Spatial prediction of caterpillar (<Emphasis Type="Italic">Ormiscodes</Emphasis>) defoliation in Patagonian <Emphasis Type="Italic">Nothofagus</Emphasis> forests

In the temperate forests of the southern Andes, southern beech species (Nothofagus), the dominant tree species of the region, experience severe defoliation caused by caterpillars of the Ormiscodes genus (Lepidoptera: Saturniidae). Despite the recent increase in defoliation frequency in some areas, there is no quantitative information on the spatial extent and dynamics of these outbreaks. This study examines the spatial patterns of O. amphimone outbreaks in relation to landscape heterogeneity. We mapped defoliation events caused by O. amphimone in northern (ca. 40–41°S) and southern Patagonian (ca. 49°S) Nothofagus forests from Landsat imagery and analyzed their spatial associations with vegetation cover type, topography (elevation, slope angle, aspect) and mean annual precipitation using overlay analyses. We used these data and relationships to develop a logistic regression model in order to generate maps of predicted susceptibility to defoliation by O. amphimone for each study area. Forests of N. pumilio are typically more susceptible to O. amphimone outbreaks than lower elevation forests of other Nothofagus species (N. dombeyi and N. antarctica). Stands located at intermediate elevations and on gentle slopes (<15°) are also more susceptible to defoliation than higher and lower elevation stands located on high angle slopes. Stands in areas with intermediate to high precipitation, relative to the distribution of Nothofagus along the precipitation gradient, are more susceptible to O. amphimone attack than are drier areas. Our study represents the first mapping and spatial analysis of insect defoliator outbreaks in Nothofagus forests in South America.     

Transient co-expression with three <Emphasis Type="Italic">O</Emphasis>-glycosylation enzymes allows production of GalNAc-<Emphasis Type="Italic">O</Emphasis>-glycosylated Granulocyte-Colony Stimulating Factor in <Emphasis Type="Italic">N. benthamiana</Emphasis>

Background

Expression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability.

Results

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification.

Conclusions

The pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.

     

A plastome primer set for comprehensive quantitative real time RT-PCR analysis of <Emphasis Type="Italic">Zea mays</Emphasis>: a starter primer set for other <Emphasis Type="Italic">Poaceae</Emphasis> species

Background  

Quantitative Real Time RT-PCR (q2(RT)PCR) is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RT)PCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species.     

Protocol: Optimised methodology for isolation of nuclei from leaves of species in the <Emphasis Type="Italic">Solanaceae</Emphasis> and <Emphasis Type="Italic">Rosaceae</Emphasis> families

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.     

Influence of patch and landscape characteristics on the distribution of the subterranean rodent <Emphasis Type="Italic">Ctenomys porteousi</Emphasis>

The understanding and prediction of the responses of animal populations to habitat fragmentation is a central issue in applied ecology. The identification of habitat variables associated to patch occupancy is particularly important when habitat quality is affected by human activities. Here, we analyze the influence of patch and landscape characteristics on patch occupancy by the subterranean herbivorous rodent Ctenomys porteousi. Patch occupancy was monitored in a network of 63 habitat patches identified by satellite imagery analysis which extends along almost the whole distributional range for C. porteousi. Suitable habitat for the occurrence of C. porteousi is highly fragmented and represents <10% of the total area in its distributional range. The distribution of C. porteousi in the patch network is affected not only by characteristics of the habitat patches, but also by those of the surrounding landscape matrix. Significant differences between occupied and empty patches were found in several environmental variables. Overall, occupied patches were larger, less vegetated, more connected, and had larger neighbor patches than empty patches. A stepwise procedure on a generalized linear model selected four habitat variables that explain patch occupancy in C. porteousi; it included the effects of habitat quality in the matrix surrounding the patch, average vegetation cover in the patch, minimum vegetation cover in the matrix surrounding the patch, and the area of the nearest neighbor patch. These results indicate that patch occupancy in C. porteousi is strongly influenced by the availability and quality of habitat both in the patch and in the surrounding landscape matrix.     

Shaken but not stirred: multiscale habitat suitability modeling of sympatric marten species (<Emphasis Type="Italic">Martes martes</Emphasis> and <Emphasis Type="Italic">Martes foina</Emphasis>) in the northern Iberian Peninsula

Context

Multispecies and multiscale habitat suitability models (HSM) are important to identify the environmental variables and scales influencing habitat selection and facilitate the comparison of closely related species with different ecological requirements.

