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Cultivation of transgenic cyanophycin-producing potatoes does not negatively affect growth, reproduction and activity of the earthworm Lumbricus terrestris (L.)
Pedobiologia 55 (2012) 161–165
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Cultivation of transgenic cyanophycin-producing potatoes does not negatively affect growth, reproduction and activity of the earthworm Lumbricus terrestris (L.) C. Emmerling a,∗ , J. Pohl a , K. Lahl a , C. Unger b , I. Broer b a b
University of Trier, FB VI – Department of Soil Science, Campus II, Behringstraße, D-54286 Trier, Germany University of Rostock, Faculty of Agronomy and Environmental Sciences, Department of Agrobiotechnology, Justus-von-Liebig Weg 8, D-18059 Rostock, Germany
a r t i c l e
i n f o
Article history: Received 1 September 2011 Received in revised form 16 December 2011 Accepted 19 December 2011 Keywords: Cyanophycin Cocoons Earthworms Hatchability Reproduction Transgenic potatoes
a b s t r a c t A microcosm experiment was performed to investigate the effects of post-harvest potato tubers from transgenic cyanophycin-producing potatoes on Lumbricus terrestris (L.) activity and biomass, number of cocoons and their hatchability as well as the remaining cyanophycin content in soil and cast samples during a period of 80 days. Potato tubers from four transgenic potato events with different cyanophycin content in a range from 0.8 to 7.5% were compared to the near isogenic, non-transgenic control (Solanum tuberosum L. cv. Albatros) and a comparative potato cultivar (S. tuberosum L. cv. Désirée). One treatment with transgenic tuber residue but without earthworms was prepared as an additional control. Potato tuber loss from the surface of the microcosms was significantly higher in the treatments with transgenic potato tubers compared with non-transgenic treatments. It can be estimated that the earthworm contribution to potato tuber loss from the soil surface was approximately 61%. Mean number of cocoons in addition to the number of hatched cocoons varied from 2.6 to 6.2 and from 7 to 15 accounting for 45.2–83.35% hatchability, respectively, but no significant differences between the treatments were found. The same was true for the development of earthworm biomass in the various treatments. The cyanophycin content in soil samples was significantly higher when earthworms were present indicating that the cyanophycin content in the upper soil layer might have been enhanced through earthworm burrowing activity. Overall, it is concluded that tubers from transgenic cyanophycin potatoes are easily degradable and neither inhibit nor stimulate earthworm growth, reproduction, and activity. © 2012 Elsevier GmbH. All rights reserved.
Introduction Over several decades, genetically modified (GM) crops have been cultivated world-wide. Besides GM crops with herbicide resistance, and resistance to plant pests and pathogens, GM crops for biodegradable polymer production have been developed. A polymer of biotechnological interest is polyaspartate, a soluble, non-toxic, biodegradable polycarboxylate (Tabata et al. 2000) which belongs to the group of poly-amino acids. It can be used as a biodegradable substitute of the synthetic polymer polyacrylate in various industrial applications (Simon 1987; Allen 1988). Cyanophycin, a water-insoluble nitrogen reserve polymer, is synthesized by many cyanobacteria and some non-cyanobacterial eubacteria (Ziegler et al. 2002) via non-ribosomal polypeptide synthesis (Ziegler et al. 1998). It consists of equimolar amounts of aspartic acid and arginine arranged as a polyaspartic acid backbone, to which arginine residues are linked to the ß-carboxyl group of each aspartate by its ␣-amino group (Simon and Weathers 1976).
∗ Corresponding author. Tel.: +49 651 201 2238. E-mail address: [email protected] (C. Emmerling). 0031-4056/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2011.12.006
The accumulation of cyanophycin in genetically modified potatoes occurs at the expense of carbohydrate storage in the tubers that causes a distinct intervention in the carbohydrate metabolism of the potatoes (Neumann et al. 2005; Hühns et al. 2009). Most studies on non-target and biodiversity risks of transgenic plants have revealed no detrimental effects of transgenic plants on non-target organisms (Andow and Zwahlen 2006). Icoz and Stotzky (2008) summarized results from numerous studies concerning the effects of insecticidal Cry proteins on non-target soil organisms. According to this synthesis, almost no significant effects on abundance, biomass and life-history traits were found. It is generally accepted, that earthworms strongly affect the turnover of soil organic matter (SOM) in agricultural ecosystems (Edwards and Fletcher 1988). Due to their high feeding and burrowing activity (Curry and Schmidt 2007) they enhance the surface of SOM, redistribute it vertically in the soil profile (Swift et al., 1979), change the size and activity of microorganisms in soil (Brown et al. 2000; Tiunov et al. 2001), and therefore strongly modify fertility and nutrient availability in soil (Aira et al. 2003; Haynes et al. 2003). Generally, litter breakdown by earthworms and microbial mineralization are strongly related to the chemical properties of the litter (Flegel and Schrader 2000; Curry 2004).
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Recently, Zeilinger et al. (2010) concluded from a four-year field study that the biomass of juveniles and adults of the earthworm species Aporrectodea caliginosa, Aporrectodea trapezoides, Aporrectodea tuberculata and Lumbricus terrestris did not differ significantly between various transgenic maize varieties (Cry1Ab and Cry3Bb1 Bt maize) and non-Bt varieties. Moreover, a microcosm study of Schrader et al. (2008) revealed a significant higher decay of Cry1Ab protein in the presence of earthworms. The digestive system of the foregut is assumed to be the most important compartment along the whole gut in terms of the fate of the Cry1Ab protein during digestion. Emmerling et al. (2011) accounted for a considerable fragmentation of Cry1Ab protein from the foregut, – midgut and hindgut to the casts. To date, studies (Milling et al. 2004; Becker et al. 2008; Gschwendtner et al. 2010) have shown no significant effects of transgenic potato tubers on soil microorganisms. However, only little information is available concerning the turnover and decomposition of genetically modified potato tuber residues in arable soil and to what extend this is governed by earthworms. Therefore, a research project was conducted in which the decomposition process of post-harvest tuber residues from various cyanophycin producing potatoes during overwintering was investigated. The present study focused on effects of GM potato tubers on the growth, reproduction and activity of earthworms. We investigated the contribution of earthworms to potato tuber loss, the development of individual earthworm biomass, earthworm reproduction and hatchability, in addition to the quantification of the remaining cyanophycin content in soil and cast material. To the best of our knowledge this is the first study on the effects of cyanophycin potato tubers on earthworm activity and life history parameters.
