverticulum of female C. carnea, 94.1% and 95.5% pollen grains, respectively, were full (Fig. 4A). In the midgut, only 14.6% of the grains remained visibly undigested, and 50.6% and 33.4% of the pollen grains were slightly and heavily digested, respectively. The proportion of undigested pollen grains further decreased to 3.6% in the hindgut. The status of the grains in the feces was similar to that in the hindgut, and no undigested pollen grains were present in the hindgut or feces of females fed in the laboratory (Fig. 4A). In the field, the proportion of undigested pollen grains in the foregut did not differ among females collected at the start, middle, and end of maize anthesis (one-way ANOVA, F2,17 = 2.51, P = 0.11); the same was true for heavily digested pollen grains in the hindgut (F2,14 = 0.04, P = 0.96). Therefore, data from the three different sampling dates were combined. The field-collected females contained a high percentage of full pollen grains in the foregut but a high percentage of heavily digested pollen grains in the hindgut (Fig. 4B). Compared to the insects reared in the laboratory, field-collected females contained 13.8% less undigested pollen grains in the foregut, and 6.4% more fully digested grains in the hindgut (Fig. 4A and B). 3.5. Fate of Cry toxin in Bt maize pollen after C. carnea gut passage During the passage through the insect gut, the pollen grains from the two Bt maize varieties lost more than half of their initial weight, with greater digestion of DKc5143Bt pollen than Compa CB1 pollen (Table 2). In addition, the Cry protein concentration in the feces decreased more with DKc5143Bt pollen (by 44.6%) than with Compa CB1 pollen (by 18.7%). Altogether, more of the initial toxin content was digested by C. carnea per pollen grain from DKc5143Bt compared to Compa CB1 (Table 2). 4. Discussion 4.1. Attractiveness of flowering maize plants to adult C. carnea In the maize field, female C. carnea were most abundant at the peak of flowering. Similar observations were reported by Sheldon and MacLeod (1971). Interestingly, abundance declined toward the end of the flowering period even though large amounts of deposited pollen were still present on maize leaves. This suggests that the insects prefer fresh pollen as a food source. Throughout the maize flowering period, only a few male C. carnea were caught, confirming previous observations that males do not require pollen as food (Li et al., 2008). Female C. lucasina, which are known to cooccur with C. carnea in Central Europe (Duelli, 2001; Villenave et al., 2006), showed a similar abundance pattern as female C. carnea (Supplementary data 1). Aphids may also attract C. carnea adults to the field because they are consumed by the larvae and produce honeydew that is consumed by the adults (Hagen et al., 1970; Principi and Canard, 1984; Hogervorst et al., 2007). In the present study, however, aphid
densities were consistently low during the maize flowering period, when most lacewings were caught. 4.2. Fitness of adult C. carnea feeding on maize pollen Adult lacewings are unable to produce eggs without feeding on a protein-rich food source, because only a limited amount of metabolites are transferred from the larvae to the adult stage (Hagen, 1950; Hagen and Tassan, 1970; Principi and Canard, 1984; Li et al., 2008). Consequently, it is not surprising that lacewings provided with sucrose solution alone survived but did not produce fertile eggs. Maize pollen can be used by C. carnea as a protein source to provide amino acids essential for reproduction. Together with sucrose solution, pollen makes an excellent diet, comparable to the artificial diet that has been considered as highly nutritious (Hagen and Tassan, 1970; Jones et al., 1977; Li et al., 2008; Li and Romeis, 2009). Our results confirm earlier reports of an increase in lacewing reproduction when pollen was supplemented with a carbohydrate-rich food source (Elbadry and Fleschner, 1965; Sundby, 1967; Sheldon and MacLeod, 1971). Interestingly, maize pollen alone is not sufficient for C. carnea to survive and to reach their full reproductive potential. Even though maize pollen contains a substantial quantity of starch (Todd and Bretherick, 1942), C. carnea adults are apparently unable to efficiently use this carbohydrate source (Sheldon and MacLeod, 1971). 