Environ. Sci. Technol. 2009, 43, 928–933
Reproductive Consequences of Paternal Genotoxin Exposure in Marine Invertebrates CERI LEWIS* AND TAMARA GALLOWAY School of Biosciences, Hatherley Laboratories, University of Exeter, Prince of Wales Road, Exeter, United Kingdom, ES4 4PS
Received August 07, 2008. Revised manuscript received December 01, 2008. Accepted December 04, 2008.
Chemicals with the potential to damage DNA are increasingly present in the marine environment; yet our understanding of the long-term consequences of DNA damage for populations remains limited. We explore the impact of paternal genotoxin exposure on the reproductive biology of two ecologically important free-spawning marine invertebrates: the polychaete Arenicola marina and the mussel Mytilus edulis. Males were exposed in vivo for 72 h to methyl methanesulfonate and benzo(a)pyrene and the impact on somatic cells and sperm assessed using the Comet assay. A strong correlation between DNA damage in somatic cells and sperm was observed after 24 h exposure (P < 0.001). Recovery in sperm was significantly lower than in coelomocytes after 72 h. The fertilization success of DNAdamaged sperm was unaffected, but a significant percentage of embryos derived from sperm with induced DNA damage exhibited severe developmental abnormalities within 24 h of fertilization with potential long-term consequences for population success. Further research is required to determine the mechanism by which paternal DNA damage causes disruption of development at this early stage.
Introduction The aquatic environment is the ultimate recipient of an increasing range of anthropogenic contaminants, one-third of which are estimated to have genotoxic properties (1-4). Almost a billion pounds of chemicals registered as carcinogenic are discharged each year in the United States alone, and while great progress has been made in understanding the implications of genotoxin exposure to human health, there is a huge gap in our understanding of the impacts of such exposures on aquatic species. Marine invertebrates express similar types of chemically induced damage to their DNA and chromosomes as those recorded in higher organisms (5), and there is now a wealth of laboratory and fieldbased evidence (6, 7) that demonstrates aquatic exposures to environmental genotoxins induce DNA damage in somatic cells of fish and invertebrate species (8-12). Various studies have described species- and cell-specific genotoxic responses of different aquatic invertebrates (12, 13) identifying both DNA strand breaks and cytogenetic damage in the somatic cells of organisms inhabiting contaminated sites (8, 10, 11). Much less is known of the implications of this damage for the long-term survival of affected populations. DNA damage to somatic cells can cause dysfunction and ultimately lead to cell death with obvious consequences for * Corresponding author phone: +44 (0)1392 264672; fax: 0(44) 1392 263700; e-mail:
[email protected]. 928
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the health of the individual. When investigating the impacts of contaminants on populations, however, it is not the health of individuals but rather the impact on population fitness (e.g., growth, survival, and reproduction) of functionally important species that is of concern (1-3). Our understanding of the ecological significance of environmentally induced DNA damage in somatic cells for natural populations is currently very limited. In contrast, genotoxic damage in germ cells has the potential to be passed on to future generations if not repaired. Recent human health studies reveal the male reproductive system is a major target of carcinogenic and genotoxic chemicals (14) and that the integrity of sperm DNA can be adversely affected by exposure to ubiquitous pollutants such as phthalates (15) and polycyclic aromatic hydrocarbons (PAHs) (16). Sperm are potentially more susceptible to contaminant-induced damage than somatic cells or oocytes since sperm are generally considered to have limited capacity for DNA repair and antioxidant defense (14, 17). There is currently no information available regarding the DNA repair capacity of spermatozoa and only very sparse information regarding environmentally induced DNA damage in spermatozoa for any marine invertebrate species (12, 18). Our preliminary investigations using the Polychaete Arenicola marina (12) revealed acute in vitro exposures of spawned spermatozoa to the direct-acting mutagen methyl methanesulfonate (MMS) resulting in significant damage to the sperm DNA. Whether paternal in vivo exposure results in similar levels of DNA damage and the consequences of this damage for the reproductive biology of animals is not currently known. Understanding the potential for such changes in marine invertebrates is essential given their important role in the structure and function of marine ecosystems. Here, we test the hypotheses that (i) paternal genotoxin exposure induces DNA damage in spermatozoa and (ii) there are consequences of sperm DNA damage on fertilization dynamics and development processes in subsequent embryos. We study two functionally important freespawning marine invertebrates: the polychaete worm Arenicola marina, which inhabits estuarine sediments where anthropogenic contamination are prevalent, and the common mussel Mytilus edulis, a filter feeder capable of concentrating contaminants from the surrounding water.