Materials and methods
the experimental soil for one week prior to the experiment. After this period earthworms were washed with distilled water, carefully dried on cellulose paper and then weighed. Afterwards, each earthworm was tagged with a visible implant elastomer (VIE, Northwest Marine Technology, Shaw Island, USA) at an individual position according to Butt and Lowe (2007). This was done in order to determine the development of the individual biomass. Subsequently, earthworms were placed on the soil surface. Mean fresh biomass of earthworms (n = 2) per microcosm varied from 5.14 (±1.0) g fresh weight (fr.w.) to 6.17 (±1.3) g. When all earthworms had entered the soil, 3 g dry weight (dr.w.) of approximately 0.5 cm × 1 cm large fragments of potato tuber residues equivalent to 5.1–5.5 g (fr.w.) was placed on the soil surface and rewetted by spraying with 5 ml tap water. The containers were covered with a close-meshed gauze (0.5 mm) to prevent possible escape of earthworms and then incubated at constant air temperature (15 ◦ C), relative air humidity (65%), and an illumination cycle (12 h dark/12 h illuminated) for 80 days. During this time, the soils in each container were moistened frequently with tap water to compensate for any loss of humidity. Supplementary residue (3 g dr.w.) was added to microcosms before all litter material was consumed or buried into the soil by earthworms. Thus, the feeding activity of earthworms was not influenced by a limited supply of potato tuber residue in the microcosms. Analyses Development of earthworm biomass For the investigation of earthworm biomass development, earthworms were removed from the microcosms at the end of the experiment after 80 days, carefully washed with tap water and subsequently dried on cellulose paper. Then, earthworms were reweighed according to their individual tag position. For statistical analyses the results of two earthworms from each of five replicates were pooled for a mean (±S.E.) weight per treatment.
Experimental design A soil microcosm experiment was performed to assess the impact of tuber residues from various genetically modified potato events on the development and activity of earthworms in soil. For comparison, tuber residues from four potato events differing in their cyanophycin content (see Table 1), their near isogenic (parental) control, Solanum tuberosum L. cv. Albatros and a comparative cultivar, S. tuberosum L. cv. Désirée, were used in the experiment. The transgenic potato events differed markedly in their cyanophycin content from 7.5% dry weight (dr.w.) in the AE39, to 0.8% (dr.w.) in the A-E23 treatment (Table 1). The two potato cultivars (Albatros and Desiree) differed markedly in intercellular protein content with nearly twice as much in the Albatros events (data not shown). The experiment consisted of five microcosms per treatment each containing two clitellate adult individuals of L. terrestris (Linnaeus, 1758). The soil within each microcosm was covered with tuber residues of the various potato cultivars and events. One treatment with residues of a cyanophycin producing potato but without earthworm inoculation (A-E12-C; Table 1) served as an additional control. For the experiment, polyethylene containers (1 l) were used. The experimental soil, a sandy loamy Fluvisol, was collected from the Ap-horizont (0–30 cm depth) from an arable field near Trier (Germany) and sieved through a 2 mm screen. Soil chemical properties were as follows: pH (CaCl2 ) 5.9, Nt 0.11%, TOC 1.50%, and CEC 11.5 cmolc kg−1 dry weight. Each container was filled with 500 g moist soil, which was wetted previously to a water content of 20% by weight and equivalent to 70% of water holding capacity. Mature earthworms were purchased from a local earthworm culturing plant (Mosella, Laufeld, Germany) and were stored in
Loss of potato tuber residues When the loss of potato tuber residues was evident but prior their complete disappearance (approximately after 60–65 days of the experiment) the remaining material was carefully sampled from the soil surface of each microcosm and weighed. Cocoon production and hatchability Earthworm cocoons per replicate were extracted from soil material by a new wet sieve method. In brief, the remaining soil material from each microcosm was placed on a cylindrical sieve (2 mm mesh size) and then transferred into a plastic dish. Subsequently, the dish was filled with water to about 1 cm in order to cover the bottom of the sieve. The dish was placed on a horizontal shaker for at least 12 min at 100 rpm, which destroyed soil aggregates while leaving cocoons undamaged. The number of cocoons was noted for each replicate. Cocoons were then transferred onto Petri-dishes on wet filter paper. The Petri-dishes were incubated at 20 ◦ C for a total of 102 days according to Lowe and Butt (2005). During this time the number of hatched cocoons of each replicate was recorded; juveniles were subsequently removed from the Petri-dishes and weighed. Quantification of cyanophycin in soil and casts Earthworm casts from the surface and soil samples from the top layer of the microcosms were collected and were immediately air-dried for subsequent cyanophycin analyses. Cyanophycin content was analysed according to Hühns et al. (2008). Briefly, 100 mg (dr.w.) of soil or cast samples was weighed in a 2 mL tube, mixed with 1 mL Tris-buffer with a vortexer and then shaken horizontally for 30 min at 220 rpm. Afterwards, samples were centrifuged at 15,000 rpm for 10 min. Pellets were extracted with 1 mL 0.1 M
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Table 1 Cyanophycin content from the tubers of different potato (Solanum tuberosum) cultivars and transgenic events used in the current experiment. Results from mean (±S.E.) cyanophycin contents (g mL−1 ) of earthworm casts and soil from different potato treatments after 80 days of the experiment are also shown. Significant differences between the treatments are shown using different letters (ANOVA and Games–Howell-test; p < 0.05, n = 5). Potato treatment
Notation
Albatros B33-PsbY-cphATe – event 39 Albatros B33-PsbY-cphATe – event 16 Albatros PsbY-cphATe – event 12 Albatros PsbY-cphATe – event 23 Albatros, near isogenic control Desiree, comparative cultivar Albatros PsbY-cphATe – event 12 a b
Cyanophycin content
A-E39 A-E16 A-E12 A-E23 A-NIC Desirée A-E12-Ca
Potatoes (% dr.w.)
Cast (g mL−1 )
Soil (g mL−1 )
7.5 6.5 0.9 0.8 0 0 0.9
4.02 ± 0.15a 3.82 ± 0.53a 5.22 ± 0.54b 3.72 ± 0.22a
3.41 3.39 3.56 3.09
b
0.36b 0.24b 0.39b 0.28a
b
b
No casts
± ± ± ±
b
2.86 ± 0.12a
Treatment A-E12 without earthworms. Below detection limit.
HCl, vortexed, also horizontally shaken at 220 rpm for 60 min, and then again centrifuged at 14,000 rpm for at least 10 min. Subsequently, 800 L of the supernatant was poured into a new 2 mL tube and mixed (vortexed) with 200 L of Coomassie Brillant Blue G-250 dye (Roti-Quant; Carl Roth, Germany) to produce a blue-coloured protein solution which could be measured photometrically with a UV-VIS photometer at OD 595 nm (Shimadzu UV 1650-PC, Duisburg, Germany). The remaining dye incubated for 5 min. A cyanophycin stock solution of 0.5 L mL−1 was used for calibration standards. Each sample, soil and casts, was measured in triplicate. Statistical analysis All results are presented as means (±S.E.) of five replicates. For comparisons between the different potato events (treatments) different statistical procedures were performed. If data were normally distributed an ANOVA was performed while non-normally distributed data was analysed using a non-parametric Kruskal–Wallis H-test. A Games–Howell test was used as a post-hoc test in combination with an ANOVA because data showed heterogeneous variances according to the Levene test. If the data set was non-normally distributed with heterogeneous variances, a nonparametric Kruskal–Wallis H-test in combination with a post-hoc Nemenyi test was used. Differences between the two treatment groups, transgenic and non-transgenic potatoes, were calculated using the non-parametric Mann–Whitney U-test. Results
non-transgenic, near-isogenic, and the comparative treatments, this variation was much lower and ranged from 0.95 to 1.66% or 1.37 to 1.53% when A-NIC or Desirée was used, respectively. However, on average, there was a significant difference in potato tuber loss between the treatments with cyanophycin potato tubers applied, compared with the controls. The mean loss per day varied between 2.3 and 4.8% compared with 1.3 and 1.5%, respectively. In the A-E12C treatment without earthworms, mean potato tuber loss was 0.9% (Table 2), which was significantly lower compared to the respective A-E12 treatment. Mean earthworm biomass (fr.wt.) of the various treatments varied from 5.14 to 6.17 g at the start of the experiment and thus, the differences in biomass between the treatments were quite small. The same was found at the end of the experiment after 80 days (Table 3). However, on average, earthworm mean biomass increased by 0.22–0.54 g or relatively about 3.70–11.58% (Table 3). Reproduction During the experimental period the mean number of cocoons in the various treatments ranged between 2.6 in the A-NIC treatment and 6.2 in A-E16 (Table 3). The highest number of cocoons (31) was found in the A-E16 treatment, followed by the A-E23 (21) and AE12 (20) treatments. Differences between the treatments were not significant (p > 0.05). During the incubation time between 7 and 15 juveniles per treatment hatched; a mean of 45 and 83% relative to total cocoons per treatment (Table 3). The mean weight of hatchlings varied between 51.5 and 63.6 mg ind.−1 . Differences between treatments were not significant (p > 0.05).