4.3. Consumption of maize pollen by female C. carnea Almost all female lacewings collected in the field during maize anthesis contained maize pollen. In addition, some insects contained small numbers of pollen grains from grasses and sunflowers and also contained fungal spores. At the peak of flowering, field-collected lacewings contained an average of approximately 5000 pollen grains, which was comparable to the amount in females fed with ad libitum maize pollen and sucrose solution in the laboratory. This number is most probably an underestimation, because we sampled the field lacewings in the afternoon, and our laboratory feeding bioassay showed that C. carnea contained more pollen in the morning than at noon or in the evening. This confirms the earlier observation that lacewings feed mainly late in the evening or early in the morning (Jones et al., 1977). Regardless, the results demonstrate that female C. carnea readily feed on maize pollen during maize anthesis in the field. The time required for female lacewings to replace their gut content was estimated with an additional laboratory study (Supplementary data 2). Females were fed maize pollen for 2 days to allow them to fill their gut. Then, they were transferred to artificial diet, and fresh feces were collected and microscopically examined every 30 min until no pollen grains were found. On average, females replaced their gut contents in 10.6 h during the day and in 12.3 h during the night. We thus expect that female C. carnea replenish their gut content approximately twice each day in a flowering maize field. Given this rate of gut passage and the
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
number of pollen grains found in the gut of field-collected insects, we estimate that on average a female lacewing can consume up to 10,000 maize pollen grains per day at the peak of flowering (5000 grains two gut passages). 4.4. Digestion of maize pollen by female C. carnea Insects have developed different ways to digest and use pollen (Roulston and Cane, 2000b; Human and Nicolson, 2003): they might (i) crack open the pollen wall mechanically; (ii) pierce the pollen wall with sharp mouthparts; (iii) dissolve the pollen wall with enzymes; (iv) induce germination or pseudo-germination; (v) burst the pollen wall through osmotic shock; and (vi) penetrate the pollen wall with digestive enzymes. Microscopic gut analyses of laboratory-fed and field-collected adult C. carnea showed that most maize pollen grains were not visibly cracked, disrupted, or germinated. This shows that lacewings, like most insects (Roulston and Cane, 2000b), are unable to break down the pollen exine. While pollen grains in the lacewing crop and diverticulum appeared undigested, almost all grains were at least partly digested in the hindgut. Apparently, most digestive activity occurs in the midgut of the lacewings. Pollen content leaking through the pores was observed with the microscope at 200 magnification. Therefore, chemical mechanisms (e.g., digestive enzymes) and diffusion processes are likely to play the major role in the digestion of pollen by lacewings.
163
In summary, flowering maize fields appear to attract females of C. carnea. These females readily consume maize pollen and are able to digest a large portion of the pollen content. In the case of transgenic varieties that express the novel insecticidal protein in the pollen, adult lacewings will be exposed to the toxin because a portion of the toxin diffuses from the grains during the gut passage. Consequently, it might be warranted to assess the risk to C. carnea resulting from such toxin exposure. Acknowledgments We thank Mario Waldburger (Agroscope Reckenholz-Ta¨nikon Research Station ART, Zurich) for providing lacewing eggs and Monsanto for providing transgenic maize seeds. We are grateful to Peter Duelli for comments on an earlier draft of this manuscript. This research was funded by the National Center of Competence in Research (NCCR) Plant Survival, a research program of the Swiss National Science Foundation.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinsphys.2009.09.011. References
4.5. Exposure of adult C. carnea to Bt protein by ingestion of transgenic maize pollen Given that maize pollen grains are not mechanically disrupted and are not completely digested during gut passage, we considered the possibility that the Cry proteins in the pollen might remain in the grains. Consequently, they would not get in contact with the insect’s gut tissue and could not bind to the gut membrane, which is a prerequisite for the toxicity of Cry proteins (De Maagd et al., 2001). Based on the number of pollen grains consumed per day by a female C. carnea in a flowering maize field (about 10,000) and the measured toxin concentration in Bt maize pollen (Table 2), a single female would ingest 22.6 ng Cry1Ab and 15.6 ng Cry3Bb1 per day in Compa CB1 and DKc5143Bt maize fields, respectively. The present study revealed that 39.5% of Cry1Ab and 21.2% of Cry3Bb1 toxin was not digested. As the toxin probably remained within the grains when Bt maize pollen passed through the lacewing gut, the absolute exposure of the insects