Methodology Collection and Maintenance of Animals. Adult specimens of the polychaete worm Arenicola marina were collected from Mothercombe estuary, South Devon, U.K (50°,18′′,41 N, 3°,56′′,45 W) during October 2006. Specimens were assessed for maturity and sex by taking coelomic samples using a 1 mL syringe fitted with a 21 g hypodermic needle and observing the samples under a compound microscope. Adult Mytilus edulis specimens (50-70 mm shell length) were collected from Port Quin, North Cornwall, U.K. (50°,35′′,20 N, 4°,52′′,04 W) during July 2007. Sex was determined at spawning since attempts to take samples induced spawning prematurely. Three times as many mussels as required were therefore exposed in order to obtain enough males. All animals were kept in well-aerated filtered (0.2 µm) seawater (FSW) in a large glass aquarium tanks at 12 °C prior to the experiments and fed an Isochrysis solution every other day. Chemicals. Methyl methanesulfonate (MMS), CAS number 66-27-3, and benzo(a)pyrene (BaP), CAS number 5032-8, were purchased from Sigma Aldrich U.K. In Vitro Exposures. Spawning was induced in mature Arenicola marina males (n ) 5) by injecting 8,11,14-eicosatrienoic acid (13 µg g-1 of worm) directly into their coeloms 10.1021/es802215d CCC: $40.75
2009 American Chemical Society
Published on Web 01/05/2009
(19). Spawning followed approximately 1 h after injection. Sperm was collected ‘dry’ as it was extruded to prevent premature activation and stored on ice until use. Samples were diluted to 105 sperm mL-1 for in vitro exposure using FSW and exposed to 52 mg L-1 MMS for 1 and 24 h in microcentrifuge tubes on ice (concentration used in previous studies 12, 20). Sperm were then washed in chilled phosphate buffer solution (PBS) three times using centrifugation at 78g and assessed for DNA damage using the Comet assay (described below). MMS is a direct acting genotoxin which induces DNA damage via methylation without any production of reactive oxygen species (ROS), which removes any confounding effects of ROS on sperm (e.g., lipid peroxidation of membranes). In Vivo Exposures. Exposures were run during November for Arenicola marina and August for Mytilus edulis to fall within the natural breeding seasons for both species. Males were exposed to 18, 32, and 52 mg L-1 MMS for 24 h (Arenicola marina only) and 72 h prior to induction of spawning together with a FSW control. In addition Mytilus edulis males were exposed to the indirect acting (and environmentally relevant) mutagen BaP at 0.01, 0.1, and 1.0 mg L-1 (concentrations used in previous studies (12)). BaP does not dissolve readily in seawater, so dimethyl sulfoxide (DMSO) was used to prepare stock solutions before addition to seawater and a solvent control (0.01%) included. Exposures were conducted in replicate (n ) 5) in individual 2 L glass beakers (i.e., one specimen per beaker). Aeration was provided, and animals were maintained at 12 °C and a 12:12 light:dark photoperiod throughout exposure, after which males were transferred to clean FSW and immediately induced to spawn. Mytilus edulis males were induced to spawn by gentle shaking in warm (25 °C) FSW. Sperm was collected as it was spawned and stored in microcentrifuge tubes on ice until use in the Comet assay and fertilization experiments. Following complete spawning somatic cell samples were also collected from each male using a 1 mL syringe fitted with a 21 g hypodermic needle, inserted into the posterior region of the coelom in Arenicola marina and the adductor muscle of Mytilus edulis. To prevent cell clumping Arenicola marina coelomocytes were collected into chilled anticoagulant at pH 7.3 as previously described (12) and Mytilus edulis hemocytes into chilled PBS. Comet Assay. All samples (sperm and somatic) were checked for cell viability prior to use with Eosin Y staining (all samples > 90% viability). The Comet assay was used to measure DNA strand breaks in sperm from all exposures and somatic cells