Bt Rice Expressing Cry2Aa Does Not Harm Cyrtorhinus lividipennis,a Main Predator of the Nontarget Herbivore Nilapavarta lugens

T2A-1 is a newly developed transgenic rice that expresses a synthesized cry2Aa gene driven by the maize ubiquitin promoter. T2A-1 exhibits high resistance against lepidopteran pests of rice. The brown planthopper, Nilapavarta lugens (Stål), is a main nontarget sap-sucking insect pest of rice, and Cyrtorhinus lividipennis (Reuter) is the major predator of the eggs and young nymphs of planthoppers. As C. lividipennis may expose to the Cry2Aa protein via N. lugens, it is therefore essential to assess the potential effects of transgenic cry2Aa rice on this predator. In the present study, three experiments were conducted to evaluate the ecological risk of transgenic cry2Aa rice to C. lividipennis: (1) a direct feeding experiment in which C. lividipennis was fed an artificial diet containing Cry2Aa at the dose of 10-time higher than that it may encounter in the realistic field condition; (2) a tritrophic experiment in which the Cry2Aa protein was delivered to C. lividipennis indirectly through prey eggs or nymphs; (3) a realistic field experiment in which the population dynamics of C. lividipennis were investigated using vacuum-suction. Both direct exposure to elevated doses of the Cry2Aa protein and prey-mediated exposure to realistic doses of the protein did not result in significant detrimental effects on the development, survival, female ratio and body weight of C. lividipennis. No significant differences in population density and population dynamics were observed between C. lividipennis in transgenic cry2Aa and nontransgenic rice fields. It may be concluded that transgenic cry2Aa rice had no detrimental effects on C. lividipennis. This study represents the first report of an assessment continuum for the effects of transgenic cry2Aa rice on C. lividipennis.     

Changes in the Spatial Configuration and Strength of Trophic Control Across a Productivity Gradient During a Massive Rodent Outbreak

Understanding the determinants of spatial and temporal differences in the relative strength of consumer–resource interactions is an important endeavour in ecology. Here, we explore the necessary conditions for temporal shifts in the relative strength of rodent–plant interactions in an area characterised by profound spatial differences in trophic control, with predator–prey interactions prevailing in productive habitats and rodent–plant interactions dominating unproductive habitats of the forest–tundra ecotone. We report data obtained during the exceptionally massive rodent outbreak of 2010–2012 in northernmost Fennoscandia, including an experimental manipulation of herbivore access to vegetation plots across a large-scale productivity gradient, multiple observational measures of plant–rodent interactions linked to rodent abundance data and a large-scale survey of breeding avian predators and mammalian predator activity. Unexpectedly, rodent grazing impacts documented during the rodent outbreak were uniformly strong across the landscape, regardless of habitat productivity. The runaway response in rodent populations was facilitated by a high population growth rate in the early phase of the outbreak due to the extended absence of predators in productive habitats, concomitant with an exceptionally long-lasting lemming outbreak in unproductive habitats. Our results showed that spatio-temporal variation in trophic control also occurs in ecosystems structured according to the exploitation ecosystems hypothesis and emphasises the importance of long-term studies to capture nonlinear and stochastic features that shape ecosystem functioning. In this context, the temporary release from top–down regulation in productive habitats caused strong grazing impacts that may be crucial for the resilience of tundra ecosystems under the threat of climate change-driven shrub encroachment.     

Expression of Caspase-3 and -7 Does Not Correlate with the Extent of Apoptosis in Primary Breast Carcinomas

Association between the rate of apoptosis and expression of the several relevant molecules (Bcl-2, pro- and active caspase-3, and caspase-7) was studied in 61 primary breast carcinomas. The rate of apoptosis detected both morphologically and by the TUNEL assay appeared to be high in 18 (30%), moderate in 14 (23%), and low in 29 (48%) carcinomas. High apoptotic index was strongly associated with advanced tumor grade and estrogen receptor positive (ER+) status but not with other investigated clinical or morphological parameters. Among the molecules studied, only the Bcl-2 protein expression demonstrated strong (inverse) correlation with the apoptotic index (p = 0.032). The data of this expected correlation was served as internal control in the study. Interestingly, high levels of the anti-apoptotic protein Bcl-2 was frequently co-incident with increased expression of pro-apoptotic molecules, such as active caspase-3 (p = 0.004) and caspase-7 (p = 0.001). However, expression of caspase-3 or caspase-7 did not show correlation with the extent of apoptosis or any clinico-morphological features, except overrepresentation of ER+ status in tumors expressing caspase-3 (p = 0.009). Thus, these findings indicate a general dysregulation of spontaneous apoptosis in primary breast tumors.