Objectives

This study explores the multiscale relationships of habitat suitability for the pine (Martes martes) and stone marten (M. foina) in northern Spain to evaluate differences in habitat selection and scaling, and to determine if there is habitat niche displacement when both species coexist.

Methods

We combined bivariate scaling and maximum entropy modeling to compare the multiscale habitat selection of the two martens. To optimize the HSM, the performance of three sampling bias correction methods at four spatial scales was explored. HSMs were compared to explore niche differentiation between species through a niche identity test.

Results

The comparison among HSMs resulted in the detection of a significant niche divergence between species. The pine marten was positively associated with cooler mountainous areas, low levels of human disturbance, high proportion of natural forests and well-connected forestry plantations, and medium-extent agroforestry mosaics. The stone marten was positively related to the density of urban areas, the proportion and extensiveness of croplands, the existence of some scrub cover and semi-continuous grasslands.

Conclusions

This study outlines the influence of the spatial scale and the importance of the sampling bias corrections in HSM, and to our knowledge, it is the first comparing multiscale habitat selection and niche divergence of two related marten species. This study provides a useful methodological framework for multispecies and multiscale comparatives.

     

The Genus <Emphasis Type="Italic">Fragaria </Emphasis>in Croatia

Only three native species of genus Fragaria (F. moschata, F. vesca and F. viridis) are recorded in three regions of Croatia. These species as well as many of their hybrids, are, or once were, cultivated for their edible fruits. The majority of cultivated strawberries in Europe belong to garden strawberries F. x ananassa (hybrids of F. chiloensis and F. virginiana). The most expanded wild strawberry species in Croatia is a woodland strawberry (F. vesca) whose berries are gathered seasonally as wild edible fruits. They often contain higher amount of nutrients and bioactive compounds in comparison to cultivated varieties. The research on the genus Fragaria species distribution in Croatia has not been carried out, and so is the case with many others wild growing fruit species in Croatia. By summing up a number of individual citations and observations, it is possible to get a perspective regarding the current state of their distribution.     

Isolation of intact sub-dermal secretory cavities from <Emphasis Type="Italic">Eucalyptus</Emphasis>

Background  

The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Eucalyptus with cavities that are relatively large and rich in essential oils.     

Optimization of Factors Affecting <Emphasis Type="Italic">In vitro</Emphasis> Establishment, <Emphasis Type="Italic">Ex vitro</Emphasis> Rooting and Hardening for Commercial Scale Multiplication of Silk Banana (<Emphasis Type="Italic">Musa</Emphasis> AAB)

Factors concerning aseptic culture establishment and hardening were studied in detail in choicest Silk Banana. Effect of size of sucker (small, medium and large), carbon concentration (10, 20 and 30 g/l), season of initiation (wet and dry) and pre-treatments such as segmentation and incision to the explants were studied during initiation. Further, hardening related factors such as substrates used for primary and secondary hardening, nursery nutrition (source and frequency of application) and pre-treatments for ex vitro rooting were also studied. Results revealed that small suckers were most suited for initiations with the least contamination, maximum establishment and higher percentage of greening. Lower concentration of sucrose, though delayed greening, resulted in 100?% establishment of explants. Initiations performed during the drier period were completely free from the fungal contamination and showed less bacterial contamination than those performed during the rainy season. Segmentation of explant into four parts during first subculture supported maximum shoot proliferation by overcoming apical dominance. Coir pith was observed to be the most congenial substrate during primary hardening, whereas coir pith alone or sand: red earth: coir pith (1:1:1) supported superior performance of plantlets during secondary hardening. Single application of mono ammonium phosphate improved growth of plantlets during secondary hardening. Pre-treatment with mono ammonium phosphate gave the best response in terms of rooting and hardening of un-rooted micro-shoots. Thus, the discussed methodology could help the industries to take up commercial scale propagation of Silk Banana.     

Timing of breeding in <Emphasis Type="Italic">Ochrotomys nuttalli</Emphasis> and <Emphasis Type="Italic">Peromyscus leucopus</Emphasis> is related to a latitudinal isotherm

Previous comparative studies on patterns of reproduction in small-mammal species focus primarily on latitudinal differences in average litter size. Few studies compare reproductive patterns among northern and southern populations at the landscape scale. Our study compares differences in seasonal patterns of reproduction in northern and southern populations of the golden mouse, Ochrotomys nuttalli, and the white-footed mouse, Peromyscus leucopus. These are remarkably similar species with regard to bioenergetics, body mass, feeding behavior, home-range size, natural history, nest-site preference, and periods of activity. Both species also exhibit very similar intraspecific seasonal patterns of reproduction across their respective geographic ranges. We found that O. nuttalli and P. leucopus switch from a summer breeding season, extending from late spring through early autumn in the north to a winter breeding season extending from late autumn through early spring in the south, near the isotherm where mean annual temperature is 15.6°C (60°F), or approximately 35° N latitude. This latitudinal isotherm provides a geographic benchmark to address future changes in patterns of reproduction attributed to climate change. Findings also suggest that length of the breeding season and patterns of reproduction between species partially explain why P. leucopus is typically more abundant than O. nuttalli in similar habitat types.     

