Abstract: In mammals, the pharmaceutical ibuprofen (IB), a non-steroidal anti-inflammatory drug, primarily functions by reversibly inhibiting the cyclooxygenase (COX) pathway in the synthesis of eicosanoids (e.g. prostaglandins). Previous studies suggest that IB may act in a similar manner to interrupt production of eicosanoids reducing reproduction in the model crustacean Daphnia magna. On this basis withdrawal of IB should lead to the recovery of D. magna reproduction. Here we test whether the effect of IB is reversible in D. magna, as it is in mammals, by observing reproduction recovery following chronic exposure. D. magna (5-days old) were exposed to a range of IB concentrations (0, 20, 40 and 80 mg l(-1)) for 10 days followed by a 10 day recovery period in uncontaminated water. During the exposure period, individuals exposed to higher concentrations produced significantly fewer offspring. Thereafter, IB-stressed individuals produced offspring faster during recovery, having similar average population growth rates (PGR) (1.15-1.28) to controls by the end of the test. It appears that maternal daphnids are susceptible to IB during egg maturation. This is the first recorded recovery of reproduction in aquatic invertebrates that suffered reproductive inhibition during chronic exposure to a chemical stressor. Our results suggest a possible theory behind the compensatory fecundity that we referred to as 'catch-up reproduction'.
Abstract: Background
Ibuprofen and other non-steroidal anti-inflammatory drugs (NSAID) have been designed to interrupt eicosanoid metabolism in mammals, but little is known of how they affect non-target organisms. Here we report a systems biology study simultaneously describing the transcriptomic and phenotypic stress responses of the model crustacean Daphnia magna following exposure to ibuprofen.
Results
Our results show intriguing similarities in the mode of action of ibuprofen between vertebrates and invertebrates, and suggest that ibuprofen has a targeted impact on reproduction at the molecular, organismal and population level in daphnids. Microarray expression and temporal real-time quantitative PCR profiles of key genes suggest early ibuprofen interruption of crustacean eicosanoid metabolism, which appears to disrupt signal transduction affecting juvenile hormone metabolism and oogenesis.
Conclusions
Combining molecular and organismal stress responses provides a guide to possible chronic consequences of environmental stress to population health. This could improve current environmental risk assessment by providing an early indication of the need for higher tier testing. Our study demonstrates the advantages of a systems approach to stress ecology, in which Daphnia will likely play a major role.
Abstract: The potential reproductive value of arbuscular mycorrhizal fungi (Glomus intraradices and Glomus invermaium), root pathogenic fungi (Rhizoctonia solani and Fusarium culmorum) and saprotrophic fungi (Penicillium hordei and Trichoderma harzianum) were examined for the collembolans Folsomia candida Willem and Folsomia fimetaria L. Dried baker's yeast (Saccharomyces cerevisiae) was used as a reference standard food in laboratory cultures. Collembolan performance was determined as final size, fecundity and population growth rate after when fed the fungal food sources for 31 days. The mycorrhizal fungi gave the least growth and fecundity compared with the other fungi, but G. intraradices gave good fecundity for F. candida. In terms of growth, Baker's yeast was a high-quality food for both adults and juveniles of both species, but it was a poorer food in terms of fecundity of F. candida. Preference of the fungi in all possible pairwise combinations showed that although F. fimetaria did not perform well on Glomus spp. and F. candida did not grow well on Glomus spp. their preference for these fungi did not reflect this. The highest fecundity was seen with the root pathogen F. culmorum. Different quality indicators such as the C:N ratio of the fungal food sources as well as other biological parameters are discussed in relation to their reproductive value and Collembola preferential feeding.
Abstract: DNA microarrays can be used to measure environmental stress responses. If they are to be predictive of environmental impact, we need to determine if altered gene expression translates into negative impacts on individuals and populations. A large cDNA microarray (14000 spots) was created to measure molecular stress responses to cadmium in Daphnia magna, the most widely used aquatic indicator species, and relate responses to population growth rate (pgr). We used the array to detect differences in the transcription of genes in juvenile D. magna (24 h old) after 24 h exposure to a control and three cadmium concentrations (6, 20, and 37 μg Cd/l). Stress responses at the population level were estimated following a further 8 days exposure. Pgr was approximately linear negative with increasing cadmium concentration over this range. The microarray profile of gene expression in response to acute cadmium exposure begins to provide an overview of the molecular responses of D. magna, especially in relation to growth and development. Of the responding genes, 29% were involved with metabolism including carbohydrate, fat and peptide metabolism, and energy production, 31% were involved with transcription/translation, while 40% of responding genes were associated with cellular processes like growth and moulting, ion transport, and general stress responses (which included oxidative stress). Our production and application of a large Daphnia magna microarray has shown that measured gene responses can be logically linked to the impact of a toxicant such as cadmium on somatic growth and development, and consequently pgr.
