ICPBR-Working Group Risks posed by dusts: overview of the area and recommendations

Background: In 2008 the poisoning of about 12000 bee colonies was reported from Germany. These poisonings were caused by the drift of dust particles containing the insecticidal substance clothianidin following the seeding of maize seeds, inadequately treated with the insecticide Poncho Pro. Results: Investigations were done on the dust load contained in seed packages of different crops, on the experimental abrasion of dust from treated seeds using the Heubach-Dustmeter as well as on the actual dust drift during the sowing operation of treated seeds with different machinery under field conditions. Resistance to abrasion of treated seeds and subsequent dust drift during sowing operations differ significantly between crops, coating recipes and facilities. Furthermore dust drift depends on particle size, sowing technology as well as on environmental conditions (e.g. wind speed, soil humidity). Conclusions: The drift of dust from treated seeds may pose a risk to honeybees, which needs to be appropriately considered within the authorization process of pesticides. The total quantity of abraded dust as well as the actual emission of dust during the sowing operation can be significantly reduced by technical means (e.g. coating recipe and facility equipment, deflector technology) and by additional mitigation measures (e.g. maximum wind speed). Keywords: honeybee, poisoning, risk, seed treatment, dust, drif

Diatom responses and geochemical feedbacks to environmental changes at Lake Rauchuagytgyn (Far East Russian Arctic)

This study is based on multiproxy data gained from a 14C-dated 6.5 m long sediment core and a 210Pb-dated 23 cm short core retrieved from Lake Rauchuagytgyn in Chukotka, Arctic Russia. Our main objectives are to reconstruct the environmental history and ecological development of the lake during the last 29 kyr and to investigate the main drivers behind bioproduction shifts. The methods comprise age-modeling, accumulation rate estimation, and light microscope diatom species analysis of 74 samples, as well as organic carbon, nitrogen, and mercury analysis. Diatoms have appeared in the lake since 21.8 ka cal BP and are dominated by planktonic Lindavia ocellata and L. cyclopuncta. Around the Pleistocene–Holocene boundary, other taxa including planktonic Aulacoseira, benthic fragilarioid (Staurosira), and achnanthoid species increase in their abundance. There is strong correlation between variations of diatom valve accumulation rates (DARs; mean 176.1×109 valves m2 a1), organic carbon accumulation rates (OCARs; mean 4.6 g m−2 a−1), and mercury accumulation rates (HgARs; mean 63.4 µg m−2 a−1). We discuss the environmental forcings behind shifts in diatom species and find moderate responses of key taxa to the cold glacial period, postglacial warming, the Younger Dryas, and the Holocene Thermal Maximum. The short-core data likely suggest recent change of the diatom community at the beginning of the 20th century related to human-induced warming but only little evidence of atmospheric deposition of contaminants. Significant correlation between DAR and OCAR in the Holocene interglacial indicates within-lake bioproduction represents bulk organic carbon deposited in the lake sediment. During both glacial and interglacial episodes HgAR is mainly bound to organic matter in the lake associated with biochemical substrate conditions. There were only ambiguous signs of increased HgAR during the industrialization period. We conclude that if increased short-term emissions are neglected, pristine Arctic lake systems can potentially serve as long-term CO2 and Hg sinks during warm climate episodes driven by insolation-enhanced within-lake primary productivity. Maintaining intact natural lake ecosystems should therefore be of interest to future environmental policy

