The costs of phenotypic adaptation to repeatedly fluctuating temperatures in a soil arthropod

Costs of phenotypic adaptation to changing environments have often been studied in morphological structures. Such structures typically are irreversible for at least some stage in the organism's life. In this study we investigated whether recurrent and reversible adaptation to changes in the thermal environment incurs a cost in terms of some key life-history traits in the collembolan Orchesella cincta. We exposed juvenile O. cincta to two treatments differing in the frequency of temperature fluctuation but with equal total temperature sums. In the high frequency treatment temperature fluctuated daily between 10 and 20 °C, while in the low frequency treatment temperature fluctuated on a weekly schedule. During the treatments we measured juvenile growth rate and juvenile mortality, and after six weeks the animals were transferred to constant 15 °C and adult starvation resistance was assessed. We found no significant differences between the treatments in juvenile growth rate or juvenile survival. Also, adults that had grown up under high frequency temperature fluctuations did not suffer from reduced starvation resistance compared to animals growing under low frequency temperature fluctuations. This finding supports the hypothesis that selection minimizes the production costs of inducible phenotypes and suggests that the development of optimal phenotypes and evolution of temperature reaction norms are not constrained by such costs.     

High throughput production of mouse monoclonal antibodies using antigen microarrays

Recent advances in proteomics research underscore the increasing need for high-affinity monoclonal antibodies, which are still generated with lengthy, low-throughput antibody production techniques. Here we present a semi-automated, high-throughput method of hybridoma generation and identification. Monoclonal antibodies were raised to different targets in single batch runs of 6-10 wk using multiplexed immunisations, automated fusion and cell-culture, and a novel antigen-coated microarray-screening assay. In a large-scale experiment, where eight mice were immunized with ten antigens each, we generated monoclonal antibodies against 68 of the targets (85%), within 6 wk of the primary immunization.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA™ kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA? kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

Determinants of intra-annual population dynamics in a tropical soil arthropod

Studies on inter-annual dynamics of populations from temperate regions have shown that density dependence and climate effects are relatively common, albeit weak. Yet, for short-lived organisms, intra-annual variation may be at least as important. Furthermore, tropical species commonly experience temperatures close to their upper thermal limit and thus may be more likely to experience heat stress. Here, we used the soil mite Rostrozetes ovulum to investigate the drivers of intra-annual population dynamics in an Amazonian rain forest. We sampled 3,600 soil cores from 20 transects during 13 months, obtaining 180 mite counts. Next, we built a dynamic N-mixture model accounting for different detection probabilities between soil types. In a Bayesian framework, we used this model to estimate (a) the strength of density dependence and (b) per capita growth rates, which were then tested against environmental variables. We found that the intra-annual population dynamics of R. ovulum were weakly density dependent. Further, per capita growth rates increased with resource supply (litterfall) and decreased with maximum temperature over much of the observed thermal range, although these effects explained relatively little variance. Yet, the seasonal correlation between these factors created a trade-off, so that realized population growth was highest when neither resource supply nor thermal suitability was optimal. Overall, our results suggest that the mechanisms shaping soil animal population dynamics may be surprisingly similar across latitudes. Our model offers a starting point for analyses of soil animal counts when extraction from soil samples is imperfect.     

High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria

Recently developed technologies have enabled multi-well measurement of O(2) consumption, facilitating the rate of mitochondrial research, particularly regarding the mechanism of action of drugs and proteins that modulate metabolism. Among these technologies, the Seahorse XF24 Analyzer was designed for use with intact cells attached in a monolayer to a multi-well tissue culture plate. In order to have a high throughput assay system in which both energy demand and substrate availability can be tightly controlled, we have developed a protocol to expand the application of the XF24 Analyzer to include isolated mitochondria. Acquisition of optimal rates requires assay conditions that are unexpectedly distinct from those of conventional polarography. The optimized conditions, derived from experiments with isolated mouse liver mitochondria, allow multi-well assessment of rates of respiration and proton production by mitochondria attached to the bottom of the XF assay plate, and require extremely small quantities of material (1-10 μg of mitochondrial protein per well). Sequential measurement of basal, State 3, State 4, and uncoupler-stimulated respiration can be made in each well through additions of reagents from the injection ports. We describe optimization and validation of this technique using isolated mouse liver and rat heart mitochondria, and apply the approach to discover that inclusion of phosphatase inhibitors in the preparation of the heart mitochondria results in a specific decrease in rates of Complex I-dependent respiration. We believe this new technique will be particularly useful for drug screening and for generating previously unobtainable respiratory data on small mitochondrial samples.     

