Impact of Bt maize pollen (MON810) on lepidopteran larvae living on accompanying weeds

Environmental risks of Bt maize, particularly pollen drift from Bt maize, were assessed for nontarget lepidopteran larvae in maize field margins. In our experimental approach, we carried out 3-year field trials on 6 ha total. Three treatments were used in a randomized block design with eight replications resulting in 24 plots: (i) near-isogenic control variety without insecticide (control), (ii) near-isogenic control variety with chemical insecticide (Baytroid) and (iii) Bt maize expressing the recombinant toxin. We established a weed strip (20 x 1 m) in every plot consisting of a Chenopodium album (goosefoot)/Sinapis alba (mustard) mixture. In these strips we measured diversity and abundance of lepidopteran larvae during maize bloom and pollen shed. C. album hosted five species but all in very low densities; therefore data were not suitable for statistical analysis. S. alba hosted nine species in total. Most abundant were Plutella xylostella and Pieris rapae. For these species no differences were detected between the Bt treatment and the control, but the chemical insecticide treatment reduced larval abundance significantly. Conclusions regarding experimental methodology and results are discussed in regard to environmental risk assessment and monitoring of genetically modified organisms.     

Farm‐scale evaluation of the impact of Cry1Ab Bt maize on canopy nontarget arthropods: a 3‐year study

The cultivation of Cry1Ab‐expressing genetically modified MON810 (Bt maize) has led to public concern in Europe, regarding its impact on nontarget arthropods (NTAs). We have assessed the potential effects of DKC 6451 YG (MON810) maize on canopy NTAs in a farm‐scale study performed in Central Spain during 3 years. The study focused on hemipteran herbivores (leafhoppers and planthoppers) and hymenopteran parasitic wasps (mymarids) collected by yellow sticky traps, which accounted for 72% of the total number of insects studied. The dynamics and abundance of these groups varied among years, but no significant differences were found between Bt and non‐Bt maize, indicating that Bt maize had no negative effect on these taxa. Nonetheless, the Cry1Ab toxin was detected in 2 different arthropods collected from Bt maize foliage, the cicadellids Zyginidia scutellaris and Empoasca spp. A retrospective power analysis on the arthropod abundance data for our field trials has determined that Z. scutellaris and the family Mymaridae have high capacity to detect differences between the Bt maize and its isogenic counterpart. The use of these canopy NTAs as surrogates for assessing environmental impacts of Bt maize is discussed.     

Field studies on the environmental fate of the Cry1Ab Bt-toxin produced by transgenic maize (MON810) and its effect on bacterial communities in the maize rhizosphere

Field studies were done to assess how much of the transgenic, insecticidal protein, Cry1Ab, encoded by a truncated cry1Ab gene from Bacillus thuringiensis (Bt), was released from Bt-maize MON810 into soil and whether bacterial communities inhabiting the rhizosphere of MON810 maize were different from those of the rhizosphere of nontransgenic maize cultivars. Bacterial community structure was investigated by SSCP (single-strand conformation polymorphism) of PCR-amplified 16S rRNA genes from community DNA. Using an improved extraction and detection protocol based on a commercially available ELISA, it was possible to detect Cry1Ab protein extracted from soils to a threshold concentration of 0.07 ng/g soil. From 100 ng of purified Cry1Ab protein added per gram of soil, only an average of 37% was extractable. At both field sites investigated, the amount of Cry1Ab protein in bulk soil of MON810 field plots was always lower than in the rhizosphere, the latter ranging from 0.1 to 10 ng/g soil. Immunoreactive Cry1Ab protein was also detected at 0.21 ng/g bulk soil 7 months after harvesting, i.e. in April of the following year. At this time, however, higher values were found in residues of leaves (21 ng/g) and of roots (183 ng/g), the latter corresponding to 12% of the Cry1Ab protein present in intact roots. A sampling 2 months later indicated further degradation of the protein. Despite the detection of Cry1Ab protein in the rhizosphere of MON810 maize, the bacterial community structure was less affected by the Cry1Ab protein than by other environmental factors, i.e. the age of the plants or field heterogeneities. The persistence of Cry1Ab protein emphasizes the importance of considering post-harvest effects on nontarget organisms.     

