A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Metabolic preconditioning of cells with AICAR-riboside: Improved cryopreservation and cell-type specific impacts on energetics and proliferation

In species whose evolutionary history has provided natural tolerance to dehydration and freezing, metabolic depression is often a pre-requisite for survival. We tested the hypothesis that preconditioning of mammalian cells with 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside (AICAR) to achieve metabolic depression will promote greater survivorship during cryopreservation. AICAR is used extensively to stimulate AMP-activated protein kinase (AMPK), which can result in downregulation of biosynthetic processes. We showed that the metabolic interconversion of AICAR was cell-type dependent. Accumulation of 5-aminoimidazole-4-carboxamide-1b-d-ribofuranosyl-5′-monophosphate (ZMP), as well as other metabolites that possess multiple phosphates (i.e., ZDP, ZTP), varied approximately 3.5-fold across the cell lines tested. AICAR treatment also significantly influenced the concentrations of cellular adenylates (ATP, ADP, and AMP). Depression of cell metabolism and proliferation with AICAR treatment differed among cell lines. Proliferation for a given cell line was negatively correlated with the fold-increase achieved in the ‘effective adenylate ratio’ ([AMP] + [ZMP])/[ATP]) after AICAR treatment. Metabolic preconditioning with AICAR promoted a significant increase in viability post-freezing in J774.A1 macrophages, HepG2/C3A cells and primary hepatocytes but not in NIH/3T3 fibroblasts or OMK cells. The effect of AICAR on viability after freezing was positively correlated (r² = 0.94) with the fold-increase in the ‘effective adenylate ratio’. Thus for each cell line, the greater the depression of metabolism and proliferation due to preconditioning with AICAR, the greater was the survivorship post-freezing.     

Improved metabolic control by depletion of Liver X Receptors in mice

Liver X Receptors (LXRs) coordinate the regulation of lipid and carbohydrate metabolism and insulin signaling. LXR-ligands lower plasma glucose in hyperglycemic rodents and have consequently been proposed as anti-diabetic agents. We investigated the metabolic effects induced by high carbohydrate diet in LXRalpha(-/-)beta(-/-) mice. Irrespective of diets, LXRalpha(-/-)beta(-/-) mice had reduced fatty acid, insulin, and C-peptide plasma levels than wild-type controls, suggesting a lower insulin production. High carbohydrate diet decreased the plasma glucose levels and the homeostasis model assessment (HOMA)-index in LXRalpha(-/-)beta(-/-) mice and increased hepatic triglyceride content and mRNA levels of lipogenic genes in wild-type and LXRalpha(-/-)beta(-/-) mice, proportionally. In wild-type mice high carbohydrate diet was associated with induced expression of LXR (1.5-fold), despite unchanged SREBP-1c expression. LXRalpha(-/-)beta(-/-) mice responded to this diet by induction of SREBP-1c. Our study suggests that in LXRalpha(-/-)beta(-/-) mice, glucose utilization seems to be privileged possibly due to reduced circulating free fatty acid levels.     

内源ABA信号水平动态调控的分子机制

从逆境信号感知、ABA合成的触发到ABA水平的动态调控,是细胞内重要的逆境信号传导途径,相对于应答ABA的下游信号事件,该领域研究滞后。研究显示,根系中ZEP、限速酶NCED、AtRGS1等合成酶基因及ABA2基因响应胁迫反应上调ABA信号水平。而7′-,8′-,9′-hydroxylase和糖基转移酶基因受逆境诱导激活,负调节ABA的积累。同时,提高的内源ABA信号水平能激活合成酶基因和代谢酶基因的表达。此外,基因表达和源库动力学分析显示,叶片ABA动态库的维持依赖根源ABA的持续供应。值得一提的是,miRNA与ABA信号起源及动态水平维持有关。进一步的代谢动力学分析揭示,ABA信号水平受合成酶基因和代谢酶基因表达的协同控制,多因素共同参与内源ABA信号水平的动态调控。     

Diversity of plant evolutionary lineages promotes arthropod diversity

Large‐scale habitat destruction and climate change result in the non‐random loss of evolutionary lineages, reducing the amount of evolutionary history represented in ecological communities. Yet, we have limited understanding of the consequences of evolutionary history on the structure of food webs and the services provided by biological communities. Drawing on 11 years of data from a long‐term plant diversity experiment, we show that evolutionary history of plant communities – measured as phylogenetic diversity – strongly predicts diversity and abundance of herbivorous and predatory arthropods. Effects of plant species richness on arthropods become stronger when phylogenetic diversity is high. Plant phylogenetic diversity explains predator and parasitoid richness as strongly as it does herbivore richness. Our findings indicate that accounting for evolutionary relationships is critical to understanding the severity of species loss for food webs and ecosystems, and for developing conservation and restoration policies.     