Potato tuber loss and earthworm biomass Potato tuber loss from the surface within the cyanophycin treatments varied markedly, for example, between 1.17 and 7.10% in A-E39 or 1.67 and 7.65% in the A-E23 treatments (Table 2). In the Table 2 Loss of potato tuber residues (n = 5) from the soil surface. Differences after 60–65 days within transgenic and non-transgenic potatoes as well as between treatments A-E12 and A-E12-C were significantly different (p < 0.05; Mann–Whitney U-test). For abbreviations see Table 1. Potato treatment
A-E39 A-E16 A-E12 A-E23 A-NIC Desirée A-E12-Ca a
Surface loss (5 d−1 ) Min
Max
Mean (±S.E.)
1.17 3.29 1.50 1.67 0.95 1.37 0.82
7.10 7.61 3.34 7.65 1.66 1.53 0.99
3.6 4.6 2.3 4.8 1.3 1.5 0.9
Treatment A-E12 without earthworms.
± ± ± ± ± ± ±
2.4 1.8 0.9 2.6 0.3 0.1 0.1
Quantification of cyanophycin in different compartments At the end of the experiment the amounts of cyanophycin in soil samples showed significant differences between the A-E12 and the A-E12-C treatment and all other treatments (Table 1). Due to a lack of earthworms only soil samples were taken from the latter treatment. Specifically, in cast samples no significant difference within the treatments were found except for significantly higher cyanophycin content in casts of the A-E12 treatment compared to all other treatments (Table 1). Unfortunately, the cyanophycin content of the various potato tuber residues could not be analysed at the end of the experiment, since it was not possible to sample residual material without adhering soil or cast material except in the A-E12-C treatment without earthworms. Compared to the initial cyanophycin content of the potato tubers in the A-E12 treatment (0.9% dr.w.), cyanophycin content of tubers in earthworm-free treatments was 0.15% dr.w. at the end of the experiment (data not shown), representing a relative decrease in cyanophycin content of 84%.
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Table 3 Mean (x ± S.E.; n = 10) earthworm biomass (g fresh mass) at the beginning of the experiment and after 80 days (end), and absolute and percentage difference (x ± S.E.) of earthworm biomass during the experimental period. Additionally, sum and mean numbers (x ± S.E.) of cocoons (after 80 days) and hatchlings (after further 102 days cocoon incubation) per treatment, and mean weight of juveniles after emergence are presented. Differences within the treatments were not significant (p > 0.05; Kruskal–Wallis H-test). For abbreviations see Table 1. A-E39 Earthworm biomass Start (day 0) End (day 80) Diff. [g] Diff. [%] Life history parameters cocoons Mean no. cocoons hatchlings Hatchlings [%] weight of juveniles [mg]
5.75 ± 0.73 6.03 ± 0.89 0.28 ± 0.42 4.79 ± 7.19 17 3.4 (±3.3) 9 52.9 56.3 (±17.4)
A-E16 5.64 ± 0.66 6.13 ± 0.48 0.49 ± 0.35 9.23 ± 7.11 31 6.2 (±2.5) 14 45.2 59.3 (±4.1)
Discussion The effects of residues from transgenic potato tubers with various contents of cyanophycin on the growth, reproduction and activity of earthworms in soil were investigated in a microcosm experiment over 80 days. Overall, mean daily potato tuber loss varied between 2.3 and 4.8% in the transgenic potato treatments compared to 1.3 and 1.5% in the non-transgenic treatments indicating that the loss of genetically modified potatoes was significantly higher (p < 0.05). These data show that mean potato tuber loss per day was nearly 1.5- to 3- fold higher in the treatments with cyanophycin residues. Decomposition of organic residues mainly depends on the palatability of the organic resource for decomposers (Satchell and Lowe 1967; Ashili et al. 2007). For example, the C-to-N ratio of the investigated potato tubers varied between 6.5 and 8.1 with no significant difference between the tubers of the different potato events. Albatros tubers are rich in starch and protein compared to Desirée tubers (data not shown). However, these two compounds had no effect on potato tuber loss within the two lines. Moreover, earthworms preferentially used cyanophycin producing potato tubers as a resource compared to the isogenic control events. Moreover mean potato tuber loss was only 0.9% in the A-E12-C treatment without earthworms. It is hypothesized, that this result indicates a considerable decay of the potato residues through microbial activity. In an experiment with transgenic maize (Bt-maize) it was also shown that microorganisms contributed significantly to surface maize litter loss (Emmerling et al. 2011). When compared to a blanc A-E12 and A-E12-C treatments it can be estimated, that the proportion of earthworms in decomposition of potato tuber residues was approximately 61%. During the experiment, earthworm biomass increased on average by 0.22–0.54 g or relatively by 3.70–11.58%, indicating that the experimental conditions were favorable for the earthworms in all treatments (Fründ et al. 2010). Thus, differences between treatments with transgenic and non-transgenic potato lines were not significant. Cocoon production of earthworms depends on a complete set of environmental life conditions, such as temperature, soil moisture content and food supply (Curry and Bolger 1984). In a laboratory experiment Butt (1991) and Grigoropoulou et al. (2007) observed the production of one cocoon per week per individual, predicting a number of 11 cocoons in 80 days. In the present study the number of cocoons was lower varying between 2.6 and 6.2 on average per treatment over 80 days. This could be due to differences in food supply (leaf litter vs. potato tuber). However, our reproductive results are comparable to those found by Butt et al. (1994). Another factor, which could be responsible for cocoon production, is the C-to-N ratio of the food resource (Meinhardt 1973; Lofs-Holmin
A-E12 5.29 ± 1.04 5.53 ± 1.14 0.24 ± 0.63 4.95 ± 11.9 20 4.0 (±2.4) 14 70.0 56.3 (±8.7)
A-E23 6.17 ± 1.30 6.39 ± 1.37 0.22 ± 0.39 3.70 ± 6.39 21 4.2 (±3.1) 12 57.1 63.6 (±11.1)
A-NIC 5.14 ± 0.98 5.68 ± 0.95 0.54 ± 0.56 11.58 ± 11.77 13 2.6 (±1.8) 7 53.9 58.2 (±0.8)
Desirée 5.71 ± 0.85 5.94 ± 0.80 0.23 ± 0.61 4.67 ± 10.89 18 3.6 (±2.8) 15 83.3 51.5 (±14.0)