High throughput screening of rooting depth in rice using buried herbicide

Root research requires high throughput phenotyping methods that provide meaningful information on root depth if the full potential of the genomic revolution is to be translated into strategies that maximise the capture of water deep in soils by crops. A very simple, low cost method of assessing root depth of seedlings using a layer of herbicide (TRIK or diuron) buried 25 or 30 cm deep in soil‐filled boxes of varying size is described that is suitable for screening hundreds or thousands of rice accessions in controlled environment conditions. Variation in cultivar sensitivity to the herbicide when injected into pots was detected but considered small in relation to the variation detected when the herbicide was buried. Using 32 rice cultivars previously characterised for root traits in rhizotron and hydroponic systems, 80% of variation in herbicide score at 35 days was explained by cultivar and herbicide score correlated strongly with rooting depth traits. Using 139 genotypes of the Bala × Azucena mapping population, heritability for herbicide symptoms reached 55% and quantitative trait loci were detected which match those previously reported in this population. In repeated experiments using different soils, the method did not always perform to its maximum potential (in terms of speed of symptom development or discrimination between cultivars). This was not due to degradation or reduced bio‐availability of the herbicide in the soil but is believed to be due to the soil water content and water release characteristics as it relates to plant water use. Therefore, when using this technique, thorough preliminary experiments to determine the best water application regime for the particular combination of soil and environmental conditions are required. The method should be applicable to seedling stage screening of rice and other crops.     

High throughput isolation of DNA and RNA in 96-well format using a paint shaker

Most steps in plant nucleic acid isolation are easily adapted to 96-well format; however, tissue disruption typically is performed on samples individually and often is the rate-limiting step in sample processing. We have found that DNA and RNA isolation from Arabidopsis tissue can be carried out in 96-well format using a paint shaker fitted with an adapter for tissue disruption.     

Effects of exposure to short-term heat stress on male reproductive fitness in a soil arthropod

Ambient temperature is a key environmental factor influencing a variety of aspects of the ecology and evolution of ectotherms. Reproductive traits have been suggested to be more sensitive to thermal stress than other life history traits. This study investigated the direct and indirect effects of heat shock on male reproductive success in the widespread springtail Orchesella cincta. Male springtails were exposed to four temperature treatments: heat hardening (35.2 °C for 1 h), heat shock (37.2 °C for 1 h), heat hardening + heat shock (35.2 °C for 1 h, followed 15 h later by 37.2 °C for 1 h), and control (20 °C). The heat shock gene Hsp70 showed high expression in all the heat treatments, indicating that the treatments indeed induced thermal stress. Significant mortality was only found in the treatment with heat shock, both with and without heat hardening. A direct effect of heat treatment was found on time to first reproduction, which was significantly longer after heat shock (with or without heat hardening) than in the control treatment. There was no difference among treatments in the number of spermatophores produced in the first reproductive instar. Heat treatment also had indirect effects on male reproductive success. Females chose significantly more spermatophores from control males than from males that received heat shock, heat hardening or both. A high percentage of spermatophores produced by heat shocked males caused reproductive failure in females, but no significant differences among treatments were found.Our results suggest that not all traits were equally affected by the heat stress. Heat hardening did not protect reproductive traits against the negative effects of heat shock. The indirect effects of heat shock on reproduction may be equally important as the direct effects.     

High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence

An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.     

