Doing what with whom? Stakeholder analysis in a large transdisciplinary research project in South-East Asia

The transdisciplinary research project LEGATO analysed the combined generation of provisioning, regulating and cultural ecosystem services in wet rice agriculture in South-East Asia and applied ecological engineering to future-proof it against global change and environmental pollution challenges. Due to its transdisciplinary character and the sheer size, a systematic stakeholder involvement was inevitable. Starting with stakeholder identification by snowballing from a limited number of contacts, we derived a multi-level stakeholder analysis and tried to involve those identified as relevant. Applying different means and aiming at different depth of involvement, the effort can be judged successful. The paper describes the methods used to identify and classify stakeholders, and key elements of the stakeholder management, guided by the BiodivERsA Stakeholder Activation Handbook. While on the local level, farmers and extension workers turned out to be influential and interested stakeholders in both countries, as well as local (PH) or provincial authorities (VN), differences were manifest on higher levels due to the divergent institutional setting. National-level agents were hard to get interested in both countries, and influential agents along the production chain, including middle men, were not really interested in collaboration.     

Effect of different culture conditions on the structural diversity of prokaryote communities in the sediment of earth ponds stocked with gilthead seabream Sparus aurata (Linnaeus, 1758)

In this study, we evaluate the use of polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) for monitoring the effect of different aquaculture practices on sediment prokaryote (Archaea and Bacteria) communities. The effect of initial fish (gilthead seabream Sparus aurata) stocking density on the structural diversity of prokaryote communities of earth ponds bottom sediments was evaluated using PCR‐DGGE after a 5 month grow‐out period. An identical approach was used to monitor the effect of supplying different fish feeds [commercial feed (CD) versus an ecofeed (ECO)]. One additional variable was the use of copper sulphate (CuSO₄) as an algicide in some of the experimental rearing tanks. The statistical analyses of prokaryote community profiles showed that the presence of fish in earth ponds significantly influenced the structure of sediment prokaryote communities, when compared with earth ponds without fish, independently of the stocking density. Our results also indicated that the structure of the prokaryote communities of earth ponds supplied with the ECO feed shared a strong similarity with that fed CD. Curiously, the use of CuSO₄ in ponds receiving the ECO feed promoted significant differences on the structural composition of the bacterial community, but not on the archaeal community. DGGE molecular fingerprints are suitable for fast evaluation of new management practices in food‐fish production on earth ponds by monitoring shifts on microbial communities in bottom sediments.     

Morphological characterization of ‘Oblačinska’ sour cherry by multivariate analysis

A wide range of ‘Obla?inska’ sour cherry germplasm was collected from the most important growing regions in Serbia. Twenty nine variables were recorded in 41 accessions to determine the overall degree of polymorphism and to detect similarities among genotypes. The highest degree of variability was observed in traits related to number and composition of fruiting twigs, fruit set and yield. The majority of significant correlation coefficients were found in the characteristics representing fruit and leaf size. Principal component analysis (PCA) makes the phenotypical diversity determination and separation of similar genotypes possible. Variables with high discriminating values were those related to fruit and leaf size, yield efficiency, blooming time and flowering density. Based on the results from this study, the genotypes XI/3 and II/10 can be recommended for cultivation. The clonal selection and conservation of the highly diverse autochthonous population of sour cherry are also recommended.     

Relationship of wild and cultivated forms of <Emphasis Type="Italic">Pisum</Emphasis> L. as inferred from an analysis of three markers,of the plastid,mitochondrial and nuclear genomes