Key Words:

Caspase-3, Caspase-7, Bcl-2, Breast carcinoma     

4-Aminopyridine Does Not Enhance Flocculus Function in Tottering,a Mouse Model of Vestibulocerebellar Dysfunction and Ataxia

The potassium channel antagonist 4-aminopyridine (4-AP) improves a variety of motor abnormalities associated with disorders of the cerebellum. The most rigorous quantitative data relate to 4-AP''s ability to improve eye movement deficits in humans referable to dysfunction of the cerebellar flocculus. Largely based on work in the ataxic mouse mutant tottering (which carries a mutation of the Cacna1a gene of the P/Q voltage-activated calcium channel), 4-AP is hypothesized to function by enhancing excitability or rhythmicity of floccular Purkinje cells. We tested this hypothesis by determining whether systemic or intrafloccular administration of 4-AP would ameliorate the eye movement deficits in tottering that are attributable to flocculus dysfunction, including the reductions in amplitude of the yaw-axis vestibulo-ocular reflex (VOR) and vision-enhanced vestibulo-ocular reflex (VVOR), and the optokinetic reflex (OKR) about yaw and roll axes. Because tottering''s deficits increase with age, both young and elderly mutants were tested to detect any age-dependent 4-AP effects. 4-AP failed to improve VOR, VVOR, and OKR gains during sinusoidal stimuli, although it may have reduced the tendency of the mutants'' responses to VOR and VVOR to decline over the course of a one-hour recording session. For constant-velocity optokinetic stimuli, 4-AP generated some enhancement of yaw OKR and upward-directed roll OKR, but the effects were also seen in normal C57BL/6 controls, and thus do not represent a specific reversal of the electrophysiological consequences of the tottering mutation. Data support a possible extra-floccular locus for the effects of 4-AP on habituation and roll OKR. Unilateral intrafloccular 4-AP injections did not affect ocular motility, except to generate mild eye elevations, consistent with reduced floccular output. Because 4-AP did not produce the effects expected if it normalized outputs of floccular Purkinje cells, there is a need for further studies to elucidate the drug''s mechanism of action on cerebellar motor dysfunction.     

Separating Lentiviral Vector Injection and Induction of Gene Expression in Time,Does Not Prevent an Immune Response to rtTA in Rats

Background

Lentiviral gene transfer can provide long-term expression of therapeutic genes such as erythropoietin. Because overexpression of erythropoietin can be toxic, regulated expression is needed. Doxycycline inducible vectors can regulate expression of therapeutic transgenes efficiently. However, because they express an immunogenic transactivator (rtTA), their utility for gene therapy is limited. In addition to immunogenic proteins that are expressed from inducible vectors, injection of the vector itself is likely to elicit an immune response because viral capsid proteins will induce “danger signals” that trigger an innate response and recruit inflammatory cells.

Methodology and Principal Findings

We have developed an autoregulatory lentiviral vector in which basal expression of rtTA is very low. This enabled us to temporally separate the injection of virus and the expression of the therapeutic gene and rtTA. Wistar rats were injected with an autoregulatory rat erythropoietin expression vector. Two or six weeks after injection, erythropoietin expression was induced by doxycycline. This resulted in an increase of the hematocrit, irrespective of the timing of the induction. However, most rats only responded once to doxycycline administration. Antibodies against rtTA were detected in the early and late induction groups.

Conclusions

Our results suggest that, even when viral vector capsid proteins have disappeared, expression of foreign proteins in muscle will lead to an immune response.     

Landscape Distribution of Microbial Activity in the McMurdo Dry Valleys: Linked Biotic Processes, Hydrology, and Geochemistry in a Cold Desert Ecosystem