Communities of ground‐dwelling arthropods in conventional and transgenic maize: background data for the post‐market environmental monitoring

To verify the validity of concerns about environmental safety of maize expressing insecticidal Cry toxins (referred to as Bt maize), we compared communities of ground beetles (Carabidae), rove beetles (Staphylinidae) and spiders (Araneae) in plots planted either with Bt maize cultivar YieldGard^® or with the non‐transgenic parental cultivar Monumental. Each cultivar was grown on 5 plots of 0.5 ha for three consecutive years. To increase the field load of Cry toxin, the fully grown maize of the first study year was shredded to small pieces that were ploughed into the soil. Arthropods were collected in pitfall traps and determined to the species level. The abundance and species richness of all studied groups greatly varied over the season and between the seasons but without statistically significant differences between the Bt and non‐Bt plots. A single spider species and three ground beetle species dominated in the catches every year, whereas a set of 1–4 most abundant rove beetle species changed every year. Frequently occurring species were typical for most of Europe. The total counts of ground beetles, rove beetles and spiders collected once or twice per season are proposed to serve as bioindicators in the post‐market environmental monitoring (PMEM).     

Evaluation of the field impacts of simulated Bacillus thuringiensis‐transgenic Pinus radiata on nontarget native Lepidoptera and their natural enemies in a New Zealand plantation forest

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An approach for post‐market monitoring of potential environmental effects of Bt‐maize expressing Cry1Ab on natural enemies

Post‐market monitoring (PMM) consistent with Swiss and European Union legislation should ensure the detection and prevention of adverse effects on the environment possibly deriving from commercial cultivation of genetically modified (GM) crops. Insect‐resistant GM crops (such as Bt‐maize) raise particular questions regarding disturbances of biological control functions provided by beneficial insects such as predators and parasitoids (so‐called natural enemies). Consensus among regulators, scientists and the agricultural biotech industry on appropriate PMM plans allowing the detection and possibly prevention of such adverse effects is still lacking. The aims of this study were to identify the necessity for PMM of Bt‐maize expressing Cry1Ab on natural enemies and to develop an appropriate PMM plan. The approach chosen consisted in determining what type of monitoring is most appropriate to address potential effects of Bt‐maize on natural enemies during commercial cultivation. This included identifying whether there remain substantial scientific uncertainties that would support case‐specific monitoring. Existing pre‐market risk assessment data indicate that Bt‐maize (Cry1Ab) comprises a negligible risk for disturbances in biological control functions of natural enemies. As a consequence, a faunistic monitoring of specific groups of natural enemies is not considered an appropriate approach to detect failures in biological control functions. Alternatively, an approach is proposed that consists in indirectly analysing biological control functions by surveying outbreaks of maize herbivores. Unusual herbivore outbreaks could indicate failures in biological control functions of natural enemies. Data could be collected via questionnaires addressed to farmers growing Bt‐maize. Significant correlations between unusual occurrences of specific maize herbivores and the cultivation of Bt‐maize would subsequently need specific studies to determine possible causalities in more detail. The here proposed approach has the advantage of covering different natural enemy groups. It represents a cost‐effective strategy to obtain scientifically sound data as a basis for regulatory decision‐making.     

Optimising the capacity of field trials to detect the effect of genetically modified maize on non‐target organisms through longitudinal sampling