Macroinvertebrate communities of non-glacial high altitude intermittent streams

1. Macroinvertebrate assemblages of five non‐glacial intermittent high altitude headwater streams (above 1400 m – Serra da Estrela, Portugal), with dry periods of different lengths (0–3 months), were investigated in nearly undisturbed conditions to (i) examine spatial differences and identify environmental variables responsible for the observed invertebrate patterns, (ii) assess the association of dry period length with invertebrate community structure and (iii) determine the influence of using different taxonomic identification levels (order, family and genus) to assess invertebrate community patterns. 2. More than 100 macroinvertebrate genera were identified. Insects clearly dominated these communities with more than 95% of total captures and around 95% of the total richness. Diptera were the most rich and abundant group with chironomid occurrences comprising over 70% of macroinvertebrate captures. 3. The highest taxon richness, diversity, EPT (Ephemeroptera + Plecoptera + Trichoptera) and OCH (Odonata + Coleoptera + Heteroptera) genus richness, the greatest number of exclusive and characteristic taxa identified by the Indicator Value (IndVal), and a distinct community structure shown by Canonical Correspondence Analyses (CCA), were found in the only stream that was never totally dry, with pools lasting over summer. Environmental gradients that spatially structured the macroinvertebrate communities were always related to flow variations. 4. Over time, the highest abundances found in these systems were also related to flow variations and maximum genus richness occurred in the connected pools or in isolated pools. Streams with longer dry periods presented a distinct recolonization phase, with higher abundance of the stonefly larvae Nemoura sp. and the presence of the chironomid larvae Krenosmittia sp., possibly arriving from the hyporheos. 5. Taxonomic level of invertebrate identification was vital for recognizing the characteristic taxa (IndVal) of streams yet was not critical for identifying streams with the highest macroinvertebrate richness/diversity or structuring environmental gradients. 6. Overall, this study emphasizes the variability of high altitude intermittent streams macroinvertebrate communities, despite spatial proximity. This variability was probably related to flow intermittency and hydrologic permanence, different vegetation covers and riverbed substrata. Consequently, the establishment of reference conditions should involve long‐term data collections and more detailed physical characterization. Also, these findings have significant implications for accurately predicting the ecological consequences of future climate change in high altitude scenarios.     

A 14C age calibration curve for the last 60 ka: the Greenland-Hulu U/Th timescale and its impact on understanding the Middle to Upper Paleolithic transition in Western Eurasia

This paper combines the data sets available today for ¹⁴C-age calibration of the last 60 ka. By stepwise synchronization of paleoclimate signatures, each of these sets of ¹⁴C-ages is compared with the U/Th-dated Chinese Hulu Cave speleothem records, which shows global paleoclimate change in high temporal resolution. By this synchronization we have established an absolute-dated Greenland-Hulu chronological framework, against which global paleoclimate data can be referenced, extending the ¹⁴C-age calibration curve back to the limits of the radiocarbon method. Based on this new, U/Th-based Greenland_Hulu chronology, we confirm that the radiocarbon timescale underestimates calendar ages by several thousand years during most of Oxygen Isotope Stage 3. Major atmospheric ¹⁴C variations are observed for the period of the Middle to Upper Paleolithic transition, which has significant implications for dating the demise of the last Neandertals. The early part of “the transition” (with ¹⁴C ages > 35.0 ka ¹⁴C BP) coincides with the Laschamp geomagnetic excursion. This period is characterized by highly-elevated atmospheric ¹⁴C levels. The following period ca. 35.0-32.5 ka ¹⁴C BP shows a series of distinct large-scale ¹⁴C age inversions and extended plateaus. In consequence, individual archaeological ¹⁴C dates older than 35.0 ka ¹⁴C BP can be age-calibrated with relatively high precision, while individual dates in the interval 35.0-32.5 ka ¹⁴C BP are subject to large systematic age-‘distortions,’ and chronologies based on large data sets will show apparent age-overlaps of up to ca. 5,000 cal years. Nevertheless, the observed variations in past ¹⁴C levels are not as extreme as previously proposed (“Middle to Upper Paleolithic dating anomaly”), and the new chronological framework leaves ample room for application of radiocarbon dating in the age-range 45.0-25.0 ka ¹⁴C BP at high temporal resolution.     

Numerical exploration of the influence of neural noise on the psychometric function at low stimulation intensity levels

The relationship between stimulus intensity and the probability of detecting the presence of the stimulus is described by the psychometrical function. The probabilistic nature of this relationship is based on the stochastic behaviour of sensory neural channels and sensory networks involved in perceptual processing (Kiang 1968). This study tries to establish a continuum of variability across different levels of integration in the central nervous system. Once the opening and closing times of ionic channels was simulated, a threshold to the collective behaviour of voltage-gated ionic channels was imposed in order to generate the spike train of a single neuron. Afterwards, the trains of spikes of different neurons were added up, simulating the activity of a sensory nerve. By adding the activity due to the stimulus to the spontaneous neural behaviour, the psychometric function was simulated using a thresholding approach. The results can replicate the stochastic resonance phenomenon, but also open up the possibility that attentional phenomena can be mediated not only by increasing neural activity (bursting or oscillatory), but also by increasing noise at the neural level.