1982). According to Suthar (2007) cocoon production of epigeic earthworm species (Eudrilus eugeniae, Perionyx excavatus, Perionyx sansibaricus) increased when C-to-N ratio of the food resource was narrow. Obviously, in the present study cocoon production was not affected by the cyanophycin or protein content since the highest number of cocoons was found in the A-L16 treatment which had the second highest cyanophycin content. The rate of hatched cocoons in the treatments varied between 45 and 83% of the number of cocoons, which was comparable to an investigation of Grigoropoulou et al. (2007), where the mean hatchling survival rate was 62%. Interestingly, in the A-E16 treatment, where the number of cocoons was highest, the rate of hatchability was lowest. However, in the A-NIC treatment, where the number of cocoons was lowest, the number of hatched cocoons fell within the mean range of all treatments. Mean weight of hatchlings also showed no significant difference between the treatments. Consequently, there seems to be little correlation between the cyanophycin content of potato tubers with the number of cocoons, the rate of hatchability or the fresh weight of juveniles. During the experiment the cyanophycin content of the potato tubers decreased by 84% which was observed in the A-E12 treatment. The remaining cyanophycin content was analysed using a new method according to Hühns et al. (2008). Due to a calibration with cyanophycin it was also possible to quantify the cyanophycin content in soil and cast samples. In the A-E12 treatment, the amount of cyanophycin in soil was higher compared to the same treatment without earthworms. This might be evidence that the cyanophycin content of the upper soil layer was enhanced by earthworm burrowing activity. The overall decrease of the cyanophycin content during the experiment can also be attributed to microbial activity. Moreover, all five replicates of the A-E12-C treatment showed an intense development of mildew immediately after exposure of the tuber residues on the soil surface with the mildew increasing in mass during the experiment. By contrast, in the A-E12 treatment and in all other earthworm treatments, a comparable initial development of mildew was found, respectively, but earthworms fed very quickly and exclusively on the mildew so that it was no longer found after several days and until the end of the experiment. According to Edwards and Fletcher (1988), Brown (1995), Bonkowski et al. (2000) and Butenschön et al. (2007) fungi serve as an important food source for earthworms and significantly affect the diet of earthworms, especially of detritivorous earthworms, like L. terrestris (Moody et al. 1995). Although not quantified, no evidence was found that mildew infection differed within the various transgenic potato lines. Finally, cyanophycin content in casts of the L-E12 treatment was significantly higher relative to the other treatments, but the reasons underlying these results remain unclear.
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Conclusions The soil microcosm experiment revealed that earthworms preferentially fed on tuber residues from transgenic cyanophycin producing potatoes compared to residues from non-transgenic potato tubers. Residue loss may be due to earthworm feeding activity and microbial activity. This enhanced activity had no significant effect on earthworm biomass, cocoon production, or on hatchability. After 80 days, nearly 3/4 of initial cyanophycin content in the residues was partly lost due to both earthworm and microbial activity. There was at least circumstantial evidence that, for the investigated earthworm parameters, no inhibiting effects of tuber residues from transgenic potatoes in soil exists. Acknowledgements Parts of the study were financially supported by the Bundesministerium für Forschung und Technologie, grant number 0315214B. The helpful comments of two reviewers are gratefully acknowledged. References Aira, M., Monroy, F., Domínguez, J., 2003. Effects of two species of earthworms (Allolobophora sp.) on soil systems: a microfaunal and biochemical analysis. Pedobiologia 47, 877–881. Allen, M.M., 1988. Inclusions: cyanophycin. Methods Enzymol. 167, 207–213. Andow, D.A., Zwahlen, C., 2006. Assessing environmental risks of transgenic plants. Ecol. Lett. 9, 196–214. Ashili, P., Tajovsky, K., Tuf, I.H., Tufova, J., 2007. Impact of ungulate browsing on leaf litter palatability for millepeds (Diplopoda). In: Tajovsky, K., et al. (Eds.), 9th Central European Workshop on Soil Zoology. Ceské Budèjovice, pp. 1–4. Becker, R., Behrendt, U., Hommel, B., Kropf, S., Ulrich, A., 2008. Effects of transgenic fructan-producing potatoes on the community structure of rhizosphere and phyllosphere bacteria. FEMS Microbiol. Ecol. 66, 411–425. Bonkowski, M., Griffiths, B.S., Ritz, K., 2000. Food preferences of earthworms for soil fungi. Pedobiologia 44, 666–676. Brown, G.G., 1995. How do earthworms affect microfloral and faunal communities? Plant Soil 170, 209–231. Brown, G.B., Barois, I., Lavelle, P., 2000. Regulation of soil organic matter dynamics and microbial activity in the drilosphere and the role of interactions with other edaphic functional domains. Eur. J. Soil Biol. 36, 177–198. Butenschön, O., Poll, C., Langel, R., Kandeler, E., Marhan, S., Scheu, S., 2007. Endogeic earthworms alter carbon translocation by fungi at the soil–litter interface. Soil Biol. Biochem. 39, 2854–2864. Butt, K.R., 1991. The effects of temperature on the intensive production of Lumbricus terrestris (Oligochaeta: Lumbricidae). Pedobiologia 35, 257–264. Butt, K.R., Frederickson, J., Morris, R.M., 1994. Effect of earthworm density on growth and reproduction of Lumbricus terrestris L. (Oligochaeta: Lumbricidae) in culture. Pedobiologia 38, 254–261. Butt, K.R., Lowe, C.N., 2007. A viable technique for tagging earthworms using visible implant elastomer. Appl. Soil Ecol. 35, 454–457. Curry, J.P., 2004. Factors affecting the abundance of earthworms in soils. In: Edwards, C.A. (Ed.), Earthworm Ecology. , 2nd. Ed. CRC Press, pp. 91–113. Curry, J.P., Bolger, T., 1984. Growth, reproduction and litter and soil consumption by Lumbricus terrestris L. in reclaimed Peat. Soil Biol. Biochem. 16, 253–257. Curry, J.P., Schmidt, O., 2007. The feeding ecology of earthworms – a review. Pedobiologia 50, 463–477. Edwards, C.A., Fletcher, K.E., 1988. Interactions between earthworms and microorganisms in organic-matter breakdown. Agric. Ecosyst. Environ. 24, 235–247. Emmerling, C., Strunk, H., Schöbinger, U., Schrader, S., 2011. Fragmentation of Cry1Ab toxin from MON810 maize through the gut of the earthworm species Lumbricus terrestris L. Eur. J. Soil Biol. 47, 160–164. Flegel, M., Schrader, S., 2000. Importance of food quality on selected enzyme activities in earthworm casts (Dendrobaena octaedra Lumbricidae). Soil Biol. Biochem. 32, 1191–1196.