To assess risks of cultivation of genetically modified crops (GMCs) on non‐target arthropods (NTAs), field tests are necessary to verify laboratory results and in situations where exposure pathways are very complex and cannot be reproduced in the laboratory. A central concern in the design of field trials for this purpose is whether the tests are capable of detecting differences in the abundance or activity of NTAs in a treated crop in comparison with a non‐treated comparator plot. The detection capacity of a trial depends on the abundance and variability of the taxon, the values assumed for type I (α) and II (β) errors, and the characteristics of the trial and statistical design. To determine the optimal trial layout and statistical analysis, 20 field trials carried out in Spain from 2000 to 2009 to assess risks of GMCs on NTAs were examined with α and β set at 0.05 and 0.20, respectively. In this article we aim to determine the optimal number of sampling dates during a season, or longitudinal samples, in the design of field trials for assessing effects of GM maize on NTAs, and the ones that contribute most to achieving detectable treatment effects (d_c) less than 50% of the mean of the control. Detection capacities are a function of the number of individual samples taken during the season but a high number of samples is rarely justified because gains of repeated sampling can be relatively low. These gains depend primarily on field tests relative experimental variability in individual samplings (i.e. experimental variability relative to the mean of the control in each sampling date) which in turn depends on the sampling method (visual counts, pitfall traps or yellow sticky traps) and the density (or abundance) of the taxon in question. Taxa showing more density (or abundance) have less relative experimental variability. The smaller the experimental variability, the lower the profit of increasing the number of sampling dates. Sticky traps have a good effect detection capacity and need very few sampling dates, whereas visual counts and pitfall traps have a poorer effect detection capacity and need more individual samples to achieve d_c values lower than 50%. In maize field trials, it is recommended to concentrate sampling efforts in certain growth stages; the optimal ones for achieving an acceptable detection capacity are variable but, in general, samples in the first half of the season render better detection capacity than samples in the second half.     

A socio‐ecological model for predicting impacts of land‐use and climate change on regional plant diversity in the Austrian Alps

Climate and land‐use change jointly affect the future of biodiversity. Yet, biodiversity scenarios have so far concentrated on climatic effects because forecasts of land use are rarely available at appropriate spatial and thematic scales. Agent‐based models (ABMs) represent a potentially powerful but little explored tool for establishing thematically and spatially fine‐grained land‐use scenarios. Here, we use an ABM parameterized for 1,329 agents, mostly farmers, in a Central European model region, and simulate the changes to land‐use patterns resulting from their response to three scenarios of changing socio‐economic conditions and three scenarios of climate change until the mid of the century. Subsequently, we use species distribution models to, first, analyse relationships between the realized niches of 832 plant species and climatic gradients or land‐use types, respectively, and, second, to project consequent changes in potential regional ranges of these species as triggered by changes in both the altered land‐use patterns and the changing climate. We find that both drivers determine the realized niches of the studied plants, with land use having a stronger effect than any single climatic variable in the model. Nevertheless, the plants' future distributions appear much more responsive to climate than to land‐use changes because alternative future socio‐economic backgrounds have only modest impact on land‐use decisions in the model region. However, relative effects of climate and land‐use changes on biodiversity may differ drastically in other regions, especially where landscapes are still dominated by natural or semi‐natural habitat. We conclude that agent‐based modelling of land use is able to provide scenarios at scales relevant to individual species distribution and suggest that coupling ABMs with models of species' range change should be intensified to provide more realistic biodiversity forecasts.     

Ecological genetics of Italian peach‐potato aphid (Myzus persicae) populations in relation to geography,dispersal and insecticide resistance as studied using microsatellite and resistance markers

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Bioenergy production and Skylark (Alauda arvensis) population abundance – a modelling approach for the analysis of land‐use change impacts and conservation options

Bioenergy production is seen as one way of meeting future energy needs. The growing demand for biomass for energy production induces the cultivation of a few fast growing and high‐yielding energy crops on vast areas of arable land. This land‐use change has been found associated with the reduction of habitat suitability for farmland birds and a decline in farmland biodiversity in general. A large number of studies have assessed the ecological effects of energy crop cultivation at the local scale of a single field. This study focuses on regional landscape changes caused by increased energy crop cultivation, which includes reduction of crop‐type richness and spatial concentration of single crop‐types. We present a spatially explicit ecological model to assess the population‐level consequences of these effects on the abundance of the farmland bird species Skylark (Alauda arvensis). We also investigate the impacts of different land‐use scenarios and aim to identify adaptive conservation options. We show that (1) the impacts of increased energy crop cultivation on Skylark population abundance depend strongly on the landscape structure; (2) impacts could be tolerated as long as a certain minimum level of crop‐type heterogeneity is retained at the landscape level and (3) conservation actions are required and effective especially on landscapes where crop‐field size is large.     