is likely to be reduced. The higher digestion rate for Cry3Bb1 than Cry1Ab might result from differences in diffusion rates through the undamaged pollen wall or the pore. If one protein diffuses more rapidly than the other, it would experience greater exposure to digestive enzymes. Evidence for this comes from the fact that pollen from the maize variety expressing the cry3Bb1 gene loses more weight during the gut passage than pollen from the cry1Ab-expressing variety. Furthermore, a supplementary study (Supplementary data 3) in which Bt maize pollen was soaked in water for 16 h revealed that 37.9% of the Cry3Bb1 from DKc5143Bt pollen but only 20.2% of the Cry1Ab from Compa CB1 pollen entered the water. Differences in rates of digestion could also result from differences in molecule stability. This is supported by the finding that Bt protein concentrations in feces for DKc5143Bt were almost half of those in fresh pollen, while they remained almost constant for Compa CB1. Similarly, Cry3Bb1 was degraded faster than Cry1Ab when leaf material from the same Bt maize varieties was incorporated in soil (Zurbru¨gg et al., 2010), and a higher digestion rate for Cry3Bb1 than for Cry1Ab was reported when two slugs, Arion lusitanicus Mabille (Mollusca: Arionidae) and Deroceras reticulatum (Mu¨ller) (Mollusca: Agriolimacidae), were fed with Bt maize leaves (Zurbru¨gg and Nentwig, 2009).
Corey, D., Kambhampati, S., Wilde, G.E., 1998. Electrophoretic analysis of Orius insidiosus (Hemiptera: Anthocoridae) feeding habits in field corn. Journal of Kansas Entomological Society 71, 11–17. De Maagd, R.A., Bravo, A., Crickmore, N., 2001. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends in Genetics 17, 193–199. Duelli, P., 2001. Lacewings in field crops. In: McEwen, P., New, T.R., Whittington, A.E. (Eds.), Lacewings in the Crop Environment. Cambridge University Press, Cambridge, pp. 158–171. Elbadry, E.A., Fleschner, C.A., 1965. The feeding habits of adults of Chrysopa californica Coq. (Neuroptera, Chrysopidae). Bulletin de la Societe entomologique d’Egypte 49, 359–366. Garcia-Alonso, M., Jacobs, E., Raybould, A., Nickson, T.E., Sowig, P., Willekens, H., van der Kouwe, P., Layton, R., Amijee, F., Fuentes, A.M., Tencalla, F., 2006. A tiered system for assessing the risk of genetically modified plants to non-target organisms. Environmental Biosafety Research 5, 57–65. Goss, J.A., 1968. Development, physiology, and biochemistry of corn and wheat pollen. The Botanical Review 34, 333–358. Hagen, K.S., 1950. Fecundity of Chrysopa californica as affected by synthetic foods. Journal of Economic Entomology 43, 101–104. Hagen, K.S., Tassan, R.L., 1970. The influence of food wheast1 and related Saccharomyces fragilis yeast products on the fecundity of Chrysopa carnea (Neuroptera: Chrysopidae). The Canadian Entomologist 120, 806–811. Hagen, K.S., Tassan, R.L., Sawall, E.F., 1970. Some ecophysiological relationships between certain Chrysopa, honeydew and yeast. Bolletino del Laboratorio di Entomologia Agraria ‘Filippo Silvestri’ 28, 113–134. Hellmich, R.L., Albajes, R., Bergvinson, D., Prasifka, J.R., Wang, Z.Y., Weiss, M.J., 2008. The present and future role of insect-resistant GM crops in maize IPM. In: Romeis, J., Shelton, A.M., Kennedy, G.G. (Eds.), Integration of Insect-resistant Genetically Modified Crops within IPM Programs. Springer Science and Business Media B. V., pp. 119–158. Henry, C.S., Brooks, S.J., Duelli, P., Johnson, J.B., 2002. Discovering the true Chrysoperla carnea (Insecta: Neuroptera: Chrysopidae) using song analysis, morphology, and ecology. Annals of the Entomological Society of America 95, 172–191. Hogervorst, P.A.M., Wa¨ckers, F.L., Romeis, J., 2007. Detecting nutritional state and food source use in field-collected insects that synthesize honeydew oligosaccharides. Functional Ecology 21, 936–946. Human, H., Nicolson, S.W., 2003. Digestion of maize and sunflower pollen by the spotted maize beetle Astylus atromaculatus (Melyridae): is there a role for osmotic shock? Journal of Insect Physiology 49, 633–643. Jones, S.L., Lingren, P.D., Bee, M.J., 1977. Diel periodicity of feeding, mating, and oviposition of adult Chrysopa carnea. Annals of the Entomological Society of America 70, 43–47. Koziel, M.G., Beland, G.L., Bowman, C., Carozzi, N.B., Crenshaw, R., Crossland, L., Dawson, J., Dasai, N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D., McPherson, K., Keghji, M.R., Merlin, E., Rhodes, R., Warren, G.W., Wright, M., Evola, S.V., 1993. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11, 194–200. Li, Y., Romeis, J., 2009. Impact of snowdrop lectin (Galanthus nivalis agglutinin; GNA) on adults of the green lacewing, Chrysoperla carnea. Journal of Insect Physiology 55, 135–142.