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Hühns, M., Neumann, K., Hausmann, T., Ziegler, K., Klemke, F., Kahmann, U., Staiger, D., Lockau, W., Pistorius, E.K., Broer, I., 2008. Plastid targeting strategies for cyanophycin synthetase to achieve high-level polymer accumulation in Nicotiana tabacum. Plant Biotechnol. J. 6, 321–336. Hühns, M., Neumann, K., Hausmann, T., Klemke, F., Lockau, W., Kahmann, U., Kopertekh, L., Staiger, D., Pistorius, E.K., Reuther, J., Waldvogel, E., Wohlleben, W., Effmert, M., Junghans, H., Neubauer, K., Kragl, U., Schmidt, K., Schmidtke, J., Broer, I., 2009. Tuber specific cphA expression to enhance cyanophycin production in potatoes. Plant Biotechnol. J. 7, 883–898. Icoz, I., Stotzky, G., 2008. Fate and effects of insect-resistant Bt crops in soil ecosystems. Soil Biol. Biochem. 40, 559–586. Lofs-Holmin, A., 1982. Reproduction and growth of common arable land and pasture species of earthworms (Lumbricidae) in laboratory cultures. Swedish J. Agric. Res. 13, 31–37. Lowe, C.N., Butt, K.R., 2005. Culture techniques for soil dwelling earthworms: a review. Pedobiologia 49, 401–413. Meinhardt, U., 1973. Vergleichende Beobachtungen zur Laboratoriumsbiologie einheimischer Regenwurmarten. 1. Haltung und Zucht. Z. Angew. Zool. 60, 233–255. Milling, A., Smalla, K., Maidl, F.X., Schloter, M., Munch, J.C., 2004. Effects of transgenic potatoes with an altered starch composition on the diversity of soil and rhizosphere bacteria and fungi. Plant Soil 266, 23–39. Moody, S.A., Briones, M.J., Piearce, T.G., Dighton, J., 1995. Selective consumption of decomposing wheat straw by earthworms. Soil Biol. Biochem. 27, 1209–1213. Neumann, K., Stephan, D.P., Ziegler, K., Hühns, M., Broer, I., Lockau, W., Pistorius, E.K., 2005. Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants. Plant Biotechnol. J. 3, 249–258. Satchell, J.E., Lowe, D.G., 1967. Selection of leaf litter by Lumbricus terrestris (L.). In: Graff, O., Satchell, J.E. (Eds.), Soil Organisms. North Holland, Amsterdam, pp. 102–119. Schrader, S., Münchenberg, T., Baumgarte, S., Tebbe, C.C., 2008. Earthworms of different functional groups affect the fate oft he Bt-toxin Cry1Ab from transgenic maize in soil. Eur. J. Soil Biol. 44, 283–289. Simon, R.D., 1987. Inclusion bodies in the cyanobacteria: cyanophycin, polyphosphate, polyhedral bodies. In: Fay, P., van Baalen, C. (Eds.), The Cyanobacteria. Elsevier, Amsterdam, New York, pp. 199–225. Simon, R.D., Weathers, P., 1976. Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in cyanobacteria. Biochim. Biophys. Acta 420, 165–176. Suthar, S., 2007. Influence of different food sources on growth and reproduction performance of composting epigeics: Eudrilus eugeniae, Perionyx excavatus and Peronyx sansibaricus. Appl. Ecol. Environ. Res. 5, 79–92. Swift, M.J., Heal, O.W., Anderson, J.M., 1979. Decomposition in Terrestrial Ecosystems. Blackwell, Oxford, pp. 372. Tabata, K., Abe, H., Doi, Y., 2000. Microbial degradation of poly(aspartic acid) by two isolated strains of Pedobacter sp. and Shingomonas sp. Biomacromolecules 1, 157–161. Tiunov, A.V., Bonkowski, M., Alphei, J., Scheu, S., 2001. Microflora, Protozoa and Nematoda in Lumbricus terrestris burrow walls: a laboratory experiment. Pedobiologia 45, 46–60. Zeilinger, A.R., Andow, D.A., Zwahlen, C., Stotzky, G., 2010. Earthworm populations in a U.S. Cornbelt soil are not affected by long-term cultivation of Bt maize expressing Cry1Ab and Cry3Bb1 proteins. Soil Biol. Biochem. 42, 1284–1292. Ziegler, K.A., Diener, A., Herpin, C., Richter, R., Deutzmann, R., Lockau, W., 1998. Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-Laspartate (cyanophycin). Eur. J. Biochem. 254, 154–159. Ziegler, K., Deutzmann, R., Lockau, W., 2002. Cyanophycin-like enzymes of noncyanobacterial Eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense. Z. Naturforsch. 57c, 522–529.
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Pedobiologia - International Journal of Soil Biology journal homepage: www.elsevier.de/pedobi
Cultivation of transgenic cyanophycin-producing potatoes does not negatively affect growth, reproduction and activity of the earthworm Lumbricus terrestris (L.) C. Emmerling a,∗ , J. Pohl a , K. Lahl a , C. Unger b , I. Broer b a b
University of Trier, FB VI – Department of Soil Science, Campus II, Behringstraße, D-54286 Trier, Germany University of Rostock, Faculty of Agronomy and Environmental Sciences, Department of Agrobiotechnology, Justus-von-Liebig Weg 8, D-18059 Rostock, Germany
a r t i c l e
i n f o
Article history: Received 1 September 2011 Received in revised form 16 December 2011 Accepted 19 December 2011 Keywords: Cyanophycin Cocoons Earthworms Hatchability Reproduction Transgenic potatoes
a b s t r a c t A microcosm experiment was performed to investigate the effects of post-harvest potato tubers from transgenic cyanophycin-producing potatoes on Lumbricus terrestris (L.) activity and biomass, number of cocoons and their hatchability as well as the remaining cyanophycin content in soil and cast samples during a period of 80 days. Potato tubers from four transgenic potato events with different cyanophycin content in a range from 0.8 to 7.5% were compared to the near isogenic, non-transgenic control (Solanum tuberosum L. cv. Albatros) and a comparative potato cultivar (S. tuberosum L. cv. Désirée). One treatment with transgenic tuber residue but without earthworms was prepared as an additional control. Potato tuber loss from the surface of the microcosms was significantly higher in the treatments with transgenic potato tubers compared with non-transgenic treatments. It can be estimated that the earthworm contribution to potato tuber loss from the soil surface was approximately 61%. Mean number of cocoons in addition to the number of hatched cocoons varied from 2.6 to 6.2 and from 7 to 15 accounting for 45.2–83.35% hatchability, respectively, but no significant differences between the treatments were found. The same was true for the development of earthworm biomass in the various treatments. The cyanophycin content in soil samples was significantly higher when earthworms were present indicating that the cyanophycin content in the upper soil layer might have been enhanced through earthworm burrowing activity. Overall, it is concluded that tubers from transgenic cyanophycin potatoes are easily degradable and neither inhibit nor stimulate earthworm growth, reproduction, and activity. © 2012 Elsevier GmbH. All rights reserved.