High throughput purification of recombinant human growth hormone using radial flow chromatography

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. Using fed-batch fermentation process, around 670 mg/L of r-hGH was produced at a cell OD600 of 35. Cell lysis followed by detergent washing resulted in semi-purified inclusion bodies with more than 80% purity. Purified inclusion bodies were homogenous in preparation having an average size of 0.6 μm. Inclusion bodies were solubilized at pH 12 in presence of 2 M urea and refolded by pulsatile dilution. Refolded protein was purified with DEAE-anion exchange chromatography using both radial and axial flow column (50 ml bed volume each). Higher buffer flow rate (30 ml/min) in radial flow column helped in reducing the batch processing time for purification of refolded r-hGH. Radial column based purification resulted in high throughput recovery of diluted refolded r-hGH in comparison to axial column. More than 40% of inclusion body protein could be refolded into bioactive form using the above method in a single batch. Purified r-hGH was analyzed by mass spectroscopy and found to be bioactive by Nb2 cell line proliferation assay. Inclusion body enrichment, mild solubilization, pulsatile refolding and radial flow chromatography worked co-operatively to improve the overall recovery of bioactive protein from inclusion bodies.     

Modeling locomotion of a soft-bodied arthropod using inverse dynamics

Most bio-inspired robots have been based on animals with jointed, stiff skeletons. There is now an increasing interest in mimicking the robust performance of animals in natural environments by incorporating compliant materials into the locomotory system. However, the mechanics of moving, highly conformable structures are particularly difficult to predict. This paper proposes a planar, extensible-link model for the soft-bodied tobacco hornworm caterpillar, Manduca sexta, to provide insight for biologists and engineers studying locomotion by highly deformable animals and caterpillar-like robots. Using inverse dynamics to process experimentally acquired point-tracking data, ground reaction forces and internal forces were determined for a crawling caterpillar. Computed ground reaction forces were compared to experimental data to validate the model. The results show that a system of linked extendable joints can faithfully describe the general form and magnitude of the contact forces produced by a crawling caterpillar. Furthermore, the model can be used to compute internal forces that cannot be measured experimentally. It is predicted that between different body segments in stance phase the body is mostly kept in tension and that compression only occurs during the swing phase when the prolegs release their grip. This finding supports a recently proposed mechanism for locomotion by soft animals in which the substrate transfers compressive forces from one part of the body to another (the environmental skeleton) thereby minimizing the need for hydrostatic stiffening. The model also provides a new means to characterize and test control strategies used in caterpillar crawling and soft robot locomotion.     

High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

Background

Short rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment.

Results

Glucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin.

Conclusions

Final glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.     

Responses of bioaugmented ryegrass to PAH soil contamination

The physiological and biochemical responses of ryegrass (Lolium multiflorum) to PAH induced stress in soils contaminated with phenanthrene and pyene were investigated, in the presence of PAH-degrading bacteria (Acinetobacteria junii) or arbuscular mycorrhizal fungi (AM fungi, Glomus mossae). The parameters monitored included chlorophyll content, chlorophyll a/b ratio, soluble-carbohydrate content, soluble-protein, malondialdehyde and electrolyte leakage, and superoxide dismutase (SOD) and peroxidase (POD) activities. Ryegrass showed good resistance and acclimation to PAH stress in soil, however, PAH contamination resulted in adverse effects such as damage of photosynthetic function and acceleration of shoot senescence. At PAH level of 100 mg kg(-1), chlorophyll contents were 14% lower than control (no PAH). Activities of SOD and POD were more sensitive indicators of PAH stress as compared to other parameters. However, all parameters showed trends based on either the bioaugmentation of the plants or PAH treatment level. It was concluded that the inoculation of AMF and PAH-degrading bacteria, especially the former, have a positive effect on alleviation of PAH toxicity to ryegrass plants. Furthermore, the inoculation of AMF increased the shoot and biomass of ryegrass by 11-19% and 18-78%, respectively. Bioaugmented ryegrass plants show promise as a host plants in the phytoremediation of PAH contaminated soils.     