Rapid metabolic profiling of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> defence responses against <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> using direct infrared laser desorption ionization mass spectrometry and principal component analysis

Background  

Successful defence of tobacco plants against attack from the oomycete Phytophthora nicotianae includes a type of local programmed cell death called the hypersensitive response. Complex and not completely understood signaling processes are required to mediate the development of this defence in the infected tissue. Here, we demonstrate that different families of metabolites can be monitored in small pieces of infected, mechanically-stressed, and healthy tobacco leaves using direct infrared laser desorption ionization orthogonal time-of-flight mass spectrometry. The defence response was monitored for 1 - 9 hours post infection.     

High-level diterpene production by transient expression in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Background

Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.

Results

Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm².

Conclusion

With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.

     

A multi-scale assessment of population connectivity in African lions (<Emphasis Type="Italic">Panthera leo)</Emphasis> in response to landscape change

Context

Habitat loss and fragmentation are among the major drivers of population declines and extinction, particularly in large carnivores. Connectivity models provide practical tools for assessing fragmentation effects and developing mitigation or conservation responses. To be useful to conservation practitioners, connectivity models need to incorporate multiple scales and include realistic scenarios based on potential changes to habitat and anthropogenic pressures. This will help to prioritize conservation efforts in a changing landscape.

Objectives

The goal of our paper was to evaluate differences in population connectivity for lions (Panthera leo) across the Kavango-Zambezi Trans-frontier Conservation Area (KAZA) under different landscape change scenarios and a range of dispersal distances.

Methods

We used an empirically optimized resistance surface, based on analysis of movement pathways of dispersing lions in southern Africa to calculate resistant kernel connectivity. We assessed changes in connectivity across nine landscape change scenarios, under each of which we explored the behavior of lions with eight different dispersal abilities.

Results

Our results demonstrate that reductions in the extent of the protected area network and/or fencing protected areas will result in large declines in the extent of population connectivity, across all modeled dispersal abilities. Creation of corridors or erection of fences strategically placed to funnel dispersers between protected areas increased overall connectivity of the population.

Conclusions

Our results strongly suggest that the most effective means of maintaining long-term population connectivity of lions in the KAZA region involves retaining the current protected area network, augmented with protected corridors or strategic fencing to direct dispersing individuals towards suitable habitat and away from potential conflict areas.

     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence,under half of the adaptive growth-irradiance,for high-throughput sensing of leaf-water deficit in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> accessions

Background

Non-invasive and high-throughput monitoring of drought in plants from its initiation to visible symptoms is essential to quest drought tolerant varieties. Among the existing methods, chlorophyll a fluorescence (ChlF) imaging has the potential to probe systematic changes in photosynthetic reactions; however, prerequisite of dark-adaptation limits its use for high-throughput screening.

Results

To improve the throughput monitoring of plants, we have exploited their light-adaptive strategy, and investigated possibilities of measuring ChlF transients under low ambient irradiance. We found that the ChlF transients and associated parameters of two contrasting Arabidopsis thaliana accessions, Rsch and Co, give almost similar information, when measured either after ~20 min dark-adaptation or in the presence of half of the adaptive growth-irradiance. The fluorescence parameters, effective quantum yield of PSII photochemistry (Φ_PSII) and fluorescence decrease ratio (R _FD) resulting from this approach enabled us to differentiate accessions that is often not possible by well-established dark-adapted fluorescence parameter maximum quantum efficiency of PSII photochemistry (F _V/F _M). Further, we screened ChlF transients in rosettes of well-watered and drought-stressed six A. thaliana accessions, under half of the adaptive growth-irradiance, without any prior dark-adaptation. Relative water content (RWC) in leaves was also assayed and compared to the ChlF parameters. As expected, the RWC was significantly different in drought-stressed from that in well-watered plants in all the six investigated accessions on day-10 of induced drought; the maximum reduction in the RWC was obtained for Rsch (16%), whereas the minimum reduction was for Co (~7%). Drought induced changes were reflected in several features of ChlF transients; combinatorial images obtained from pattern recognition algorithms, trained on pixels of image sequence, improved the contrast among drought-stressed accessions, and the derived images were well-correlated with their RWC.

