Sonication-assisted extraction of chitin from North Atlantic shrimps (Pandalus borealis)

The influence of sonication during extraction of chitin from North Atlantic shrimp (NAS) shells (Pandalus borealis) on chitin yield, purity, and crystallinity was investigated. Shells were peeled, washed, lyophilized, ground, and suspended for 4 h in 0.25 M HCl (1:40) at 40 degrees C followed by ultrasonication at 41 W/cm(2) for 0, 1, and 4 h, respectively. Demineralized shells were lyophilized, resuspended in 0.25 M NaOH (1:40), and ultrasonicated at 41 W/cm(2) for 0, 1, and 4 h to remove proteins. The yield and mineral and protein contents were determined after each processing step. The purity of extracted chitin was determined from the total amount of glucosamine. The crystallinity index and size of crystals were calculated from wide-angle X-ray scattering measurements. Scanning electron microscope images were recorded to evaluate morphological changes in samples. The yield of chitin from NAS decreased from 16.5 to 11.4% for 0 and 1 h sonicated samples, respectively, which was attributed to increased concentrations of depolymerized materials in the wash water. Sonication did not enhance the removal of minerals. The application of ultrasound enhanced the removal of proteins from 39.8 to 10.6, 8.3, and 7.3% after 0, 1, and 4 h of sonication treatments. The crystallinity index of chitin decreased from 87.6 to 79.1 and 78.5% after 1 and 4 h of sonication, yielding chitosans with crystallinity indices of 76.7, 79.5, and 74.8% after deacetylation, respectively. Fourier transform infrared spectroscopy scans indicated that the degree of acetylation of chitins was unaffected by sonication. Comparison of the extraction results of NAS with that from freshwater prawns indicated that more impurities were left in NAS chitin, suggesting that composition and structural arrangement of chitin in shells influence the efficiency of ultrasound-assisted extraction.     

Linking life history traits to pollinator loss in fragmented calcareous grasslands

To gain insight into the drivers of pollinator loss, a holistic approach to land-use change including habitat size, isolation, habitat quality and the surrounding landscape matrix is necessary. Moreover, species’ responses to land-use change may differ depending on their life history traits such as dispersal ability, trophic level, or sociality. We assessed species richness and life history traits of wild bees in 32 calcareous grasslands in central Germany that differ in size, connectivity, resource availability and landscape context. Declining habitat area and, to a lesser degree, reduced diversity of the surrounding landscape were the key factors negatively influencing species richness. In the community-wide analysis, small body size and solitary reproduction were traits that made species particularly vulnerable to habitat loss. Contrary to our expectations, cleptoparasitic species were not more affected by reduced habitat area and landscape diversity than nest-building species. We performed further detailed trait analyses within the family Halictidae to prevent possible confounding effects due to trait correlations across families. Here, social as opposed to solitary species were more affected by habitat loss. We conclude that the opposite pattern observed for all social bees was mainly caused by large-sized social bumblebee species with high mobility and large foraging distances. Our results demonstrate the risks of concealed trait interference when analyzing community-wide patterns of life history traits. As a consequence, conservation requirements of small social bee species might be overlooked by generalizations from community responses.     

Impact of surface-active compounds on physicochemical and oxidative properties of edible oil

The physical properties of lipids can have a major influence on lipid oxidation reactions. Edible oils contain surface-active compounds and water that can form physical structures such as reverse micelles. This study used the fluorescence probe, 5-dodecanoylaminofluorescein (DAF), to study both the physical and the chemical properties of stripped corn oil containing oleic acid and phosphatidylcholine. The fluorescence intensity of DAF increased with increasing water concentration in the edible oil. The addition of oleic acid decreased DAF fluorescence due to the ability of the free fatty acid to decrease the pH of the aqueous phase of the bulk oil. Phosphatidylcholine increased DAF fluorescence due to its ability to increase DAF exposure to the aqueous phase. Oleic acid had no impact on interactions between DAF and water-soluble peroxyl radicals, while phosphatidylcholine decreased peroxyl radical degradation of DAF. These results suggest that DAF could be a useful analytical tool to study the impact of the aqueous environment of bulk oil on lipid oxidation.     