Abstract: In this study, we assessed the influence of prey quality and prey biomass during a standardized 3-week test on adult survival and reproductive output of the predatory mite Hypoaspis aculeifer when fed one of six different diets: springtails (Folsomia candida and Folsomia fimetaria), a storage mite (Caloglyphus cf. michaeli), an oligochaete (Enchytraeus crypticus), a nematode (Turbatrix silusiae), and a 1:1:1 mix of F. candida:F. fimetaria:E. crypticus. Our results revealed that a single prey species may be nutritionally sufficient for a 3-week period, as H. aculeifer performed equally well, or better, on a diet based on a 1:1:1 mix of F. candida:F. fimetaria:E. crypticus. However, when fed C. cf. michaeli, H. aculeifer had a poor reproductive output (<200 juveniles) and a reduced survival (60–70%). Thus, investigators should validate their choice of prey prior to testing H. aculeifer performance during toxicant exposure.
Abstract: Genetically modified (GM) crops may affect earthworms either directly through the plant, its root exudates and litter, or indirectly through the agricultural management changes that are associated with GM plant production. In order to investigate such possible effects, we established two field studies of Bacillus thuringiensis (Bt) maize and a glufosinate ammonium tolerant maize and included a reduced tillage (RT) treatment and a conventional tillage (CT) treatment as examples of a likely concomitant change in the agricultural practise. At a French study site at Varois, (Bourgogne), a field grown with the Bt-toxin producing transgenic maize line MON810 was studied for 1 year. At a Danish study site, Foulum (Jutland), 1 year of Bt maize was followed by 2 years of herbicide tolerant (HT) maize. At the French study site, the most prominent effects observed were due to the tillage method where RT significantly reduced the earthworm populations to levels about half of CT. At the Danish study site effects of CT complied with known reduction of anecic earthworms due to this technique and likewise effects of RT were observed for endogeic earthworms. Earthworm populations were diminished with the HT crop, probably due to exposure to the herbicide Basta® during the two consecutive autumn seasons. This study confirms the importance of including the tillage techniques and pesticide usage when evaluating the environmental effects of new agricultural technologies.
Abstract: The non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) is a widely used pharmaceutical that can be found in several freshwater ecosystems. Acute toxicity studies with Daphnia magna suggest that the 48h EC(50) (immobilisation) is 10-100 mg IB/l. However, there are currently no chronic IB toxicity data on arthropod populations, and the aquatic life impacts of such analgesic drugs are still undefined. We performed a 14-day exposure of D. magna to IB as a model compound (concentration range: 0, 20, 40 and 80 mgIBl(-1)) measuring chronic effects on life history traits and population performance. Population growth rate was significantly reduced at all IB concentrations, although survival was only affected at 80 mg IB/l. Reproduction, however, was affected at lower concentrations of IB (14-day EC(50) of 13.4 mg IB/l), and was completely inhibited at the highest test concentration. The results from this study indicate that the long-term crustacean population consequences of a chronic IB exposure at environmentally realistic concentrations (ng/l to ug/l) would most likely be of minor importance. We discuss our results in relation to recent genomic studies, which suggest that the potential mechanism of toxicity in Daphnia is similar to the mode of action in mammals, where IB inhibits eicosanoid biosynthesis.
Abstract: The interactions of genetically modified (GM) crops with soil species and ecosystems is complex, requiring both specific and broad spectrum assessments. In the ECOGEN project we undertook experiments at three scales of increasing complexity, using Bt maize expressing the Cry1Ab protein from Bacillus thuringiensis as an example. Test species were selected for laboratory-scale experiments to represent taxonomic groups that we could also monitor at glasshouse and field scales (e.g., nematodes, protozoa, micro-arthropods, earthworms, and snails). In the laboratory, single species were exposed to purified Cry1Ab protein or to Bt maize leaf powder incorporated into simplified diets under controlled conditions. In the glasshouse, multiple test species and soil microbial communities taken from ECOGEN's field sites were exposed to Bt maize plants growing under glasshouse or mesocosm conditions. In the field, evaluations were conducted on our selected indicator groups over multiple sites and growing seasons. Field evaluation included assessment of effects due to the local environment, crop type, seasonal variation and conventional crop management practice (tillage and pesticide use), which cannot be assessed in the glasshouse. No direct effects of Cry1Ab protein or Bt leaf residues were detected on our laboratory test organisms, but some significant effects were detected in the glasshouse. Total nematode and protozoan numbers increased in field soil under Bt maize relative to conventional maize, whilst microbial community structure and activity were unaffected. Field results for the abundance of nematodes and protozoa showed some negative effects of Bt maize, thus contradicting the glasshouse results. However, these negative results were specific to particular field sites and sampling times and therefore were transient. Taking the overall variation found in maize ecosystems at different sites into account, any negative effects of Bt maize at field scale were judged to be indirect and no greater than the impacts of crop type, tillage and pesticide use. Although the ECOGEN results were not predictive between the three experimental scales, we propose that they have value when used with feedback loops between the scales. This holistic approach can used to address questions raised by results from any level of experimentation and also for putting GM crop risk:benefit into context with current agricultural practices in regionally differing agro-ecosystems.
