How do climate change experiments alter plot-scale climate?

To understand and forecast biological responses to climate change, scientists frequently use field experiments that alter temperature and precipitation. Climate manipulations can manifest in complex ways, however, challenging interpretations of biological responses. We reviewed publications to compile a database of daily plot-scale climate data from 15 active-warming experiments. We find that the common practices of analysing treatments as mean or categorical changes (e.g. warmed vs. unwarmed) masks important variation in treatment effects over space and time. Our synthesis showed that measured mean warming, in plots with the same target warming within a study, differed by up to 1.6  ∘ C (63% of target), on average, across six studies with blocked designs. Variation was high across sites and designs: for example, plots differed by 1.1  ∘ C (47% of target) on average, for infrared studies with feedback control (n = 3) vs. by 2.2  ∘ C (80% of target) on average for infrared with constant wattage designs (n = 2). Warming treatments produce non-temperature effects as well, such as soil drying. The combination of these direct and indirect effects is complex and can have important biological consequences. With a case study of plant phenology across five experiments in our database, we show how accounting for drier soils with warming tripled the estimated sensitivity of budburst to temperature. We provide recommendations for future analyses, experimental design, and data sharing to improve our mechanistic understanding from climate change experiments, and thus their utility to accurately forecast species' responses.

Design and optimization of a novel reverse transcription linear-after-the-exponential PCR for the detection of foot-and-mouth disease virus.

AIMS: A novel molecular assay for the detection of foot-and-mouth disease virus (FMDV) was developed using linear-after-the-exponential polymerase chain reaction (LATE-PCR). METHODS AND RESULTS: Pilot experiments using synthetic DNA targets demonstrated the ability of LATE-PCR to quantify initial target concentration through endpoint detection. A two-step protocol involving reverse transcription (RT) followed by LATE-PCR was then used to confirm the ability of the assay to detect FMDV RNA. Finally, RT and LATE-PCR were combined in a one-step duplex assay for co-amplification of an FMDV RNA segment and an internal control comprised of an Armored RNA. In that form, each of the excess primers in the reaction mixture hybridize to their respective RNA targets during a short pre-incubation, then generate cDNA strands during a 3-min RT step at 60°C, and the resulting cDNA is amplified by LATE-PCR without intervening sample processing. CONCLUSIONS: The RT-LATE-PCR assay generates fluorescent signals at endpoint that are proportional to the starting number of RNA targets and can detect a range of sequence variants using a single mismatch-tolerant probe. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to offering improvements over current laboratory-based molecular diagnostic assays for FMDV, this new assay is compatible with a novel portable ('point-of-care') device, the BioSeeq II, designed for the rapid diagnosis of FMD in the field.

Modeling the fast gating mechanism in the ClC-0 chloride channel.

A simplified three-dimensional model ClC-0 chloride channel is constructed to couple the permeation of Cl- ions to the motion of a glutamate side chain that acts as the putative fast gate in the ClC-0 channel. The gate is treated as a single spherical particle attached by a rod to a pivot point. This particle moves in a one-dimensional arc under the influence of a bistable potential, which mimics the isomerization process by which the glutamate side chain moves from an open state (not blocking the channel pore) to a closed state (blocking the channel pore, at a position which also acts as a binding site for Cl- ions moving through the channel). A dynamic Monte Carlo (DMC) technique is utilized to perform Brownian dynamics simulations to investigate the dependence of the gate closing rate on both internal and external chloride concentration and the gate charge as well. To accelerate the simulation of gate closing to a time scale that can be accommodated with current methodology and computer power, namely, microseconds, parameters that govern the motion of the bare gate (i.e., in the absence of coupling to the permeating ions) are chosen appropriately. Our simulation results are in qualitative agreement with experimental observations and consistent with the "foot-in-the-door" mechanism (Chen et al. J. Gen. Physiol. 2003, 122, 641; Chen and Miller J. Gen. Physiol. 1996, 108, 237), although the absolute time scale of gate closing in the real channel is much longer (millisecond time scale). A simple model based on the fractional occupation probability of the Cl- binding site that is ultimately blocked by the fast gate suggests straightforward scalability of simulation results for the model channel considered herein to experimentally realistic time scales.

Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus.

Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan assay, RT-LAMP appears to be sensitive, rapid, specific, and cost-effective, with the potential for field deployment and use by developing countries for FMDV surveillance.

Odorant receptor gene expression changes during the parr-smolt transformation in Atlantic salmon.

The ability of salmon to home accurately to their natal stream to spawn has long intrigued biologists and has important consequences for the maintenance of population structure in these species. It is known that olfaction is crucial to homing, and that the transition from the freshwater to the marine environment (the parr-smolt transformation; PST) is a period of increased olfactory sensitivity and learning, resulting in a permanent memory of natal site odours that is retained, at least in part, in peripheral sensory neurones. These odours are then used as cues by sexually maturing fish on their homeward migration. We used quantitative polymerase chain reaction techniques to demonstrate transient increases in expression of odorant receptor transcripts (of up to fifty-fold over pre-PST levels) coincident with PST. Both olfactory (SORB) and vomeronasal receptors (SVRA and SVRC) are involved, which suggests that the fish learn both environmental odours and semiochemicals (pheromones). Receptor expression varies between families and changes over time indicating both genetic differences in odour stimuli and multiple periods of olfactory sensitivity. We suggest that changes in OR gene expression may have a role in homing behaviour and thus the maintenance of population structure in Atlantic salmon.

Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA analyses.

BACKGROUND: Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability. METHODS: Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems. RESULTS: All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments. CONCLUSIONS: Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.

BVA 2 mosquito larvicide--a new surface oil larvicide for mosquito control.

BVA 2 mosquito larvicide was evaluated in laboratory pan tests against 3rd-instar Aedes taeniorhynchus (Wied.), Culex quinquefasciatus Say, and Culex nigripalpus Theobald larvae. BVA 2 was as effective as the standard, GB-1111, at 14 liters/ha (> 99.1% vs. 99.8%). In small field plot tests BVA 2 mosquito larvicide applied at 28 liters/ha was as effective as GB-1111 (99.0% vs. 99.8%) 24 h posttreatment. Operationally, applied by helicopter at 46.8 liters/ha, BVA 2 mosquito larvicide was more effective (> 90%) in the slightly less vegetated site than in the heavily vegetated site. As a pupicide applied at 14 liters/ha in laboratory pan tests, no significant differences were noted between BVA 2 mosquito larvicide and GB-1111 against Ae. taeniorhynchus, Cx. quinquefasciatus, and Cx. nigripalpus pupae.

Downwind drift and deposition of malathion on human targets from ground ultra-low volume mosquito sprays.

Malathion was sprayed using a truck-mounted ultra-low volume (ULV) aerosol generator. The generator was operated at 41.4 kPa (6 psi) at flow rate of 128 ml (4.3 fl. oz.) per min. Malathion concentrations were measured at selected positions on live, stationary human subjects wearing protective clothing and placed along a transect at right angles to the path of the truck. Two standing subjects were exposed downwind to the malathion spray at 7.6 and 15.2 m. A third subject was exposed while jogging in the same direction as the spray vehicle and 1.5 m from the spray path. No significant differences (P > 0.05) in total amount of malathion deposited on subjects was demonstrated. During the last 4 sprays, average amounts of malathion deposited on ground level at 15.2, 30.4 and 91.2 m were not significantly different (P > 0.05). Malathion dermal residues were compared with the acute LD50 value (4,100 mg/kg) for a 70 kg adult male. Calculated malathion dermal exposures were less than the acute lethal dose for a human subject by 4 orders-of-magnitude or more.

A comparison of Teflon slides and the Army Insecticide Measuring System for sampling aerosol clouds.

The effects of method of droplet analysis, reader of Teflon slides and distance on mass median diameter of a Cythion aerosol cloud were examined in the calibration of an Army Insecticide Measuring System (AIMS). There were no significant differences in results among readers and between the AIMS and readers. There were slight but statistically significant differences between readers of Teflon slides and between the methods of analysis. Data supports the manufacturer's recommendation that, for the AIMS, the distance at which an aerosol generator air blast is between 3 and 7 m3 s-1 must be determined.

At the cutting edge.

In the second of two articles, Jim Dukes and Rosemary Stewart look at how protocols can be implemented successfully.

Patient protocols: commissioning, design and use.

The author begins by considering existing definitions of 'protocol' and related terms and later develops this into a discussion of how they work. Among other things, we learn from this article that methods of designing protocols have a significant influence on their implementation, creating a problem for organizations attempting to introduce protocols to promote uniform treatment. While the advantages and disadvantages of protocols are summarized, such a summary is to some extent unrevealing. It has to be appreciated that medicine is practised by social groups governed by informal arrangements which protocols may disturb. Furthermore, by making medical procedures explicit protocols may open up social and moral problems which have previously been concealed.

Evaluation of three candidate cypermethrin-piperonyl butoxide formulations compared with Scourge against adult Culex quinquefasciatus.

Cypermethrin-piperonyl butoxide formulations (1:1, 1:5, 1:10) were evaluated in a laboratory wind tunnel against colony reared, susceptible adult Culex quinquefasciatus mosquitoes. Scourge (4% resmethrin:12% piperonyl butoxide) was used as the standard for comparison. Based on the LC90 data, the 3 cypermethrin-piperonyl butoxide formulations were 2.6-3.7x more toxic than Scourge 24-h posttreatment.

Control of Aedes taeniorhynchus and Culex quinquefasciatus emergence with sustained release Altosid sand granules and pellets in saltwater and freshwater test plots.

The efficacy of sustained release Altosid sand granules to control adult Aedes taeniorhynchus and Culex quinquefasciatus emergence was investigated. Sand granules applied at a 7-day preflood application rate of 5.6 kg/ha controlled 99% of the Ae. taeniorhynchus emergence in saltwater plots for 44 days posttreatment and 35% of Cx. quinquefasciatus in freshwater plots. A 5.6 kg/ha rate controlled 100% of the Ae. taeniorhynchus emergence for 30 days posttreatment in semi-permanent saltwater plots. Altosid pellets were used at the label rate for comparison. Sand granules applied at 11.2, 16.8 and 22.4 kg/ha against Cx. quinquefasciatus in freshwater plots gave 98% emergence inhibition at the 22.4 kg/ha rate 37 days posttreatment, 93% at 16.8 kg/ha for 22 days, and 100% at 11.2 kg/ha rate for 16 days.