Eighty-nine accessions of wild and cultivated peas (12 Pisum fulvum Sibth. et Smith., 7 P. abyssinicum A. Br., 31 wild and 42 cultivated forms of P. sativum L.) were analysed for presence of the variants of three functionally unrelated polymorphic markers referring to different cellular genomes. The plastid gene rbcL either contains or not the recognition site for restriction endonuclease AspLEI (rbcL+ vs. rbcL−); the mitochondrial gene cox1 either contains or not the recognition site for restriction endonuclease PsiI (cox1+ vs. cox1−); the nuclear encoded seed albumin SCA is represented by slow (SCA^S) or fast (SCA^F) variant. Most of the accessions possessed either of two marker combinations: 24 had SCA^F cox1+ rbcL+ (combination A) and 49 accessions had SCA^S cox1− rbcL− (combination B), 16 accessions represented 5 of the rest 6 possible combinations. All accessions of P. fulvum and P. abyssinicum had combination A, the overwhelming majority of cultivated forms of P. sativum had combination B while wild representatives of P. sativum had both combinations A and B, as well as rare combinations. This pattern indicates that combination A is the ancestral state in the genus Pisum L., inherited by P. fulvum and P. abyssinicum, while combination B seems to have arisen in some lineage of wild P. sativum which rapidly fixed mutational transitions of the three markers studied, most probably via a bottleneck effect during the Pleistocene. Then this ‘lineage B’ spread over Mediterranean and also gave rise to cultivated forms of P. sativum. Rare combinations may have resulted from occasional crosses between ‘lineage A’ and ‘lineage B’ in nature or during cultivation, or represent intermediate evolutionary lineages. The latter explanation seems relevant for an Egyptian cultivated form ‘Pisum jomardii Schrank’ (SCA^F cox1− rbcL−) which is here given a subspecies rank. Wild representatives of P. sativum could be subdivided in two subspecies corresponding to ‘lineage A’ and ‘lineage B’ but all available subspecies names seem to belong to lineage B only. Presently all wild forms would better be considered within a fuzzy paraphyletic subspecies P. sativum subsp. elatius (Bieb.) Schmalh. s. l.     

Microsatellite data suggest significant population structure and differentiation within the malaria vector Anopheles darlingi in Central and South America

Background

Anopheles darlingi is the most important malaria vector in the Neotropics. An understanding of A. darlingi's population structure and contemporary gene flow patterns is necessary if vector populations are to be successfully controlled. We assessed population genetic structure and levels of differentiation based on 1,376 samples from 31 localities throughout the Peruvian and Brazilian Amazon and Central America using 5–8 microsatellite loci.

Results

We found high levels of polymorphism for all of the Amazonian populations (mean R _S = 7.62, mean H _O = 0.742), and low levels for the Belize and Guatemalan populations (mean R _S = 4.3, mean H _O = 0.457). The Bayesian clustering analysis revealed five population clusters: northeastern Amazonian Brazil, southeastern and central Amazonian Brazil, western and central Amazonian Brazil, Peruvian Amazon, and the Central American populations. Within Central America there was low non-significant differentiation, except for between the populations separated by the Maya Mountains. Within Amazonia there was a moderate level of significant differentiation attributed to isolation by distance. Within Peru there was no significant population structure and low differentiation, and some evidence of a population expansion. The pairwise estimates of genetic differentiation between Central America and Amazonian populations were all very high and highly significant (F _ST = 0.1859 – 0.3901, P < 0.05). Both the D _A and F _ST distance-based trees illustrated the main division to be between Central America and Amazonia.

Conclusion

We detected a large amount of population structure in Amazonia, with three population clusters within Brazil and one including the Peru populations. The considerable differences in N _e among the populations may have contributed to the observed genetic differentiation. All of the data suggest that the primary division within A. darlingi corresponds to two white gene genotypes between Amazonia (genotype 1) and Central America, parts of Colombia and Venezuela (genotype 2), and are in agreement with previously published mitochondrial COI gene sequences interpreted as incipient species. Overall, it appears that two main factors have contributed to the genetic differentiation between the population clusters: physical distance between the populations and the differences in effective population sizes among the subpopulations.     

Identification of genomic islands in Chilean Piscirickettsia salmonis strains and analysis of gene expression involved in virulence

Piscirickettsia salmonis, an agent of Piscirickettsiosis, is the cause of major losses in the Chilean salmon industry. We identified, characterized and bioinformatically analysed genomic islands in field strains of P. Salmonis, using the bioinformatic software PIPS, that uses the characteristics of the islands of pathogenicity to identify them. We analysed nine partially sequenced genomes in different new field strains, and compared them with the LF‐89 (Type strain) genome, selecting a genomic island present in all of them. We then evaluated the relative expression of three genes present in that island. From the obtained results, we conclude that the expression of the tcf gene is directly proportional to the cytopathogenicity in vitro of the bacteria; the product of the dnsa gene could contribute to its pathogenicity, but would be potentiated by one or more factors. The product of the gene liso is necessary for the virulence process and could have functions in early stages of infection. Regarding the strains, the IBM‐040 strain showed a significant increase in the expression of all the genes in the study. Contrarily, LF‐89 only presented a significant increase in expression of the gene liso, which correlates with the cytopathogenicity in vitro observed in the SHK‐1 cells.     