from the in vivo experiments using previously described methods (12). Samples were centrifuged (somatic cells at 78g, sperm at 7826g) for 4 min and the excess fluid removed. Cell concentrate was gently mixed with 1% low melting point agarose (heated to 37 °C) and dropped onto slides previously coated with 1% normal melting point agarose. The Comet assay was performed using alkaline conditions at 5 °C. Briefly, 2 h lysis for sperm or 1 h lysis for somatic cells, followed by 45 min denaturation in electrophoresis buffer (0.3 M NaOH and 1 mM EDTA), then electrophoresis for 30 min at 25 V and 300 mA followed by neutralization. Slides were stained with 20 mg L-1 ethidium bromide and examined using a fluorescent microscope (excitation ) 420-490 nm; emission ) 520 nm). The percentage of DNA in the comet tail (caused by DNA strand breaks) in one hundred cells per preparation was quantified using Kinetic V COMET Software. Fertilizations. Female Arenicola marina were induced to spawn by injecting homogenized prostomia from five donor females (method previously described (19)) into five gravid (unexposed) females the night before they were needed and then leaving them overnight in FSW at 12 °C. For Mytilus edulis five unexposed females were induced to spawn by
gentle shaking and warm (25 °C) FSW. Spawned oocytes were immediately collected using a pipet, washed with FSW, and stored on ice until use. Fertilizations were conducted using previously developed methods (21). Sperm concentrations for each male (from 72 h exposures) and oocyte densities for each female were determined (21). Optimum sperm concentrations and sperm: oocyte ratio for fertilization success in Mytilus edulis (22) and Arenicola marina (23) of 106 sperm/mL and 104 sperm/ oocyte were used. All sperm samples were checked for viability prior to use, and only samples with >90% active sperm were used. A volume equivalent to 1000 oocytes (pooled from 5 unexposed females) was pipetted into experimental Petri dishes containing 10 mL of FSW at 12 °C (one dish per male plus a no-sperm control per treatment). A volume equivalent to 107 sperm from each exposed (or control) male was added to each Petri dish, giving a ratio of 104 sperm per oocyte and a final concentration of 106 sperm/ mL. Petri dishes were then placed into environmental cabinets at 12 °C for 10 min, removed briefly while oocytes were carefully washed with FSW at 12 °C, and then returned to the environmental cabinets overnight. Twenty-four hours post-fertilization subsamples of oocytes/ embryos were taken from each dish, fixed in 5% ethanol in seawater, and observed under a compound microscope. Fifty oocytes/embryos per replicate were scored as fertilized/not fertilized according to the presence or absence of a fertilization membrane (21). Development was assessed visually, the developmental stage was recorded, and embryos showing signs of irregular cleavage, incomplete blastula development, necrosis, or discoloration were recorded as ‘abnormal’. Recovery Experiment. Mature Mytilus edulis males were exposed to 52 mg L-1 MMS for a period of 3 days. At the end of the exposure period one-half of the mussels were transferred to FSW and induced to spawn while the other half were transferred to FSW to ‘recover’ for a further 3 days. At the end of the recovery period these males were also induced to spawn. Following complete spawning hemolymph samples were also collected from each male as described above. The Comet assay was then performed on all sperm and hemocytes samples. Statistics. Percentage fertilization data was normalized using the arcsine transformation to enable the use of parametric statistics. Bartlett’s and Levene’s tests were used to confirm homogeneity of variance between treatments and then one-way ANOVAs performed to compare treatments. A posteriori analysis was performed using Fisher’s Pairwise Comparisons using the statistical package Minitab Inc. A t test was used for the recovery experiment.