Conclusions

We demonstrate here that ChlF transients and associated parameters measured even in the presence of low ambient irradiance preserved its features comparable to that of measured after dark-adaptation and discriminated the accessions having differential geographical origin; further, in combination with combinatorial image analysis tools, these data may be readily employed for early sensing and mapping effects of drought on plant’s physiology via easy and fully non-invasive means.

     

How landscape,pollen intake and pollen quality affect colony growth in <Emphasis Type="Italic">Bombus terrestris</Emphasis>

Context

Abundance and diversity of bumblebees have been declining over the past decades. To successfully conserve bumblebee populations, we need to understand how landscape characteristics affect the quantity and quality of floral resources collected by colonies and subsequently colony performance.

Objectives

We therefore investigated how amount and composition of pollen collected by buff-tailed bumblebee Bombus terrestris colonies was affected by the surrounding landscape (i.e. the proportion of forest, urban, semi-natural habitats) and how they were related to colony growth.

Methods

Thirty B. terrestris colonies were placed at grassland sites differing in surrounding landscape. Colonies were established in spring when availability of flowering plants was highest, and their weight gain was monitored for 1 month. We additionally recorded the quantity and compared plant taxonomic composition and nutritional quality (i.e. amino acid composition) of pollen stored.

Results

Bumblebee colonies varied little in the pollen spectra collected despite differences in surrounding landscape composition. They collected on average 80 % of pollen from woody plants, with 34 % belonging to the genus Acer. Early colony growth positively correlated with total amount of woody pollen and protein collected and decreased with increasing proportions of semi-natural habitats and total amino acid concentrations.

Conclusions

Our results suggest that woody plant species represent highly important pollen sources for the generalist forager B. terrestris early in the season. We further show that colony growth of B. terrestris is predominantly affected by the quantity, not quality, of forage, indicating that several abundant plant species flowering throughout the bumblebees’ foraging season may cover the colonies’ nutritional needs.

     

Spatial scaling and multi-model inference in landscape genetics: <Emphasis Type="Italic">Martes americana</Emphasis> in northern Idaho

Individual-based analyses relating landscape structure to genetic distances across complex landscapes enable rigorous evaluation of multiple alternative hypotheses linking landscape structure to gene flow. We utilize two extensions to increase the rigor of the individual-based causal modeling approach to inferring relationships between landscape patterns and gene flow processes. First, we add a univariate scaling analysis to ensure that each landscape variable is represented in the functional form that represents the optimal scale of its association with gene flow. Second, we use a two-step form of the causal modeling approach to integrate model selection with null hypothesis testing in individual-based landscape genetic analysis. This series of causal modeling indicated that gene flow in American marten in northern Idaho was primarily related to elevation, and that alternative hypotheses involving isolation by distance, geographical barriers, effects of canopy closure, roads, tree size class and an empirical habitat model were not supported. Gene flow in the Northern Idaho American marten population is therefore driven by a gradient of landscape resistance that is a function of elevation, with minimum resistance to gene flow at 1500 m.     

Genetic Diversity Among <Emphasis Type="Italic">Juglans Regia</Emphasis> Genotypes Based on Morphological Characters of Nut

Walnut germplasm from three different sites in Oltenia region, located in the southwestern part of Romania, was evaluated to determine the variability and to identify promising types. A total of 64 genotypes were evaluated using morphological characteristics of nuts. Considerable genetic variation in the measured traits was found in native trees which were of seedling origin. Variability found was between 7.62–20.90?g in nut weight, between 31.17 and 53.60?mm in nut length, between 24.81 and 37.48?mm in nut diameter I, and between 23.64 and 36.18?mm in nut diameter II. Separation of the three areas in multivariate space was assessed by calculating the Mahalanobis distance. The highest inter-populations value was observed between Bechet and Ostroveni population (1.83), followed by Sadova and Ostroveni population (1.08), which indicates a wide genetic distance between these populations. Cluster analyses showed a considerable diversity within the same germplasm population. The results indicated that most of genotypes were suitable in terms of morphological traits of nut. These data demonstrated large phenotypic diversity of nut among the selected genotypes that can be considered as a valuable gene stock for breeding programs.     

Editing of the urease gene by CRISPR-Cas in the diatom <Emphasis Type="Italic">Thalassiosira pseudonana</Emphasis>

Background

CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas.

Results

A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate.

Conclusions

CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.