Abstract: Here we describe a novel, inexpensive and simple method for preserving RNA that reduces handling stress in aquatic invertebrates following ecotoxicogenomic experimentation. The application of the method is based on transcriptomic experiments conducted on Daphnia magna, but may easily be applied on a range of other aquatic organisms of a particular size with e.g. amphipod Gammarus pulex representing an upper size limit. We explain in detail how to apply this new method, named the "Cylindrical Sieve (CS) system", and highlight its advantages and disadvantages.
Abstract: A glasshouse experiment was undertaken to provide baseline data on the variation between conventional maize (Zea mays L.) varieties and genetically modified maize plants expressing the insecticidal Bacillus thuringiensis protein (Bt, Cry1Ab). The objective was to determine whether the variation in soil parameters under a range of conventional maize cultivars exceeded the differences between Bt and non-Bt maize cultivars. Variations in plant growth parameters (shoot and root biomass, percentage carbon, percentage nitrogen), Bt protein concentration in shoots, roots and soil, soil nematode abundance and soil microbial community structure were determined. Eight paired varieties (i.e. varieties genetically modified to express Bt protein and their near-isogenic control varieties) were investigated, together with a Bt variety for which no near-isogenic control was available (NX3622, a combined transformant expressing both Bt and herbicide tolerance) and a conventional barley (Hordeum vulgare L.) variety which was included as a positive control. The only plant parameter which showed a difference between Bt varieties and near-isogenic counterparts was the shoot carbon to nitrogen ratio; this was observed for only two of the eight varieties, and so was not attributable to the Bt trait. There were no detectable differences in the concentration of Bt protein in plant or soil with any of the Bt-expressing varieties. There were significant differences in the abundance of soil nematodes, but this was not related to the Bt trait. Differences in previously published soil nematode studies under Bt maize were smaller than these varietal effects. Soil microbial community structure, as determined by phospholipid fatty acid (PLFA) analysis, was strongly affected by plant growth stage but not by the Bt trait. The experimental addition of purified Cry1Ab protein to soil confirmed that, at ecologically relevant concentrations, there were no measurable effects on microbial community structure.
Abstract: BACKGROUND: Transcriptomic techniques are now being applied in ecotoxicology and toxicology to measure the impact of stressors and develop understanding of mechanisms of toxicity. Microarray technology in particular offers the potential to measure thousands of gene responses simultaneously. However, it is important that microarrays responses should be validated, at least initially, using real-time quantitative polymerase chain reaction (QPCR). The accurate measurement of target gene expression requires normalisation to an invariant internal control e.g., total RNA or reference genes. Reference genes are preferable, as they control for variation inherent in the cDNA synthesis and PCR. However, reference gene expression can vary between tissues and experimental conditions, which makes it crucial to validate them prior to application. RESULTS: We evaluated 10 candidate reference genes for QPCR in Daphnia magna following a 24 h exposure to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) at 0, 20, 40 and 80 mg IB/l. Six of the 10 candidates appeared suitable for use as reference genes. As a robust approach, we used a combination normalisation factor (NF), calculated using the geNorm application, based on the geometric mean of three selected reference genes: glyceraldehyde-3-phosphate dehydrogenase, ubiquitin conjugating enzyme and actin. The effects of normalisation are illustrated using as target gene leukotriene B4 12-hydroxydehydrogenase (Ltb4dh), which was up-regulated following 24 h exposure to 63-81 mg IB/l. CONCLUSIONS: As anticipated, use of the NF clarified the response of Ltb4dh in daphnids exposed to sublethal levels of ibuprofen. Our findings emphasise the importance in toxicogenomics of finding and applying invariant internal QPCR control(s) relevant to the study conditions.