Results In Vitro Exposure. In vitro exposures of freshly spawned Arenicola marina spermatozoa to the direct acting genotoxin MMS (Figure 1) resulted in significant and dose-dependent DNA damage measured as DNA strand breaks using the Comet assay (one-way ANOVA; 1 h, P < 0.001; 24 h, P < 0.001). Linear dose-response relationships between sperm DNA damage and MMS concentration were measured for both time points (1 h, R2 ) 78.4%, P < 0.001; 24 h, R2 ) 81.9%, P < 0.001). The amount of DNA damage measured after 24 h exposure was slightly (not significant) higher than that measured after 1 h exposure in all treatments. In Vivo Exposures. In vivo exposure of Arenicola marina males to MMS for 3 days prior to spawning induced significant DNA damage in their spermatozoa (one-way ANOVA P < 0.001, Figure 1). Regression analysis revealed a significant linear relationship between DNA damage and MMS concentration (P < 0.001, R2 ) 62.5%). The percentage of tail DNA in sperm after 3 days exposure was higher than after 1 and 24 h exposures, but this was not significant (P ) 0.153). VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. DNA damage in Arenicola marina spermatozoa (% DNA in Comet tail) after 1 h, 24 h (in vitro), and 3 day (paternal in vivo) exposure periods to MMS (data as mean ( S.E., n ) 5). FSW ) filtered seawater control. (*) Significantly different from control. (**) Significantly different from lowest treatment concentration.
FIGURE 3. Relationship between the percentage DNA damage measured in somatic cells and spermatozoa for individual male (a) Arenicola marina and (b) Mytilus edulis.
FIGURE 2. DNA damage in Mytilus edulis spermatozoa (% DNA in Comet tail) from males exposed in vivo for 72 h to (A) MMS (n ) 5) and (B) BaP (n ) 5) (data as mean ( S.E.). FSW ) filtered seawater control. (*) Significantly different from control. (**) Significantly different from lowest treatment concentration. A similar result was recorded for Mytilus edulis (Figure 2a and 2b) with a significant increase in DNA damage measured in sperm in both the MMS (one-way ANOVA P < 0.01) and BaP exposures (one-way ANOVA P < 0.01). Significant dose responses in sperm DNA damage were measured for MMS (linear relationship P < 0.001, R2 ) 89.8%) and BaP exposures (second-order polynomial relationship P < 0.01, R2 ) 84.3%). In Arenicola marina males exposed to MMS a significant relationship between DNA damage measured in sperm and coelomocytes of the same individuals was observed after 24 and 72 h of exposure (Figure 3a; 24 h, Pearson’s correlation 930
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coefficient ) 0.747, P < 0.001; 72 h, Pearson’s correlation coefficient ) 0.885, P < 0.001). No such relationship was observed after 1 h exposure (Pearson’s correlation coefficient ) 0.321, P ) 0.243). In Mytilus edulis (Figure 3b) a significant correlation between DNA damage in hemocytes and sperm (Pearson’s correlation coefficient ) 0.929, P < 0.001) was recorded after 72 h exposure. Fertilization and Post-fertilization Development. In Arenicola marina fertilization success was not significantly affected by DNA damage in sperm induced through paternal exposure to MMS (Figure 4a, one-way ANOVA P ) 0.104); however, post-fertilization development was significantly disrupted (Figure 4b, one-way ANOVA P < 0.01). Similarly, in Mytilus edulis no effect of paternal exposure to MMS was observed on fertilization success (Figure 4c, one-way ANOVA P ) 0.655) but a significant disruption of post-fertilization development was recorded (Figure 4d, one-way ANOVA P < 0.01). The same results were observed after paternal exposure to BaP (Figure 4e, one-way ANOVA P ) 0.806; Figure 4f oneway ANOVA P < 0.01). Abnormal embryos comprised embryos exhibiting irregular cleavage, incomplete blastula development, necrosis, or discoloration. Strong negative correlations between the percentage of normally developing embryos and sperm DNA damage were observed for both Arenicola marina (Figure 5a, Pearson’s correlation coefficient ) -0.750, P < 0.001) and Mytilus edulis (Figure 5b, Pearson’s correlation coefficient ) -0.881, P ) 0.002). Recovery Experiment. In the recovery experiment using Mytilus edulis (Figure 6) no significant difference in DNA damage was observed between hemocytes and spermatozoa of either unexposed males (t test, P ) 0.271) or males exposed in vivo to 52 mg L-1 MMS for 72 h prior to spawning (t test, P ) 0.291). In Mytilus edulis allowed to recover in FSW for 72 h after exposure and prior to spawning hemocytes showed significantly better recovery from DNA damage than spermatozoa (t test, P < 0.001).