164
Y. Li et al. / Journal of Insect Physiology 56 (2010) 157–164
Li, Y., Meissle, M., Romeis, J., 2008. Consumption of Bt maize pollen expressing Cry1Ab or Cry3Bb1 does not harm adult green lacewings, Chrysoperla carnea (Neuroptera: Chrysopidae). PLoS ONE 3 (8), e2909 doi:10.1371/journal. pone.0002909. Lundgren, J.G., Wiedenmann, R.N., 2004. Nutritional suitability of corn pollen for the predator Coleomegilla maculata (Coleoptera: Coccinellidae). Journal of Insect Physiology 50, 567–575. Meissle, M., Romeis, J., 2009a. The web-building spider Theridion impressum (Araneae: Theridiidae) is not adversely affected by Bt maize resistant to corn rootworms. Plant Biotechnology Journal 7, 645–656. Meissle, M., Romeis, J., 2009b. Insecticidal activity of Cry3Bb1 expressed in Bt maize on larvae of the Colorado potato beetle, Leptinotarsa decemlineata. Entomologia Experimentalis et Applicata 131, 308–319. Monsanto, 2004. Petition for the determination of nonregulated status for Mon 88017 corn. Monsanto Petition # 04-CR-108U; OECD Unique Identifier: MON88017-3. http://www.aphis.usda.gov/brs/aphisdocs/04_12501p.pdf. New, T.R., 1975. The biology of Chrysopidae and Hemerobiidae (Neuroptera), with reference to their usage as biocontrol agents: a review. Transactions of the Royal Entomological Society of London 127, 115–140. Obrist, L.B., Dutton, A., Albajes, R., Bigler, F., 2006. Exposure of arthropod predators to Cry1Ab toxin in Bt maize fields. Ecological Entomology 31, 143–154. Pilcher, C.D., Obrycki, J.J., Rice, M.E., Lewis, L.C., 1997. Preimaginal development, survival, and field abundance of insect predators on transgenic Bacillus thuringiensis corn. Environmental Entomology 26, 446–454. Principi, M.M., Canard, M., 1984. Feeding habits. In: Canard, M., Se´me´ria, Y., New, T.R. (Eds.), Biology of Chrysopidae. Dr. W. Junk Publishers, The Hague, pp. 76–92. Romeis, J., Dutton, A., Bigler, F., 2004. Bacillus thuringiensis toxin (Cry1Ab) has no direct effect on larvae of the green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae). Journal of Insect Physiology 50, 175–183. Romeis, J., Meissle, M., Bigler, F., 2006. Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology 24, 63–71. Romeis, J., Van Driesche, R.G., Barratt, B.I.P., Bigler, F., 2008a. Insect-resistant transgenic crops and biological control. In: Romeis, J., Shelton, A.M., Kennedy, G.G. (Eds.), Integration of Insect-resistant Genetically Modified Crops within IPM Programs. Springer Science and Business Media B. V., pp. 87–117. Romeis, J., Bartsch, D., Bigler, F., Candolfi, M.P., Gielkens, M.M.C., Hartley, S.E., Hellmich, R.L., Huesing, J.E., Jepson, P.C., Layton, R., Quemada, H., Raybould, A., Rose, R.I., Schiemann, J., Sears, M.K., Shelton, A.M., Sweet, J., Vaituzis, Z., Wolt, J.D., 2008b. Assessment of risk of insect-resistant transgenic crops to nontarget arthropods. Nature Biotechnology 26, 203–208. Romeis, J., Meissle, M., Raybould, A., Hellmich, R.L., 2009. Impact of insectresistant transgenic crops on above-ground non-target arthropods. In: Ferry,