Assessment of risks to honey bees posed by guttation

Background: Besides their nectar and pollen collecting activities, honey bees also forage water. Guttation droplets may be used as a water source. Measurements of high residue levels of some intrinsically highly toxic, systemic insecticides in guttation droplets triggered research activities on the potential risk for honey bees. Since 2009, a large number of studies have been conducted on the environmental conditions and factors favoring guttation, foraging of guttation, the occurrence of guttation in different crops, the frequency of guttation events and residue measurements in guttation droplets in different crops, at different growth stages and with different active ingredients. Different approaches of laboratory, semi-field and field studies were set up to address the potential risk of guttation to bees and to gain clarification whether and how this concern would need to be specifically addressed in the risk assessment for bees. Results: Occasionally increased mortalities of worker bees were reported from single events in some trials, when colonies were placed directly next to the sown maize crop treated with a systemic insecticide. However, there were no long-term colony effects (e.g. on colony strength and brood development) reported from any of the realistic worst case exposure trials conducted by either public research institutes or industry. Conclusion: The potential risk for bees is in the first instance dependent on the distance of the colonies to treated crops. Maize is considered as the worst case crop in terms of frequency, duration and intensity of guttation and of residue level of compounds found in guttation liquid. Though increased worker bee mortality on individual days was seen in some of the field studies where hives were placed directly at guttating maize fields, adverse effects to colony vitality, colony and brood development were never observed. Keywords: Guttation, risk assessment, pesticides, honey bees

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in <i>Saccharomyces cerevisiae</i>

Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L(-1) of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity

EasyClone: method for iterative chromosomal integration of multiple genes in <em>Saccharomyces cerevisiae</em>

Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log(10) mean +/- 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out

Natural computation meta-heuristics for the in silico optimization of microbial strains

<p>Abstract</p> <p>Background</p> <p>One of the greatest challenges in Metabolic Engineering is to develop quantitative models and algorithms to identify a set of genetic manipulations that will result in a microbial strain with a desirable metabolic phenotype which typically means having a high yield/productivity. This challenge is not only due to the inherent complexity of the metabolic and regulatory networks, but also to the lack of appropriate modelling and optimization tools. To this end, Evolutionary Algorithms (EAs) have been proposed for <it>in silico </it>metabolic engineering, for example, to identify sets of gene deletions towards maximization of a desired physiological objective function. In this approach, each mutant strain is evaluated by resorting to the simulation of its phenotype using the Flux-Balance Analysis (FBA) approach, together with the premise that microorganisms have maximized their growth along natural evolution.</p> <p>Results</p> <p>This work reports on improved EAs, as well as novel Simulated Annealing (SA) algorithms to address the task of <it>in silico </it>metabolic engineering. Both approaches use a variable size set-based representation, thereby allowing the automatic finding of the best number of gene deletions necessary for achieving a given productivity goal. The work presents extensive computational experiments, involving four case studies that consider the production of succinic and lactic acid as the targets, by using <it>S. cerevisiae </it>and <it>E. coli </it>as model organisms. The proposed algorithms are able to reach optimal/near-optimal solutions regarding the production of the desired compounds and presenting low variability among the several runs.</p> <p>Conclusion</p> <p>The results show that the proposed SA and EA both perform well in the optimization task. A comparison between them is favourable to the SA in terms of consistency in obtaining optimal solutions and faster convergence. In both cases, the use of variable size representations allows the automatic discovery of the approximate number of gene deletions, without compromising the optimality of the solutions.</p

High-mass star-forming cloud G0.38+0.04 in the Galactic center dust ridge contains H2CO and SiO masers

We have discovered a new H2CO (formaldehyde) 11,0−11,1 4.82966 GHz maser in Galactic center Cloud C, G0.38+0.04. At the time of acceptance, this is the eighth region to contain an H2CO maser detected in the Galaxy. Cloud C is one of only two sites of confirmed high-mass star formation along the Galactic center ridge, affirming that H2CO masers are exclusively associated with high-mass star formation. This discovery led us to search for other masers, among which we found new SiO vibrationally excited masers, making this the fourth star-forming region in the Galaxy to exhibit SiO maser emission. Cloud C is also a known source of CH3OH Class-II and OH maser emission. There are now two known regions that contain both SiO and H2CO masers in the CMZ, compared to two SiO and six H2CO in the Galactic disk, while there is a relative dearth of H2O and CH3OH Class-II masers in the CMZ. SiO and H2CO masers may be preferentially excited in the CMZ, perhaps because of higher gas-phase abundances from grain destruction and heating, or alternatively H2O and CH3OH maser formation may be suppressed in the CMZ. In any case, Cloud C is a new testing ground for understanding maser excitation conditions