FIGURE 4. Effects of paternal genotoxin exposure on fertilization success and postfertilization development in Arenicola marina exposed to MMS (A and B) and Mytilus edulis exposed to MMS (C and D) and BaP (E and F). Data as mean ( SE (n ) 5). FSW ) filtered seawater control. (*) Significantly different from control. (**) Significantly different from lowest treatment concentration.
Discussion This work clearly demonstrates that marine invertebrate spermatozoa are susceptible to DNA damage from paternal genotoxin exposures and exhibit similar responses to genotoxic insult as those previously described for somatic cells and human and mammalian sperm. Both in vitro exposures of spawned sperm and in vivo exposures of male Arenicola marina to the direct acting genotoxin MMS resulted in significant damage to their sperm DNA, measured as DNA strand breaks, which increased with dose. A similar result was observed in sperm from Mytilus edulis males exposed in vivo to MMS and BaP. This adds to the growing evidence from human biomonitoring studies that exposures to environmental contaminants such as polycyclic aromatic hydrocarbons (16) induce DNA damage in sperm. In our experiments paternal genotoxin exposure had no impact on fertilization success in either Arenicola marina or Mytilus edulis, suggesting that swimming ability and behavior of sperm is not affected by DNA damage. Previous studies have investigated the impacts of irradiation-induced DNA damage in bovine sperm on the fertilization reaction, and similarly found sperm carrying DNA damage were capable of normal fertilization and did not affect the first cleavage stages (24). The only existing report of environmentally induced sperm DNA damage in marine invertebrates (18) used samples collected straight from the hemolymph of Mytilus edulis, without inducing the animals to spawn, using an unreliable and unvalidated method relying on nuclei size alone to
distinguish between somatic cells and sperm in their Comet analysis (18). Using freshly spawned sperm our study clearly and unambiguously demonstrates induction of DNA damage in sperm from paternal exposures in marine invertebrates. There are implications associated with sperm DNA damage and the lack of any associated impact on fertilization success that are specific to free spawning marine animals. The marine environment poses particular challenges in terms of sperm function and susceptibility to environmental damage since the majority of larger marine invertebrates reproduce by releasing their sperm (and/or eggs) freely into the water column so that fertilization takes place externally. Invertebrates generally lack the blood-gonad barrier found in mammals, meaning sperm are unprotected and exposed to environmental contaminants not only during spawning but also during their development and maturation. Gametes released freely into a turbulent aquatic environment yield highly unpredictable distributions of sperm and egg concentrations (25), and although on average sperm will be more concentrated than eggs, evidence strongly suggests that population dynamics are often sperm limited (25, 26) with fertilization success rarely 100% even under optimal conditions (23). This lack of sperm competition means that even ‘poor quality’ sperm, such as one carrying DNA damage, may still fertilize an oocyte under natural conditions, particularly if swimming behavior is not affected. A highly significant negative relationship was revealed between DNA damage in sperm and the percentage of embryos developing VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Linking the amount of DNA damage in sperm with the resulting percentage of abnormal development observed in (A) Arenicola marina and (B) Mytilus edulis.