In desert ecosystems, microbial activity and associated nutrient cycles are driven primarily by water availability and secondarily by nutrient availability. This is especially apparent in the extremely low productivity cold deserts of the McMurdo Dry Valleys, Antarctica. In this region, sediments near streams and lakes provide the seasonally wet conditions necessary for microbial activity and nutrient cycling and thus transfer energy to higher organisms. However, aside from a few studies of soil respiration, rates of microbial activity throughout the region remain unexplored. We measured extracellular enzyme activity potentials (alkaline phosphatase, leucine-aminopeptidase, beta-glucosidase, phenol oxidase, and peroxidase) in soils adjacent to lakes and streams, expecting activity to be primarily related to soil water content, as well as time of season and organic matter supply. Phosphatase and beta-glucosidase activities were higher in shoreline than upland soils; however, potential rates were not correlated with soil water content. Instead, soil organic matter, salinity, and pH were the best predictors of microbial activity. Microbial nutrient limitation metrics estimated from extracellular enzyme activity were correlated with pH and salinity and exhibited similar patterns to previously published trends in soil P and N content. Compared to other terrestrial ecosystems, organic matter specific rates for leucine-aminopeptidase and oxidative enzyme activities were high, typical of alkaline desert soils. Phosphatase activity was close to the global mean whereas beta-glucosidase activity was extremely low, which may reflect the lack of vascular plant derived organic matter in the Dry Valleys. In this cold desert ecosystem, water availability promotes microbial activity, and microbial nutrient cycling potentials are related to soil geochemistry. Author contributions:   LHZ performed research, analyzed data, and wrote the paper; RLS contributed new methods and wrote the paper; JEB conceived/designed study, performed research and analyzed data; MNG conceived/designed study and performed research; CTV conceived/designed study and performed research.     

Primary Productivity of Phytoplankton,Environmental Covariates and Fish Growth in West Bengal Ponds under a Polyculture System

Measurements of primary production by phytoplankton in four fish ponds over a period of one year revealed a distinct spatial variation in rate. The ponds were used for polyculture, monoculture, or a traditional system of fish culture. The rates of gross photosynthesis were highly correlated with temperature, free CO₂, oxygen, inorganic nitrogen (NH₄⁺, NO₂⁻, and NO₃⁻) and PO₄-P. It was shown by step-wise multiple regression analysis that in the polyculture pond with a predominantly autotrophic metabolism, gross photosynthesis was the most significant factor correlated with fish growth. In another polyculture system in which the metabolism was mainly hetero-trophic, the influence of the photosynthesis rate was slight.     

Application of a Hybrid Forest Growth Model to Evaluate Climate Change Impacts on Productivity,Nutrient Cycling and Mortality in a Montane Forest Ecosystem

Climate change introduces considerable uncertainty in forest management planning and outcomes, potentially undermining efforts at achieving sustainable practices. Here, we describe the development and application of the FORECAST Climate model. Constructed using a hybrid simulation approach, the model includes an explicit representation of the effect of temperature and moisture availability on tree growth and survival, litter decomposition, and nutrient cycling. The model also includes a representation of the impact of increasing atmospheric CO₂ on water use efficiency, but no direct CO₂ fertilization effect. FORECAST Climate was evaluated for its ability to reproduce the effects of historical climate on Douglas-fir and lodgepole pine growth in a montane forest in southern British Columbia, Canada, as measured using tree ring analysis. The model was subsequently used to project the long-term impacts of alternative future climate change scenarios on forest productivity in young and established stands. There was a close association between predicted sapwood production and measured tree ring chronologies, providing confidence that model is able to predict the relative impact of annual climate variability on tree productivity. Simulations of future climate change suggest a modest increase in productivity in young stands of both species related to an increase in growing season length. In contrast, results showed a negative impact on stemwood biomass production (particularly in the case of lodgepole pine) for established stands due to increased moisture stress mortality.     

The Mammalian Orthologs of Drosophila Lgd,CC2D1A and CC2D1B,Function in the Endocytic Pathway,but Their Individual Loss of Function Does Not Affect Notch Signalling

CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.     

BopA Does Not Have a Major Role in the Adhesion of Bifidobacterium bifidum to Intestinal Epithelial Cells,Extracellular Matrix Proteins,and Mucus