Introduction Over several decades, genetically modified (GM) crops have been cultivated world-wide. Besides GM crops with herbicide resistance, and resistance to plant pests and pathogens, GM crops for biodegradable polymer production have been developed. A polymer of biotechnological interest is polyaspartate, a soluble, non-toxic, biodegradable polycarboxylate (Tabata et al. 2000) which belongs to the group of poly-amino acids. It can be used as a biodegradable substitute of the synthetic polymer polyacrylate in various industrial applications (Simon 1987; Allen 1988). Cyanophycin, a water-insoluble nitrogen reserve polymer, is synthesized by many cyanobacteria and some non-cyanobacterial eubacteria (Ziegler et al. 2002) via non-ribosomal polypeptide synthesis (Ziegler et al. 1998). It consists of equimolar amounts of aspartic acid and arginine arranged as a polyaspartic acid backbone, to which arginine residues are linked to the ß-carboxyl group of each aspartate by its ␣-amino group (Simon and Weathers 1976).
∗ Corresponding author. Tel.: +49 651 201 2238. E-mail address: [email protected] (C. Emmerling). 0031-4056/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2011.12.006
The accumulation of cyanophycin in genetically modified potatoes occurs at the expense of carbohydrate storage in the tubers that causes a distinct intervention in the carbohydrate metabolism of the potatoes (Neumann et al. 2005; Hühns et al. 2009). Most studies on non-target and biodiversity risks of transgenic plants have revealed no detrimental effects of transgenic plants on non-target organisms (Andow and Zwahlen 2006). Icoz and Stotzky (2008) summarized results from numerous studies concerning the effects of insecticidal Cry proteins on non-target soil organisms. According to this synthesis, almost no significant effects on abundance, biomass and life-history traits were found. It is generally accepted, that earthworms strongly affect the turnover of soil organic matter (SOM) in agricultural ecosystems (Edwards and Fletcher 1988). Due to their high feeding and burrowing activity (Curry and Schmidt 2007) they enhance the surface of SOM, redistribute it vertically in the soil profile (Swift et al., 1979), change the size and activity of microorganisms in soil (Brown et al. 2000; Tiunov et al. 2001), and therefore strongly modify fertility and nutrient availability in soil (Aira et al. 2003; Haynes et al. 2003). Generally, litter breakdown by earthworms and microbial mineralization are strongly related to the chemical properties of the litter (Flegel and Schrader 2000; Curry 2004).
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Recently, Zeilinger et al. (2010) concluded from a four-year field study that the biomass of juveniles and adults of the earthworm species Aporrectodea caliginosa, Aporrectodea trapezoides, Aporrectodea tuberculata and Lumbricus terrestris did not differ significantly between various transgenic maize varieties (Cry1Ab and Cry3Bb1 Bt maize) and non-Bt varieties. Moreover, a microcosm study of Schrader et al. (2008) revealed a significant higher decay of Cry1Ab protein in the presence of earthworms. The digestive system of the foregut is assumed to be the most important compartment along the whole gut in terms of the fate of the Cry1Ab protein during digestion. Emmerling et al. (2011) accounted for a considerable fragmentation of Cry1Ab protein from the foregut, – midgut and hindgut to the casts. To date, studies (Milling et al. 2004; Becker et al. 2008; Gschwendtner et al. 2010) have shown no significant effects of transgenic potato tubers on soil microorganisms. However, only little information is available concerning the turnover and decomposition of genetically modified potato tuber residues in arable soil and to what extend this is governed by earthworms. Therefore, a research project was conducted in which the decomposition process of post-harvest tuber residues from various cyanophycin producing potatoes during overwintering was investigated. The present study focused on effects of GM potato tubers on the growth, reproduction and activity of earthworms. We investigated the contribution of earthworms to potato tuber loss, the development of individual earthworm biomass, earthworm reproduction and hatchability, in addition to the quantification of the remaining cyanophycin content in soil and cast material. To the best of our knowledge this is the first study on the effects of cyanophycin potato tubers on earthworm activity and life history parameters.
Materials and methods
the experimental soil for one week prior to the experiment. After this period earthworms were washed with distilled water, carefully dried on cellulose paper and then weighed. Afterwards, each earthworm was tagged with a visible implant elastomer (VIE, Northwest Marine Technology, Shaw Island, USA) at an individual position according to Butt and Lowe (2007). This was done in order to determine the development of the individual biomass. Subsequently, earthworms were placed on the soil surface. Mean fresh biomass of earthworms (n = 2) per microcosm varied from 5.14 (±1.0) g fresh weight (fr.w.) to 6.17 (±1.3) g. When all earthworms had entered the soil, 3 g dry weight (dr.w.) of approximately 0.5 cm × 1 cm large fragments of potato tuber residues equivalent to 5.1–5.5 g (fr.w.) was placed on the soil surface and rewetted by spraying with 5 ml tap water. The containers were covered with a close-meshed gauze (0.5 mm) to prevent possible escape of earthworms and then incubated at constant air temperature (15 ◦ C), relative air humidity (65%), and an illumination cycle (12 h dark/12 h illuminated) for 80 days. During this time, the soils in each container were moistened frequently with tap water to compensate for any loss of humidity. Supplementary residue (3 g dr.w.) was added to microcosms before all litter material was consumed or buried into the soil by earthworms. Thus, the feeding activity of earthworms was not influenced by a limited supply of potato tuber residue in the microcosms. Analyses Development of earthworm biomass For the investigation of earthworm biomass development, earthworms were removed from the microcosms at the end of the experiment after 80 days, carefully washed with tap water and subsequently dried on cellulose paper. Then, earthworms were reweighed according to their individual tag position. For statistical analyses the results of two earthworms from each of five replicates were pooled for a mean (±S.E.) weight per treatment.