Assessment of the antifungal activity of selected biocontrol agents and their secondary metabolites against <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Fusarium graminearum (teleomorph: Gibberella zeae) is the causal agent of several destructive diseases in cereal crops worldwide. In the present study we have evaluated the potential of two strains of Trichoderma sp. (T23, and T16), a strain of Paecilomyces sp. (PS1), and their secondary metabolites (SMs) in suppressing F. graminearum. Results from dual culture experiments show that in the presence of either Trichoderma sp., or Paecilomyces sp. mycelial growth of F. graminearum is considerably inhibited. Strain T23 causes the greatest inhibition (83.8%), followed by strain T16 (72.2%), and strain PS1 (61.9%). Likewise, mycelial growth of the pathogen is completely inhibited (≥ 98%) when grown under exposure to volatile metabolites excreted from Trichoderma cultures. Bioautographic analyses using culture filtrates revealed that several antifungal SMs are excreted. Among five metabolites tested, 6-pentyl-alpha-pyrone (6PAP) from strain T23, and PF3 from strain PS1 exhibit pronounced antifungal activity against F. graminearum. A new method for mass production of perithecia of F. graminearum which is simple and more effective than traditional methods was developed, which allows an increase in perithecial formation of more than 5-fold. Using this method, we found, that in the presence of SMs perithecial formation was negatively affected. Perithecial production was suppressed by 81.4% and 76.6% using 200 μg ml^?1 of either 6PAP or PF3, respectively. Moreover, ascospore discharge was significantly suppressed (67.0%) when perithecia were exposed to the metabolite F116 produced by T16. Including 6PAP or PF3 in conidial suspensions impeded germination of conidia completely. Similarly, both metabolites strongly inhibited ascospore germination (? 90%).     

Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh

Avian rotaviruses (AvRVs) represent a diverse group of intestinal viruses, which are suspected as the cause of several diseases in poultry with symptoms of diarrhoea, growth retardation or runting and stunting syndrome (RSS). To assess the distribution of AvRVs in chickens and turkeys, we have developed specific PCR protocols. These protocols were applied in two field studies investigating faecal samples or intestinal contents of diseased birds derived from several European countries and Bangladesh. In the first study, samples of 166 chickens and 33 turkeys collected between 2005 and 2008 were tested by PAGE and conventional RT-PCR and AvRVs were detected in 46.2%. In detail, 16.1% and 39.2% were positive for AvRVs of groups A or D, respectively. 11.1% of the samples contained both of them and only four samples (2.0%) contained rotaviruses showing a PAGE pattern typical for groups F and G. In the second study, samples from 375 chickens and 18 turkeys collected between 2009 and 2010 were analyzed using a more sensitive group A-specific and a new group D-specific real-time RT-PCR. In this survey, 85.0% were AvRV-positive, 58.8% for group A AvRVs, 65.9% for group D AvRVs and 38.9% for both of them. Although geographical differences exist, the results generally indicate a very high prevalence of group A and D rotaviruses in chicken and turkey flocks with cases of diarrhoea, growth retardation or RSS. The newly developed diagnostic tools will help to investigate the epidemiology and clinical significance of AvRV infections in poultry.     

Multi-scale mismatches between urban sprawl and landscape fragmentation create windows of opportunity for conservation development

Context

Urban sprawl and the expanding transportation infrastructure drive land consumption and landscape fragmentation, causing environmental deterioration and loss of species. Current understanding of how these drivers interact to shape landscape fragmentation is still poor. However, a strong correlation between urban sprawl and landscape fragmentation patterns is commonly assumed.

Objectives

Our main objective was to test the strength, non-stationarity, and scale-dependency of the relationship between urban sprawl and landscape fragmentation patterns (‘sprawl-fragmentation relationship’). Subsequently, we propose an extended framework for the links between urban sprawl, expansion of transport infrastructure, and landscape fragmentation.

Methods

We quantified spatial patterns of urban sprawl and landscape fragmentation for mainland Spain at multiple scales. We then fitted global regression models and geographically weighted regression models with metrics of landscape fragmentation and urban sprawl.

Results

Most variation in landscape fragmentation values (almost 80 % on average) is not explained by urban sprawl metrics through global modeling. Local models show substantial improvements in model performance, with an average of 37 % of the variance remaining unexplained. The contribution of urban sprawl to landscape fragmentation patterns varies locally and depends on scale, with higher contributions at coarser scales and at higher organizational levels.

Conclusions

Our investigation revealed three critical characteristics of the sprawl-fragmentation relationship: it does not prevail, is non-stationary, and scale-dependent. We propose four mechanisms that may have resulted in this mismatch: scale, time-lagged development, spatial arrangement of development, and other external variables including teleconnections. These spatial mismatches provide windows of opportunity for conservation through better development strategies.