The ability of bifidobacteria to adhere to the intestine of the human host is considered to be important for efficient colonization and achieving probiotic effects. Bifidobacterium bifidum strains DSM20456 and MIMBb75 adhere well to the human intestinal cell lines Caco-2 and HT-29. The surface lipoprotein BopA was previously described to be involved in mediating adherence of B. bifidum to epithelial cells, but thioacylated, purified BopA inhibited the adhesion of B. bifidum to epithelial cells in competitive adhesion assays only at very high concentrations, indicating an unspecific effect. In this study, the role of BopA in the adhesion of B. bifidum was readdressed. The gene encoding BopA was cloned and expressed without its lipobox and hydrophobic signal peptide in Escherichia coli, and an antiserum against the recombinant BopA was produced. The antiserum was used to demonstrate the abundant localization of BopA on the cell surface of B. bifidum. However, blocking of B. bifidum BopA with specific antiserum did not reduce adhesion of bacteria to epithelial cell lines, arguing that BopA is not an adhesin. Also, adhesion of B. bifidum to human colonic mucin and fibronectin was found to be BopA independent. The recombinant BopA bound only moderately to human epithelial cells and colonic mucus, and it failed to bind to fibronectin. Thus, our results contrast the earlier findings on the major role of BopA in adhesion, indicating that the strong adhesion of B. bifidum to epithelial cell lines is BopA independent.     

Patterns in Temporal Variability of Temperature,Oxygen and pH along an Environmental Gradient in a Coral Reef

Spatial and temporal environmental variability are important drivers of ecological processes at all scales. As new tools allow the in situ exploration of individual responses to fluctuations, ecologically meaningful ways of characterizing environmental variability at organism scales are needed. We investigated the fine-scale spatial heterogeneity of high-frequency temporal variability in temperature, dissolved oxygen concentration, and pH experienced by benthic organisms in a shallow coastal coral reef. We used a spatio-temporal sampling design, consisting of 21 short-term time-series located along a reef flat-to-reef slope transect, coupled to a long-term station monitoring water column changes. Spectral analyses revealed sharp gradients in variance decomposed by frequency, as well as differences between physically-driven and biologically-reactive parameters. These results highlight the importance of environmental variance at organismal scales and present a new sampling scheme for exploring this variability in situ.     

Drosophila Rbp6 Is an Orthologue of Vertebrate Msi-1 and Msi-2, but Does Not Function Redundantly with dMsi to Regulate Germline Stem Cell Behaviour

The vertebrate RNA-binding proteins, Musashi-1 (Msi-1) and Musashi-2 (Msi-2) are expressed in multiple stem cell populations. A role for Musashi proteins in preventing stem cell differentiation has been suggested from genetic analysis of the Drosophila family member, dMsi, and both vertebrate Msi proteins function co-operatively to regulate neural stem cell behaviour. Here we have identified a second Drosophila Msi family member, Rbp6, which shares more amino acid identity with vertebrate Msi-1 and Msi-2 than dMsi. We generated an antibody that detects most Rbp6 splice isoforms and show that Rbp6 is expressed in multiple tissues throughout development. However, Rbp6 deletion mutants generated in this study are viable and fertile, and show only minor defects. We used Drosophila spermatogonial germline stem cells (GSC’s) as a model to test whether Drosophila Msi proteins function redundantly to regulate stem cell behaviour. However, like vertebrate Msi-1 and Msi-2, Rbp6 and Msi do not appear to be co-expressed in spermatogenic GSC’s and do not function co-operatively in the regulation of GSC maintenance. Thus while two Msi family members are present in Drosophila, the function of the family members have diverged.     

Comparison Between a Live,Attenuated Anticoccidial Vaccine and an Anticoccidial Ionophore,on Performance of Broilers Raised with or without a Growth Promoter,in an Initially Eimeria-Free Environment

An experiment was carried out to study the effects of vaccination with Paracox®, a live, attenuated vaccine against avian coccidiosis, on broilers isolated from extraneous Eimeria parasites. The study involved 3200 broiler chickens raised in floor pens similar to commercial conditions, but in an initially Eimeria-free environment. Forty percent of the chickens were vaccinated at 3 days of age and given either a basal unmedicated feed or a feed supplemented with the feed antibiotic virginiamycin. Unvaccinated birds were given either the basal feed or feed supplemented either with virginiamycin or the anticoccidial ionophore narasin. At slaughter at 36 days of age vaccinated birds had a lower live weight than non-vaccinated birds. The difference was 4.6% in unmedicated, and 6.0% in virginiamycin medicated chickens. Feed conversion ratio at slaughter was 2.5% higher for unmedicated vaccinated birds, and 1.3% higher for virginiamycin medicated vaccinated birds, compared to respective non-vaccinated groups. There was no significant difference in overall performance of unvaccinated birds given narasin as compared to virginiamycin. At 10 days post vaccination vaccinated birds had a higher number of Clostridium perfringens in the caeca, but there was no difference thereafter. Throughout the experiment, caecal clostridial counts were considerably higher in vaccinated unmedicated birds than in unvaccinated birds given narasin. The number of oocysts shed in the vaccinated groups was very low, but during a subsequent challenge with E. maxima and E. tenella the birds’ immunity was found to be satisfactory.     