Experimental design A soil microcosm experiment was performed to assess the impact of tuber residues from various genetically modified potato events on the development and activity of earthworms in soil. For comparison, tuber residues from four potato events differing in their cyanophycin content (see Table 1), their near isogenic (parental) control, Solanum tuberosum L. cv. Albatros and a comparative cultivar, S. tuberosum L. cv. Désirée, were used in the experiment. The transgenic potato events differed markedly in their cyanophycin content from 7.5% dry weight (dr.w.) in the AE39, to 0.8% (dr.w.) in the A-E23 treatment (Table 1). The two potato cultivars (Albatros and Desiree) differed markedly in intercellular protein content with nearly twice as much in the Albatros events (data not shown). The experiment consisted of five microcosms per treatment each containing two clitellate adult individuals of L. terrestris (Linnaeus, 1758). The soil within each microcosm was covered with tuber residues of the various potato cultivars and events. One treatment with residues of a cyanophycin producing potato but without earthworm inoculation (A-E12-C; Table 1) served as an additional control. For the experiment, polyethylene containers (1 l) were used. The experimental soil, a sandy loamy Fluvisol, was collected from the Ap-horizont (0–30 cm depth) from an arable field near Trier (Germany) and sieved through a 2 mm screen. Soil chemical properties were as follows: pH (CaCl2 ) 5.9, Nt 0.11%, TOC 1.50%, and CEC 11.5 cmolc kg−1 dry weight. Each container was filled with 500 g moist soil, which was wetted previously to a water content of 20% by weight and equivalent to 70% of water holding capacity. Mature earthworms were purchased from a local earthworm culturing plant (Mosella, Laufeld, Germany) and were stored in
Loss of potato tuber residues When the loss of potato tuber residues was evident but prior their complete disappearance (approximately after 60–65 days of the experiment) the remaining material was carefully sampled from the soil surface of each microcosm and weighed. Cocoon production and hatchability Earthworm cocoons per replicate were extracted from soil material by a new wet sieve method. In brief, the remaining soil material from each microcosm was placed on a cylindrical sieve (2 mm mesh size) and then transferred into a plastic dish. Subsequently, the dish was filled with water to about 1 cm in order to cover the bottom of the sieve. The dish was placed on a horizontal shaker for at least 12 min at 100 rpm, which destroyed soil aggregates while leaving cocoons undamaged. The number of cocoons was noted for each replicate. Cocoons were then transferred onto Petri-dishes on wet filter paper. The Petri-dishes were incubated at 20 ◦ C for a total of 102 days according to Lowe and Butt (2005). During this time the number of hatched cocoons of each replicate was recorded; juveniles were subsequently removed from the Petri-dishes and weighed. Quantification of cyanophycin in soil and casts Earthworm casts from the surface and soil samples from the top layer of the microcosms were collected and were immediately air-dried for subsequent cyanophycin analyses. Cyanophycin content was analysed according to Hühns et al. (2008). Briefly, 100 mg (dr.w.) of soil or cast samples was weighed in a 2 mL tube, mixed with 1 mL Tris-buffer with a vortexer and then shaken horizontally for 30 min at 220 rpm. Afterwards, samples were centrifuged at 15,000 rpm for 10 min. Pellets were extracted with 1 mL 0.1 M
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Table 1 Cyanophycin content from the tubers of different potato (Solanum tuberosum) cultivars and transgenic events used in the current experiment. Results from mean (±S.E.) cyanophycin contents (g mL−1 ) of earthworm casts and soil from different potato treatments after 80 days of the experiment are also shown. Significant differences between the treatments are shown using different letters (ANOVA and Games–Howell-test; p < 0.05, n = 5). Potato treatment
Notation
Albatros B33-PsbY-cphATe – event 39 Albatros B33-PsbY-cphATe – event 16 Albatros PsbY-cphATe – event 12 Albatros PsbY-cphATe – event 23 Albatros, near isogenic control Desiree, comparative cultivar Albatros PsbY-cphATe – event 12 a b
Cyanophycin content
A-E39 A-E16 A-E12 A-E23 A-NIC Desirée A-E12-Ca
Potatoes (% dr.w.)
Cast (g mL−1 )
Soil (g mL−1 )
7.5 6.5 0.9 0.8 0 0 0.9
4.02 ± 0.15a 3.82 ± 0.53a 5.22 ± 0.54b 3.72 ± 0.22a
3.41 3.39 3.56 3.09
b
0.36b 0.24b 0.39b 0.28a
b
b
No casts
± ± ± ±
b
2.86 ± 0.12a
Treatment A-E12 without earthworms. Below detection limit.
HCl, vortexed, also horizontally shaken at 220 rpm for 60 min, and then again centrifuged at 14,000 rpm for at least 10 min. Subsequently, 800 L of the supernatant was poured into a new 2 mL tube and mixed (vortexed) with 200 L of Coomassie Brillant Blue G-250 dye (Roti-Quant; Carl Roth, Germany) to produce a blue-coloured protein solution which could be measured photometrically with a UV-VIS photometer at OD 595 nm (Shimadzu UV 1650-PC, Duisburg, Germany). The remaining dye incubated for 5 min. A cyanophycin stock solution of 0.5 L mL−1 was used for calibration standards. Each sample, soil and casts, was measured in triplicate. Statistical analysis All results are presented as means (±S.E.) of five replicates. For comparisons between the different potato events (treatments) different statistical procedures were performed. If data were normally distributed an ANOVA was performed while non-normally distributed data was analysed using a non-parametric Kruskal–Wallis H-test. A Games–Howell test was used as a post-hoc test in combination with an ANOVA because data showed heterogeneous variances according to the Levene test. If the data set was non-normally distributed with heterogeneous variances, a nonparametric Kruskal–Wallis H-test in combination with a post-hoc Nemenyi test was used. Differences between the two treatment groups, transgenic and non-transgenic potatoes, were calculated using the non-parametric Mann–Whitney U-test. Results
non-transgenic, near-isogenic, and the comparative treatments, this variation was much lower and ranged from 0.95 to 1.66% or 1.37 to 1.53% when A-NIC or Desirée was used, respectively. However, on average, there was a significant difference in potato tuber loss between the treatments with cyanophycin potato tubers applied, compared with the controls. The mean loss per day varied between 2.3 and 4.8% compared with 1.3 and 1.5%, respectively. In the A-E12C treatment without earthworms, mean potato tuber loss was 0.9% (Table 2), which was significantly lower compared to the respective A-E12 treatment. Mean earthworm biomass (fr.wt.) of the various treatments varied from 5.14 to 6.17 g at the start of the experiment and thus, the differences in biomass between the treatments were quite small. The same was found at the end of the experiment after 80 days (Table 3). However, on average, earthworm mean biomass increased by 0.22–0.54 g or relatively about 3.70–11.58% (Table 3). Reproduction During the experimental period the mean number of cocoons in the various treatments ranged between 2.6 in the A-NIC treatment and 6.2 in A-E16 (Table 3). The highest number of cocoons (31) was found in the A-E16 treatment, followed by the A-E23 (21) and AE12 (20) treatments. Differences between the treatments were not significant (p > 0.05). During the incubation time between 7 and 15 juveniles per treatment hatched; a mean of 45 and 83% relative to total cocoons per treatment (Table 3). The mean weight of hatchlings varied between 51.5 and 63.6 mg ind.−1 . Differences between treatments were not significant (p > 0.05).
Potato tuber loss and earthworm biomass Potato tuber loss from the surface within the cyanophycin treatments varied markedly, for example, between 1.17 and 7.10% in A-E39 or 1.67 and 7.65% in the A-E23 treatments (Table 2). In the Table 2 Loss of potato tuber residues (n = 5) from the soil surface. Differences after 60–65 days within transgenic and non-transgenic potatoes as well as between treatments A-E12 and A-E12-C were significantly different (p < 0.05; Mann–Whitney U-test). For abbreviations see Table 1. Potato treatment
A-E39 A-E16 A-E12 A-E23 A-NIC Desirée A-E12-Ca a
Surface loss (5 d−1 ) Min
Max
Mean (±S.E.)
1.17 3.29 1.50 1.67 0.95 1.37 0.82
7.10 7.61 3.34 7.65 1.66 1.53 0.99
3.6 4.6 2.3 4.8 1.3 1.5 0.9
Treatment A-E12 without earthworms.
± ± ± ± ± ± ±
2.4 1.8 0.9 2.6 0.3 0.1 0.1
Quantification of cyanophycin in different compartments At the end of the experiment the amounts of cyanophycin in soil samples showed significant differences between the A-E12 and the A-E12-C treatment and all other treatments (Table 1). Due to a lack of earthworms only soil samples were taken from the latter treatment. Specifically, in cast samples no significant difference within the treatments were found except for significantly higher cyanophycin content in casts of the A-E12 treatment compared to all other treatments (Table 1). Unfortunately, the cyanophycin content of the various potato tuber residues could not be analysed at the end of the experiment, since it was not possible to sample residual material without adhering soil or cast material except in the A-E12-C treatment without earthworms. Compared to the initial cyanophycin content of the potato tubers in the A-E12 treatment (0.9% dr.w.), cyanophycin content of tubers in earthworm-free treatments was 0.15% dr.w. at the end of the experiment (data not shown), representing a relative decrease in cyanophycin content of 84%.