FIGURE 6. Recovery experiment in Mytilus edulis comparing DNA recovery in somatic cells and spermatozoa after exposure to 52 mg L-1 MMS. FSW ) filtered seawater control. (*) Significant difference between the cell types. normally for both Arenicola marina and Mytilus edulis. Embryos derived from sperm carrying DNA damage exhibited highly irregular cleavage patterns, incomplete blastula development, necrosis, and discoloration. Marine invertebrates generally produce very high numbers of larvae in any one breeding episode, but mortality and competition is high and form strong selective pressures on their life history evolution (27). Larval quality is known to be a major factor controlling intra- and interspecific competitive interactions 932
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at settlement and therefore population structure for both polychaetes (28) and mussels (29). Hence, any impacts of pollutant exposure on larval quality have the potential to alter population structure. Disruption of early postfertilization development due to sperm DNA damage is particularly interesting since the paternal genome is not widely considered to have a role in controlling early cleavage. Oocytes contain a stockpile of maternally derived mRNAs which govern embryogenesis through cleavage to the blastula stage. Following formation of the blastula, zygotic gene transcription is activated, which carries the embryo through the rest of embryogenesis. Disruption of development observed here was observed within the first 24 h post-fertilization. In both species studied the majority of embryos reach the blastula stage within this 24 h period; therefore, the underlying mechanism by which paternal DNA damage causes disruption of development at this early stage cannot be determined from these experiments and requires further investigation. Use of MMS as a positive control means that the only affect of exposure on sperm will have been through direct damage to the DNA via methylation since MMS does not produce ROS, adding to the evidence that the DNA damage measured in the sperm caused the observed developmental abnormalities. A significant correlation between DNA damage in somatic cells and DNA damage in sperm for individuals of both species was also observed. Sperm showed lower levels of DNA damage than coelomocytes from the same males for Arenicola marina, although this difference in damage levels between the two cell types decreased with increased exposure time. The tightly compacted nature of sperm chromatin may offer a certain level of protection; however, damage was measured as DNA strand breaks, which will transiently be present during DNA repair via base or nucleotide excision. The lower levels of DNA damage measured in sperm compared to coelomocytes could therefore be related to a lack of DNA repair enzymes in mature spermatozoa, i.e., not all of the DNA adducts present are converted to strand breaks and so are not measured as Tail DNA. The change in relationship between DNA damage in somatic cells and sperm over exposure time observed in Arenicola marina may also be due to differences in repair capabilities with sperm accumulating damage over time while somatic cells repair damage. Levels of DNA damage increased slightly but not significantly with exposure time in sperm from the lower two exposure concentrations. No difference was measured with time at the highest MMS treatment, possibly due to apoptotic responses in sperm with higher levels of DNA damage. In Mytilus edulis similar levels of DNA damage were recorded in sperm and somatic cells, but measurements were only taken at one time point, and differences may accumulate over time depending on differences in any repair capabilities of the cells. Sperm are considered to be particularly susceptible to oxidative damage due to the abundance of polyunsaturated fatty acids acting as substrates for ROS (29) and a perceived lack of DNA repair mechanisms (17, 29, 30). Mammalian studies have revealed that while the stem cells that produce sperm have highly effective repair as developing spermatids undergo meiosis their capacity for DNA repair is reduced and their ability to respond to DNA damage by undergoing programmed cell death is progressively lost (14), although some evidence of base excision repair in the sperm of rats has been presented (31). Similar studies have also demonstrated that paternal germ cells are more susceptible to contaminant-induced mutation than maternal germ cells (32). Nothing is currently known regarding the DNA repair capabilities of invertebrate spermatozoa. Our results for Mytilus edulis demonstrate significant recovery in sperm DNA integrity, suggesting some repair capability is present; however, this was significantly reduced compared to somatic cells.
These experiments used acute exposures to quite high concentrations of two chemicals with well-described mechanisms of genotoxicity. While these concentrations were much higher than would be considered environmentally relevant, the levels of DNA damage induced in the somatic cells from these exposure concentrations have actually been measured in somatic cells of natural populations, for example, in a population of the polychaete Nereis virens from a contaminated site in Cornwall, U.K (12). It is therefore vital to determine whether this level of DNA damage to germ cells also occurs in populations from contaminated sites and what the consequences of this damage are for population reproductive success. Additionally, any lack of DNA repair enzymes in spermatozoa would make them far more susceptible to accumulated DNA damage from chronic environmental exposures to much lower concentrations of genotoxins than somatic cells. At present we know very little about the nature of xenobiotic-metabolizing enzymes for spermatozoa for any species and thus the potential that different groups of compounds have for inducing genetic damage is uncertain (14). This work has revealed DNA damage to sperm is not restricted to human and mammalian species but will also affect aquatic animals. The susceptibility of sperm to environmentally induced DNA damage has wide-ranging implications for both human health and the long-term health of natural populations. Further research into the defense and repair capabilities of sperm and the role of paternal DNA damage in the disruption of early developmental processes is vital in order to increase our understanding of the consequences of genotoxic exposures on the reproductive health of all species.
Acknowledgments This research was funded via the EU 6th Framework directive as part of the FACE-iT Project (FP6-2004-Global-3.3.111.3.1), contract 018391. The authors thank Chris Pook, Jo Hagger, and Trevor Worsey for their help and technical support and Awadhesh Jha for his insightful advice. The authors declare that they have no competing financial interests.
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