Abstract: Studies on the effect of genetically modified Bacillus thuringiensis (Bt) crops on true soil dwelling non-target arthropods are scarce. The objective of this study was to assess the influence of a 4-week exposure to two Bt maize varieties (Cry1Ab) Cascade and MEB307 on the collembolan Protaphorura armata. For comparison three non-Bt maize varieties, Rivaldo (isogenic to Cascade), Monumental (isogenic to MEB307) and DK242, and two control diets based on baker's yeast (uncontaminated and contaminated with Bt toxin Cry1Ab) were also tested. Due to a lower C:N ratio, individuals reared on yeast performed significantly better in all of the measured endpoints than those reared on maize. P. armata performed equally well when reared on two Bt and three non-Bt maize varieties. Although there were no negative effects of Bt maize in this experiment, we recommend future studies on Bt crops to focus on species interactions in long-term, multi-species experiments.
Abstract: The impact of the pyrethroid lambda-cyhalothrin was studied in an in-stream mesocosm placed in a natural riffle of a Danish stream. Twice during summer 2002, the natural macroinvertebrate community was exposed in situ to a 30-min pulse of lambda-cyhalothrin. During exposure, nets caught macroinvertebrates in drift. Exposed and unexposed individuals of the amphipod Gammarus pulex (L) were transferred to aquaria in the laboratory and monitored for biochemical changes (ie biomarkers), pre-copulatory behaviour and mortality. Biochemical biomarkers were identified by screening extracts of exposed and unexposed G pulex using high-performance planar chromatography (HPPC). Biochemical biomarkers were detected 3 h after pulse-exposure, and one biomarker was persistent up to 7 days after exposure. Pre-copulatory behaviour (ie pair formation) was significantly impaired up to 5 days after exposure, and had not fully recovered at the end of the observation period. EC10(0.5 h) and EC50(0.5 h) values for pre-copulatory behaviour were 0.04 and 0.20 microg litre(-1), respectively. Mortality was significant at 0.35 microg litre(-1) with an LC50(0.5 h) of 5.69 microg litre(-1). There was a significant relationship between two biomarkers and mortality. The study demonstrated that pulse-exposure at expected environmental concentrations can reduce local populations of G pulex, resulting in severe impact on populations with limited possibilities of re-colonisation.
Abstract: Toxicants may affect juveniles more than adults because of physiological and behavioral aspects. When developing toxicity tests, this issue is often not addressed, and the most sensitive end point may be ignored. The topic was raised by a European working group aiming to develop a standard toxicity test with the predatory mite, Hypoaspis (Geolaelaps) aculeifer Canestrini, as this species was included in the EU Guidance documentfortesting of pesticides. To assess whether the juvenile life stages are the most susceptible, we examined the acute toxicity of dimethoate on larvae, protonymphs, deutonymphs, males, and females of H. aculeifer. The mites were exposed to 0, 2, 4, and 6 mg dimethoate kg(-1) for 7 days in an OECD artificial soil (5% organic matter). Total juvenile biomass, reproduction, mortality, and population growth rate (pgr) lambda were assessed at the end of the test. A comparison of mortality ranked the sensitivity of the life stages: Larvae (LC50 = 3.8 mg kg(-1)) > protonymphs (LC50 = 5.3 mg kg(-1)) > males (LC50 = 5.6 mg kg(-1)) > deutonymphs (LC50 = 7.1 mg kg(-1)) > females (LC50 = 7.6 mg kg(-1)). Effects on reproduction and pgr were significant at 2 mg dimethoate kg(-1), with population decline starting at this concentration. Thus, a test system with H. aculeifer including reproduction as end point is a rational approach, as reproduction will encompass juvenile mortality, at least with respect to dimethoate. Moreover, we suggest that pgr should be included in chronic standard tests because of high ecological relevance and the feasibility of applying it.
Abstract: Pesticides are constantly being applied to agricultural catchments, but little is known about their impact on aquatic biota during natural exposure. In the present study, the impact of the pyrethroid lambda-cyhalothrin was studied in an in-stream mesocosm setup. Twice during the summer of 2002, the natural macroinvertebrate community was exposed in situ to a 30-min pulse of lambda-cyhalothrin. Pyrethroid doses were released through a modified drip set with nominal concentrations of 0.10, 1.00, and 10.0 microg L(-1) during the first exposure and 0.05, 0.50, and 5.00 microg L(-1) in the second exposure. Before, during, and after exposure, drifting macroinvertebrates were caught in nets. Quantitative benthic samples were taken both before and on two occasions after exposure. Macroinvertebrate drift increased immediately after the pulse exposure, with total drift being significantly higher at all concentrations. Gammarus pulex, various Ephemeroptera, Leuctra sp., and Simuliidae were some of the taxa showing the most pronounced drift response. Structural change in the community was found only at 5.00 and 10.0 microg L(-1), and recovery occurred within approximately two weeks. The present study may be valuable in assessing extrapolations based on laboratory results as well as in evaluating pyrethroid impact on natural freshwater environments.