Bt maize fed-prey mediated effect on fitness and digestive physiology of the ground predator Poecilus cupreus L. (Coleoptera: Carabidae)

We investigated the effects of a Bt maize hybrid on fitness and digestive physiology of the ground-dwelling predator Poecilus cupreus L., as compared with the near-isogenic hybrid. A tritrophic assay revealed that there was a great decline in the detection of Cry1Ab toxin through the trophic chain, the concentration of the toxin being 945, 349 and 37 ng g⁻¹ of fresh weight in Bt maize leaves, Spodoptera littoralis (Boisduval) larvae and P. cupreus larvae, respectively. Moreover, the toxin was only detected in 8% of the P. cupreus adults collected from fields growing Bt maize. Developmental time of both larvae and pupae of P. cupreus was not adversely affected by the Cry1Ab toxin via fed-prey. To elucidate potential detrimental effects due to a reduction in the quality of the prey, we assessed the digestive proteolytic activities of P. cupreus adults from a laboratory culture and insects collected in commercial Bt and non-Bt maize fields. Field-collected P. cupreus adults had higher proteolytic activities than those reared in the laboratory, whereas no significant differences were found between P. cupreus adults reared on Bt and non-Bt maize fed-S. littoralis or between P. cupreus adults collected in commercial Bt and non-Bt maize fields.     

Two functionally distinct members of the MATE (multi‐drug and toxic compound extrusion) family of transporters potentially underlie two major aluminum tolerance QTLs in maize

Crop yields are significantly reduced by aluminum (Al) toxicity on acidic soils, which comprise up to 50% of the world’s arable land. Al‐activated release of ligands (such as organic acids) from the roots is a major Al tolerance mechanism in plants. In maize, Al‐activated root citrate exudation plays an important role in tolerance. However, maize Al tolerance is a complex trait involving multiple genes and physiological mechanisms. Recently, transporters from the MATE family have been shown to mediate Al‐activated citrate exudation in a number of plant species. Here we describe the cloning and characterization of two MATE family members in maize, ZmMATE1 and ZmMATE2, which co‐localize to major Al tolerance QTL. Both genes encode plasma membrane proteins that mediate significant anion efflux when expressed in Xenopus oocytes. ZmMATE1 expression is mostly concentrated in root tissues, is up‐regulated by Al and is significantly higher in Al‐tolerant maize genotypes. In contrast, ZmMATE2 expression is not specifically localized to any particular tissue and does not respond to Al. [¹⁴C]‐citrate efflux experiments in oocytes demonstrate that ZmMATE1 is a citrate transporter. In addition, ZmMATE1 expression confers a significant increase in Al tolerance in transgenic Arabidopsis. Our data suggests that ZmMATE1 is a functional homolog of the Al tolerance genes recently characterized in sorghum, barley and Arabidopsis, and is likely to underlie the largest maize Al tolerance QTL found on chromosome 6. However, ZmMATE2 most likely does not encode a citrate transporter, and could be involved in a novel Al tolerance mechanism.     

Rapid molecular methods for in‐field and laboratory identification of the yellow‐legged Asian hornet (Vespa velutina nigrithorax)