Long-Term Overexpression of Hsp70 Does Not Protect against Cardiac Dysfunction and Adverse Remodeling in a MURC Transgenic Mouse Model with Chronic Heart Failure and Atrial Fibrillation

Previous animal studies had shown that increasing heat shock protein 70 (Hsp70) using a transgenic, gene therapy or pharmacological approach provided cardiac protection in models of acute cardiac stress. Furthermore, clinical studies had reported associations between Hsp70 levels and protection against atrial fibrillation (AF). AF is the most common cardiac arrhythmia presenting in cardiology clinics and is associated with increased rates of heart failure and stroke. Improved therapies for AF and heart failure are urgently required. Despite promising observations in animal studies which targeted Hsp70, we recently reported that increasing Hsp70 was unable to attenuate cardiac dysfunction and pathology in a mouse model which develops heart failure and intermittent AF. Given our somewhat unexpected finding and the extensive literature suggesting Hsp70 provides cardiac protection, it was considered important to assess whether Hsp70 could provide protection in another mouse model of heart failure and AF. The aim of the current study was to determine whether increasing Hsp70 could attenuate adverse cardiac remodeling, cardiac dysfunction and episodes of arrhythmia in a mouse model of heart failure and AF due to overexpression of Muscle-Restricted Coiled-Coil (MURC). Cardiac function and pathology were assessed in mice at approximately 12 months of age. We report here, that chronic overexpression of Hsp70 was unable to provide protection against cardiac dysfunction, conduction abnormalities, fibrosis or characteristic molecular markers of the failing heart. In summary, elevated Hsp70 may provide protection in acute cardiac stress settings, but appears insufficient to protect the heart under chronic cardiac disease conditions.     

Oxygen-18 Content of Atmospheric Oxygen Does Not Affect the Oxygen Isotope Relationship between Environmental Water and Cellulose in a Submerged Aquatic Plant, Egeria densa Planch

We determined that the oxygen isotopic composition of cellulose synthesized by a submerged plant, Egeria densa Planch., is related to the isotopic composition of environmental water by a linear function, δ¹⁸O cellulose = 0.48 δ¹⁸O water + 24.1%‰. The observation of a slope of less than 1 indicates that a portion of cellulose oxygen is derived from an isotopically constant source other than water. We tested whether this source might be molecular oxygen by growing plants in the presence of high concentrations of ¹⁸O in the form of O₂ bubbled into the bottom of an aquarium. Cellulose synthesized during this experiment did not have significantly different oxygen isotope ratios than that synthesized by control plants exposed to O₂ of normal ¹⁸O abundance. We propose that oxygen in organic matter recycled from senescent portions of the plant is incorporated into cellulose. Our findings indicate that paleoclimatic models linking the oxygen isotope composition of environmental water to cellulose from fossil plants will have to be modified to account for contributions of oxygen from this or other sources besides water.     

Oceanographic Currents and Local Ecological Knowledge Indicate,and Genetics Does Not Refute,a Contemporary Pattern of Larval Dispersal for The Ornate Spiny Lobster,Panulirus ornatus in the South-East Asian Archipelago

Here we utilize a combination of genetic data, oceanographic data, and local ecological knowledge to assess connectivity patterns of the ornate spiny lobster Panulirus ornatus (Fabricius, 1798) in the South-East Asian archipelago from Vietnam to Australia. Partial mitochondrial DNA control region and 10 polymorphic microsatellites did not detect genetic structure of 216 wild P. ornatus samples from Australia, Indonesia and Vietnam. Analyses show no evidence for genetic differentiation among populations (mtDNA control region sequences Φ_ST = -0.008; microsatellite loci F_ST = 0.003). A lack of evidence for regional or localized mtDNA haplotype clusters, or geographic clusters of microsatellite genotypes, reveals a pattern of high gene flow in P. ornatus throughout the South-East Asian Archipelago. This lack of genetic structure may be due to the oceanography-driven connectivity of the pelagic lobster larvae between spawning grounds in Papua New Guinea, the Philippines and, possibly, Indonesia. The connectivity cycle necessitates three generations. The lack of genetic structure of P. ornatus population in the South-East Asian archipelago has important implications for the sustainable management of this lobster in that the species within the region needs to be managed as one genetic stock.