Fatty acid composition and extreme temperature tolerance following exposure to fluctuating temperatures in a soil arthropod

Ectotherms commonly adjust their lipid composition to ambient temperature to counteract detrimental thermal effects on lipid fluidity. However, the extent of lipid remodeling and the associated fitness consequences under continuous temperature fluctuations are not well-described. The objective of this study was to investigate the effect of repeated temperature fluctuations on fatty acid composition and thermal tolerance. We exposed the springtail Orchesella cincta to two constant temperatures of 5 and 20 °C, and a continuously fluctuating treatment between 5 and 20 °C every 2 days. Fatty acid composition differed significantly between constant low and high temperatures. As expected, animals were most cold tolerant in the low temperature treatment, while heat tolerance was highest under high temperature. Under fluctuating temperatures, fatty acid composition changed with temperature initially, but later in the experiment fatty acid composition stabilized and closely resembled that found under constant warm temperatures. Consistent with this, heat tolerance in the fluctuating temperature treatment was comparable to the constant warm treatment. Cold tolerance in the fluctuating temperature treatment was intermediate compared to animals acclimated to constant cold or warmth, despite the fact that fatty acid composition was adjusted to warm conditions. This unexpected finding suggests that in animals acclimated to fluctuating temperatures an additional underlying mechanism is involved in the cold shock response. Other aspects of homeoviscous adaptation may protect animals during extreme cold. This paper forms a next step to fully understand the functioning of ectotherms in more thermally variable environments.     

Habitat-specific differences in thermal plasticity in natural populations of a soil arthropod

When populations experience substantial variation in environmental conditions, they may evolve phenotypic plasticity in response to these varying selection pressures. Evolutionary theory predicts differentiation in the level of phenotypic plasticity among different habitats. We evaluated temperature-induced phenotypic responses in juvenile growth rate in natural populations of the springtail Orchesella cincta , inhabiting forest and heathland. These habitats typically co-occur but differ strongly with respect to, for example, thermal regime, relative humidity, and structure. Offspring of females from the two habitats were reared at different temperatures in climate rooms and the temperature response of juvenile growth rate and egg size was measured. We found a habitat-specific difference in plasticity of juvenile growth rate. The reaction norms of the forest populations were steeper than the reaction norms for heath populations at two replicated sampling sites. Egg weight itself was demonstrated to be a plastic trait with a higher egg weight at low temperatures, but the thermal response did not differ between habitats. We conclude that these populations have diverged due to strong local natural selection. Our results support the argument that the level of phenotypic plasticity itself can be under selection and that differentiation in reaction norms can occur even in neighbouring habitats with no barrier to gene flow.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 265–271.     

亚热带森林凋落叶分解过程土壤节肢动物群落的变化特征

土壤动物群落结构和多样性可能随凋落物分解进程和基质质量的变化不断改变。为了解亚热带森林凋落叶分解过程中土壤节肢动物群落变化特征,以四川盆地亚热带森林麻栎（Quercus acutissima）和柳杉（Cryptomeria fortunei）凋落叶为对象,于2011-2015年采用分解袋法研究了2种凋落叶分解过程中土壤节肢动物组成、结构和多样性动态变化。整个研究期间,柳杉和麻栎凋落叶分解袋中共捕获土壤节肢动物3855只,分属于16目51科,且均以等节跳科和棘跳科为优势类群;麻栎凋落叶中土壤节肢动物的个体密度随分解进程呈现增加趋势,在分解的1079天达最高值后降低,而柳杉凋落叶则在分解的156天急剧增加后快速降低,2种凋落叶中土壤节肢动物类群数量具有相似的动态变化过程;2种凋落叶中土壤节肢动物总体以菌食性数量比例最高,腐食性最低,且随凋落叶分解进程,植食性土壤节肢动物占比明显下降,菌食性则上升;非度量多维尺度（NMDS）分析显示,2种凋落叶中土壤节肢动物群落组成具有显著差异,聚类分析表明,2种凋落叶土壤节肢动物群落结构相似性随分解进程不断降低。亚热带森林凋落叶分解过程中土壤节肢动物群落组成、结构和多样性受凋落叶类型影响。     

High throughput sequencing approaches to mutation discovery in the mouse

Phenotype-driven approaches in mice are powerful strategies for the discovery of genes and gene functions and for unravelling complex biological mechanisms. Traditional methods for mutation discovery are reliable and robust, but they can also be laborious and time consuming. Recently, high-throughput sequencing (HTS) technologies have revolutionised the process of forward genetics in mice by paving the way to rapid mutation discovery. However, successful application of HTS for mutation discovery relies heavily on the sequencing approach employed and strategies for data analysis. Here we review current HTS applications and resources for mutation discovery and provide an overview of the practical considerations for HTS implementation and data analysis.