The yellow‐legged Asian hornet (Vespa velutina nigrithorax) is an invasive species that presents a threat to apiculture in Europe; first introduced into France in 2004, it has subsequently spread into neighbouring European countries. There is a risk of invasion and establishment in the UK, and in 2016, nests were found and destroyed in Alderney in the Channel Islands, and in Tetbury, Gloucestershire, illustrating a need for screening of suspect specimens so that invading hornets can be rapidly identified, and their nests destroyed. In this study, loop‐mediated isothermal amplification (LAMP) and real‐time PCR assays were developed to enable both in‐field and laboratory testing. Species‐specific identification assays and generic invertebrate control assays were developed. All the assays were validated according to the European Plant Protection Organisation standard PM 7/98. The assays were tested successfully against V. velutina nigrithorax obtained from France, Asia and the UK. Eight non‐target species, that were closely related or morphologically similar to the Asian hornet, gave negative results with the species‐specific assays, and positive results with the control assays. The assays could be used to detect target DNA at concentrations as low as 5 pg per reaction. LAMP was rapid, and cable of generating positive results within 10 min. Using simplified sample homogenization protocols that could be performed in the field, the LAMP assay was successful when tested against all developmental stages and nest samples, assisting with identification of samples that cannot be determined morphologically and allowing detection away from the laboratory. These assays provide a valuable tool for fast and reliable detection of this invasive species, offering the ability to identify damaged/incomplete specimens and immature life‐stages.     

Chemiluminescence method for the determination of piroxicam by the enhancement of the tris‐(4,7‐diphenyl‐1,10‐phenanthrolinedisulphonic acid) ruthenium(II) (RuBPS)–cerium(IV) system and its application

A simple, rapid chemiluminescence (CL) method was described for the determination of piroxicam, a commonly used analgesic agent drug. A strong CL signal was detected when cerium(IV) sulphate was injected into tris‐(4,7‐diphenyl‐1,10‐phenanthrolinedisulphonic acid) ruthenium(II) (RuBPS)–piroxicam solution. The CL signal was proportional to the concentration of piroxicam in the range 2.8 × 10^–8–1.2 × 10^–5 mol/L. The detection limit was 2 × 10^–8 mol/L and the relative standard deviation (RSD) was 3.7% (c = 7.0 × 10^–7 mol/L piroxicam; n = 11). The proposed method was applied to the determination of piroxicam in pharmaceutical preparations in capsules, spiked serum and urine samples with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

A simple photograph‐based approach for discriminating between free‐ranging long‐finned (Globicephala melas) and short‐finned (G. macrorhynchus) pilot whales off the east coast of the United States

Line transect based abundance estimation is complicated for long‐finned (LFPW, Globicephala melas) and short‐finned (SFPW, G. macrorhynchus) pilot whales because of their similarity in appearance and their overlapping summertime range in some areas. We developed a photograph‐based approach to distinguish between species of free‐ranging pilot whales in the northwest Atlantic. We collected skin samples and photographs during the summers of 2004–2007 and used skin samples to distinguish species based on mitochondrial DNA. Relative morphometric measurements from photographs were examined using mixed‐effect models and logistic regression. The best model among 94 candidate models had an overall classification error rate of 2.5%. We tested the presence/absence of pigmentation in four regions of the dorsal body (melon, eye, cape, and saddle) for differences. Pigmentation was present in all four regions in 100% of the SFPWs sampled. Melon patch, blaze, and saddle patch pigmentation were present in 6%, 68%, and 50%, respectively, of the LFPWs, but the cape was completely absent. Both types of analyses provided positive species discrimination of free‐ranging animals. We created a cost‐effective, simple tool which could ultimately assist in providing appropriate management, mitigation, and conservation strategies for both northwest Atlantic species of pilot whales.     

Synthesis of dendrimer‐type chiral stationary phases based on the selector of (1S,2R)‐(+)‐2‐Amino‐1,2‐diphenylethanol derivate and their enantioseparation evaluation by HPLC