     

Evaluation of serological tests for detecting tick-borne encephalitis virus (TBEV) antibodies in animals

Tick-borne encephalitis (TBE) in animals is not well understood yet. TBE virus (TBEV) serology in several host species could be valuable for epidemiological analyses in the field as well as for the detection of clinical cases. However, performance and suitability of the available test systems are not well assessed. Therefore, we evaluated two commercial TBEV-ELISA kits in a pilot study and compared them for their suitability in veterinary applications. For this purpose, we tested 163 field collected goat sera and evaluated the results by serum neutralization test (SNT) as "gold standard". Twenty-eight SNT positive sera (17.2%) were detected. The best suited ELISA kit was used for determination of a species-specific cutoff for horses, cattle, sheep, goats, pigs, mice, dogs, rabbits and monkeys with defined sera from animals without known or with improbable contact to TBEV. The level of non-specific ELISA results does not only differ between animal species but may also be influenced by the age of the tested animals. The number of sera which tested false positive by ELISA was higher in older than in young sheep. In order to obtain defined polyclonal sera as references, two dogs, cattle, goats, sheep, rabbits and pigs each, as well as one horse and 90 mice were immunized four times with a commercially available TBEV vaccine. In conclusion, our results demonstrated that commercial TBEV-ELISA kits are suitable for application in veterinary medicine for both, verification of clinical TBE cases and epidemiological screening. However, positive ELISA results should be verified by SNT. Only a very low number of false negative ELISA-results were found.     

The Colletotrichum higginsianum secreted effector protein ChEC91 induces plant cell death

ChEC91, a novel cell death-inducing effector protein from the fungal pathogen Colletotrichum higginsianum, causal agent of crucifer anthracnose disease, is described. Both transient expression of ChEC91 and infiltration of purified recombinant protein induced necrotic lesions in Nicotiana benthamiana leaves. The recombinant protein also induced electrolyte leakage and callose deposition in Arabidopsis thaliana leaf tissue and the expression of defence marker genes. Moreover, fungal mutants constitutively over-expressing ChEC91 in C. higginsianum were impaired in appressorial penetration on Brassica rapa cotyledons. These results suggest that inappropriate expression of ChEC91 might negatively affect the early stage of C. higginsianum infection by inducing plant defence responses. Protein domain deletion analysis showed that the C-terminal region of ChEC91 was necessary, but not sufficient, for activity in N. benthamiana. Homologous effector proteins cloned from C. gloeosporioides, Fusarium graminearum, and Pyricularia oryzae differed in their cell death-inducing activity, which appeared related to sequence variations in the C-terminal region of these proteins. Moreover, this region contained amino acid residues that were well conserved within Colletotrichum species. These results suggest that the amino acid residues in the C-terminal region may be important for inducing cell death in plants.

     

Evaluation of yield–density relationships and optimization of intercrop compositions of field-grown pea–oat intercrops using the replacement series and the response surface design

In a 2-year field experiment (2002/2003) on a loess soil near Göttingen/Germany, pea (Pisum sativum L.) and oat (Avena sativa L.) were grown alone and intercropped at a range of densities. Shoot biomass, grain yields and amount of N in grain were evaluated and optimized using two different replacement series and a hyperbolic yield–density equation describing a response surface to address the following questions: (i) what is the optimal composition of the pea–oat intercrop with regard to maximum yields, (ii) which intercropping design is most suitable to describe competition effects in pea–oat intercrops and the optimal intercrop compositions and (iii) which intercropping design is best suited for the evaluation of field data. For (i), the optimal intercrop compositions varied depending on the growth conditions for the crops. Furthermore, optimal intercrop compositions were found above the recommended sole crop densities. The density of oat had to be reduced more than that of pea, especially when optimal grain-N yields were desired and soil-N content was high. For maximum grain-N yields, pea could be sown at high densities in combination with 5–50% of the recommended density of oat. Thus, density can be used as a yield regulator for specific purposes such as a high N yield. The effects of competition at final harvest were described equally by both designs (ii). Oat was the clearly stronger and pea the inferior competitor. In contrast to the replacement series design, the hyperbolic yield–density equation was capable of adding valuable information about the extent of intra and interspecific competition. As intraspecific competition was consistently more important than interspecific competition, resource complementarity could be hold responsible for intercrop advantages. The highest intercrop advantage was found when total intraspecific competition was low, as shown by the relative yield total (RYT) and niche differentiation index (NDI) values >1. However, due to the RYT dependence on sole crops and total densities, the replacement series design led to misleading interpretations of the yield advantages. Both experimental designs were able to describe the field-data reliably (iii), but the response surface design had the advantage of being unaffected by insufficient field emergences, as it is not based on total densities. Numbers of plants m⁻² instead of seeds m⁻² can be used for the evaluation. Data from sole crops are not needed for the response surface design and thus the feared high experimental effort of this design can be reduced. However, when using the replacement series design, experimental effort should be greater than normal, as different sole crop densities and more intercrop compositions within a replacement series can lead to a more precise interpretation of the competition effects.