N., Gatehouse, A.M.R. (Eds.), Environmental Impact of Genetically Modified Crops. CABI Publishing, Wallingford, pp. 165–198. Roulston, T.H., Buchmann, S.L., 2000a. A phylogenetic reconsideration of the starchpollination correlation. Evolutionary Ecology Research 2, 627–643. Roulston, T.H., Cane, J.H., 2000b. Pollen nutritional content and digestibility for animals. Plant Systematics and Evolution 222, 187–209. Sheldon, J.K., MacLeod, E.G., 1971. Studies on the biology of the Chrysopidae. II. The feeding behavior of the adult of Chrysopa carnea (Neuroptera). Psyche 78, 107–121. Stanley, R.G., Linskens, H.F., 1974. Pollen: Biology, Biochemistry, Management. Springer–Verlag, New York. Sundby, R.A., 1967. Influence of food on the fecundity of Chrysopa carnea Stephens (Neuroptera, Chrysopidae). Entomophaga 12, 475–479. Todd, F.E., Bretherick, O., 1942. The composition of pollens. Journal of Economic Entomology 35, 312–317. Treu, R., Emberlin, J., 2000. Pollen dispersal in the crops maize (Zea mays) oil seed rape (Brassica napus spp. oleifera), potatoes (Solanum tuberosum), sugar beet (Beta valgaris ssp. Valgaris) and wheat (Triticum aestivum) In: Report for the Soil Association, National Pollen Research Unit, University College Worcester, UK. Vaughn, T., Cavato, T., Brar, G., Coombe, T., DeGooyer, T., Ford, S., Groth, M., Howe, A., Johnson, S., Kolacz, K., Pilcher, C., Purcell, J., Romano, C., English, L., Pershing, J., 2005. A method of controlling corn rootworm feeding using a Bacillus thuringiensis protein expressed in transgenic maize. Crop Science 45, 931–938. Villenave, J., Thierry, D., Mamun, A.A., Lode´, T., Rat-Morris, E., 2005. The pollens consumed by common green lacewings Chrysoperla spp. (Neuroptera: Chrysopidae) in cabbage crop environment in western France. European Journal of Entomology 102, 547–552. Villenave, J., Deutsch, B., Lode´, T., Rat-Morris, E., 2006. Pollen preference of the Chrysoperla species (Neuroptera: Chrysopidae) occurring in the crop environment in western France. European Journal of Entomology 103, 771–777. Wolfenbarger, L.L., Naranjo, S.E., Lundgren, J.G., Bitzer, R.J., Watrud, L.S., 2008. Bt crops effects on functional guilds of non-target arthropods: a meta-analysis. PLoS ONE 3 (5), e2118, doi:10.1371/journal.pone.0002118. Woolfolk, S.W., Inglis, G.D., 2003. Microorganisms associated with field-collected Chrysoperla rufilabris (Neuroptera: Chrysopidae) adults with emphasis on yeast symbionts. Biological Control 29, 155–168. Zurbru¨gg, C., Nentwig, W., 2009. Ingestion and excretion of two transgenic Bt corn varieties by slugs. Transgenic Research 18, 215–225. Zurbru¨gg, C., Ho¨nemann, L., Meissle, M., Romeis, J., Nentwig, W., 2010. Decomposition dynamics and structural plant components of genetically modified Bt maize leaves do not differ from leaves of conventional hybrids. Transgenic Research 19, doi:10.1007/s11248-009-9304-x.