In our recent work, a series of dendritic chiral stationary phases (CSPs) were synthesized, in which the chiral selector was L‐2‐(p‐toluenesulfonamido)‐3‐phenylpropionyl chloride (selector I), and the CSP derived from three‐generation dendrimer showed the best separation ability. To further investigate the influence of the structures of dendrimer and chiral selector on enantioseparation ability, in this work, another series CSPs ( CSPs 1‐4 ) were prepared by immobilizing (1S,2R)‐1,2‐diphenyl‐2‐(3‐phenylureido)ethyl 4‐isocyanatophenylcarbamate (selector II) on one‐ to four‐generation dendrimers that were prepared in previous work. CSPs 1 and 4 demonstrated the equivalent enantioseparation ability. CSPs 2 and 3 showed the best and poorest enantioseparation ability respectively. Basically, these two series of CSPs exhibited the equivalent enantioseparation ability although the chiral selectors were different. Considering the enantioseparation ability of the CSP derived from aminated silica gel and selector II is much better than that of the one derived from aminated silica gel and selector I, it is believed that the dendrimer conformation essentially impacts enantioseparation. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Life history and initial assessment of fishing impacts on the by‐catch species Dules auriga (Teleostei: Serranidae) in southern Brazil

The life history of Dules auriga, a small hermaphrodite serranid species inhabiting deep waters and a frequent component of the discarded catch of bottom trawling in southern Brazil, was studied to assess the fishery effects on the stock through the estimation of the remaining spawning‐potential ratio. Sampling was conducted throughout a year and included specimens to determine sex, maturity and age. Age was validated by the edge type and marginal‐increment analysis. The oldest and the largest individuals were 9 years and 195 mm total length. Growth parameters fitted to the von Bertalanffy equation were L_∞ = 178·34 mm, k = 0·641 year^?1 and t₀ = ?0·341 years. Length and age at first maturity were 140·72 mm and 2 years, respectively. The reproductive season was throughout the austral spring and summer. The assessment of the effects of fishing showed that it may have resulted in a loss of 50% of the spawning potential. This loss may be higher when taking into account the uncertainty in the life‐history parameters and could be considered of concern for the population. Fast growth, moderate longevity, long spawning season, small size and age at maturity make D. auriga relatively resilient to the removal of biomass by fishing. When considering the uncertainty, however, the losses of the spawning potential have been severely reducing the population resilience in the face of ecosystem changes.     

Comparison of five different scoring methods in the evaluation of inflammatory infiltration (tumor‐infiltrating lymphocytes) in superficial spreading and nodular melanoma

The objective of our study was to compare the five different scoring methods of tumor‐infiltrating lymphocytes (TILs) assessment in a group of 213 cases of superficial spreading and nodular melanoma. The scoring methods include (a) Clark scoring; (b) Melanoma Institute Australia system; (c) scoring system used in the study of Saldanha et al.; (d) scoring system used in the TCGA study and modified by Park et al.; and (e) the system recently proposed by the “International Immuno‐Oncology Biomarker Working Group” for TILs scoring in all solid tumors. Prediction of survival with three main outcomes—disease‐specific‐free survival, local recurrence‐free survival, and distant metastasis‐free survival—was evaluated. The prognostic value of TILs showed statistical significance in univariate analysis regarding all three of the outcomes only for three of the five evaluated methods; the Clark scoring, the Melanoma Institute Australia system, and the system proposed by the “International Immuno‐Oncology Biomarker Working Group”. However, in multivariate analysis with covariants including Breslow thickness, type of melanoma, location, sex, and age, we did not find TILs to be an independent prognostic factor.     

Unusual Temperature‐Induced Retention Behavior of Constrained β‐Amino Acid Enantiomers on the Zwitterionic Chiral Stationary Phases ZWIX(+) and ZWIX(–)

The effects of temperature on the chiral recognition of cyclic β‐amino acid enantiomers on zwitterionic [Chiralpak ZWIX(+) and ZWIX(–)] chiral stationary phases were investigated. Experiments were performed at different mobile phase compositions and under 10°C column temperature increments in the temperature range 10–50°C. Apparent thermodynamic parameters and T_iso values were calculated from plots of ln k and ln α versus 1/T, respectively. Unusual temperature behavior was observed, especially on the ZWIX(–) column, where the application of MeOH/MeCN (50/50 v/v) containing 25 mM triethylamine and 50 mM formic acid as mobile phase led to nonlinear van't Hoff plots and increasing retention time with increasing temperature. On both columns, both enthalpically and entropically driven separations were observed. Chirality 26:385–393, 2014. © 2014 Wiley Periodicals, Inc.