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Table 3 Mean (x ± S.E.; n = 10) earthworm biomass (g fresh mass) at the beginning of the experiment and after 80 days (end), and absolute and percentage difference (x ± S.E.) of earthworm biomass during the experimental period. Additionally, sum and mean numbers (x ± S.E.) of cocoons (after 80 days) and hatchlings (after further 102 days cocoon incubation) per treatment, and mean weight of juveniles after emergence are presented. Differences within the treatments were not significant (p > 0.05; Kruskal–Wallis H-test). For abbreviations see Table 1. A-E39 Earthworm biomass Start (day 0) End (day 80) Diff. [g] Diff. [%] Life history parameters cocoons Mean no. cocoons hatchlings Hatchlings [%] weight of juveniles [mg]
5.75 ± 0.73 6.03 ± 0.89 0.28 ± 0.42 4.79 ± 7.19 17 3.4 (±3.3) 9 52.9 56.3 (±17.4)
A-E16 5.64 ± 0.66 6.13 ± 0.48 0.49 ± 0.35 9.23 ± 7.11 31 6.2 (±2.5) 14 45.2 59.3 (±4.1)
Discussion The effects of residues from transgenic potato tubers with various contents of cyanophycin on the growth, reproduction and activity of earthworms in soil were investigated in a microcosm experiment over 80 days. Overall, mean daily potato tuber loss varied between 2.3 and 4.8% in the transgenic potato treatments compared to 1.3 and 1.5% in the non-transgenic treatments indicating that the loss of genetically modified potatoes was significantly higher (p < 0.05). These data show that mean potato tuber loss per day was nearly 1.5- to 3- fold higher in the treatments with cyanophycin residues. Decomposition of organic residues mainly depends on the palatability of the organic resource for decomposers (Satchell and Lowe 1967; Ashili et al. 2007). For example, the C-to-N ratio of the investigated potato tubers varied between 6.5 and 8.1 with no significant difference between the tubers of the different potato events. Albatros tubers are rich in starch and protein compared to Desirée tubers (data not shown). However, these two compounds had no effect on potato tuber loss within the two lines. Moreover, earthworms preferentially used cyanophycin producing potato tubers as a resource compared to the isogenic control events. Moreover mean potato tuber loss was only 0.9% in the A-E12-C treatment without earthworms. It is hypothesized, that this result indicates a considerable decay of the potato residues through microbial activity. In an experiment with transgenic maize (Bt-maize) it was also shown that microorganisms contributed significantly to surface maize litter loss (Emmerling et al. 2011). When compared to a blanc A-E12 and A-E12-C treatments it can be estimated, that the proportion of earthworms in decomposition of potato tuber residues was approximately 61%. During the experiment, earthworm biomass increased on average by 0.22–0.54 g or relatively by 3.70–11.58%, indicating that the experimental conditions were favorable for the earthworms in all treatments (Fründ et al. 2010). Thus, differences between treatments with transgenic and non-transgenic potato lines were not significant. Cocoon production of earthworms depends on a complete set of environmental life conditions, such as temperature, soil moisture content and food supply (Curry and Bolger 1984). In a laboratory experiment Butt (1991) and Grigoropoulou et al. (2007) observed the production of one cocoon per week per individual, predicting a number of 11 cocoons in 80 days. In the present study the number of cocoons was lower varying between 2.6 and 6.2 on average per treatment over 80 days. This could be due to differences in food supply (leaf litter vs. potato tuber). However, our reproductive results are comparable to those found by Butt et al. (1994). Another factor, which could be responsible for cocoon production, is the C-to-N ratio of the food resource (Meinhardt 1973; Lofs-Holmin
A-E12 5.29 ± 1.04 5.53 ± 1.14 0.24 ± 0.63 4.95 ± 11.9 20 4.0 (±2.4) 14 70.0 56.3 (±8.7)
A-E23 6.17 ± 1.30 6.39 ± 1.37 0.22 ± 0.39 3.70 ± 6.39 21 4.2 (±3.1) 12 57.1 63.6 (±11.1)
A-NIC 5.14 ± 0.98 5.68 ± 0.95 0.54 ± 0.56 11.58 ± 11.77 13 2.6 (±1.8) 7 53.9 58.2 (±0.8)
Desirée 5.71 ± 0.85 5.94 ± 0.80 0.23 ± 0.61 4.67 ± 10.89 18 3.6 (±2.8) 15 83.3 51.5 (±14.0)
1982). According to Suthar (2007) cocoon production of epigeic earthworm species (Eudrilus eugeniae, Perionyx excavatus, Perionyx sansibaricus) increased when C-to-N ratio of the food resource was narrow. Obviously, in the present study cocoon production was not affected by the cyanophycin or protein content since the highest number of cocoons was found in the A-L16 treatment which had the second highest cyanophycin content. The rate of hatched cocoons in the treatments varied between 45 and 83% of the number of cocoons, which was comparable to an investigation of Grigoropoulou et al. (2007), where the mean hatchling survival rate was 62%. Interestingly, in the A-E16 treatment, where the number of cocoons was highest, the rate of hatchability was lowest. However, in the A-NIC treatment, where the number of cocoons was lowest, the number of hatched cocoons fell within the mean range of all treatments. Mean weight of hatchlings also showed no significant difference between the treatments. Consequently, there seems to be little correlation between the cyanophycin content of potato tubers with the number of cocoons, the rate of hatchability or the fresh weight of juveniles. During the experiment the cyanophycin content of the potato tubers decreased by 84% which was observed in the A-E12 treatment. The remaining cyanophycin content was analysed using a new method according to Hühns et al. (2008). Due to a calibration with cyanophycin it was also possible to quantify the cyanophycin content in soil and cast samples. In the A-E12 treatment, the amount of cyanophycin in soil was higher compared to the same treatment without earthworms. This might be evidence that the cyanophycin content of the upper soil layer was enhanced by earthworm burrowing activity. The overall decrease of the cyanophycin content during the experiment can also be attributed to microbial activity. Moreover, all five replicates of the A-E12-C treatment showed an intense development of mildew immediately after exposure of the tuber residues on the soil surface with the mildew increasing in mass during the experiment. By contrast, in the A-E12 treatment and in all other earthworm treatments, a comparable initial development of mildew was found, respectively, but earthworms fed very quickly and exclusively on the mildew so that it was no longer found after several days and until the end of the experiment. According to Edwards and Fletcher (1988), Brown (1995), Bonkowski et al. (2000) and Butenschön et al. (2007) fungi serve as an important food source for earthworms and significantly affect the diet of earthworms, especially of detritivorous earthworms, like L. terrestris (Moody et al. 1995). Although not quantified, no evidence was found that mildew infection differed within the various transgenic potato lines. Finally, cyanophycin content in casts of the L-E12 treatment was significantly higher relative to the other treatments, but the reasons underlying these results remain unclear.
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