Interactions of Protein and Starch Studied Through Amyloglucosidase Action

The access of amyloglucosidase to the carbohydrate molecule was taken as a measure of interactions occurring among starch, amylose, amylopectin, β-dextrin and purified gluten, gliadins, high molecular weight glutenin subunits, or bovine serum albumin when they were mixed together and gelatinized before digestion. The most relevant decrease in liberated glucose, denoting coverage of some reaction sites for amyloglucosidase, occurred when gliadins were mixed with the carbohydrates. Other proteins were not as effective as gliadins. Amylopectin was not affected by any protein. Comparison of results allows some hypothesis to be formulated about the influence of structure on molecular interactions.     

Impact of Degree of Ionization and PEGylation on the Stability of Nanoparticles of Chitosan Derivatives at Physiological Conditions

Nowadays, the therapeutic efficiency of small interfering RNAs (siRNA) is still limited by the efficiency of gene therapy vectors capable of carrying them inside the target cells. In this study, siRNA nanocarriers based on low molecular weight chitosan grafted with increasing proportions (5 to 55%) of diisopropylethylamine (DIPEA) groups were developed, which allowed precise control of the degree of ionization of the polycations at pH 7.4. This approach made obtaining siRNA nanocarriers with small sizes (100–200 nm), positive surface charge and enhanced colloidal stability (up to 24 h) at physiological conditions of pH (7.4) and ionic strength (150 mmol L⁻¹) possible. Moreover, the PEGylation improved the stability of the nanoparticles, which maintained their colloidal stability and nanometric sizes even in an albumin-containing medium. The chitosan-derivatives displayed non-cytotoxic effects in both fibroblasts (NIH/3T3) and macrophages (RAW 264.7) at high N/P ratios and polymer concentrations (up to 0.5 g L⁻¹). Confocal microscopy showed a successful uptake of nanocarriers by RAW 264.7 macrophages and a promising ability to silence green fluorescent protein (GFP) in HeLa cells. These results were confirmed by a high level of tumor necrosis factor-α (TNFα) knockdown (higher than 60%) in LPS-stimulated macrophages treated with the siRNA-loaded nanoparticles even in the FBS-containing medium, findings that reveal a good correlation between the degree of ionization of the polycations and the physicochemical properties of nanocarriers. Overall, this study provides an approach to enhance siRNA condensation by chitosan-based carriers and highlights the potential of these nanocarriers for in vivo studies.     

Leaching of dissolved and particulate phosphorus via preferential flow pathways in a forest soil: An approach using zero‐tension lysimeters

Phosphorus (P) fluxes from forest soils are not well understood. For temperate zone forests, there is some evidence from watershed studies that P export occurs mainly in preferential flow pathways during storm events after dry periods. Therefore, we tested the practical applicability of a sampling approach, which should allow for quantifying P concentrations and fluxes. We used zero‐tension lysimeters, which were installed beneath the topsoil and in the subsoil at plots in the Tharandt Forest (Saxony, East Germany). Two storm events after dry periods were simulated by means of experimental sprinkler irrigation. Preferential flow water collected with the zero‐tension lysimeters was analyzed for total P (TP), particulate P (PP), dissolved organic P (DOP) and dissolved inorganic P (DIP), and the respective P fluxes were calculated. The results indicate that the experimental approach is applicable to estimate fluxes of different P fractions in preferential flow. Measured data point toward a distinct flushing of P in the first hours of heavy rainfall events with PP fraction playing a dominant role. In general, P concentrations decreased quickly during the first 2 h of irrigation and then remained constant. Initial concentrations and cumulative fluxes were highest in the subsoil samplers. For a better understanding of underlying processes, further research with a special focus on the PP fraction is necessary.     

Partial lateral corpectomy for ventral extradural thoracic spinal cord compression in a cat

A 7-year-old, female spayed, domestic shorthair cat was presented for ambulatory paraparesis. No trauma history was reported. Myelography and subsequent computed tomography revealed multiple ventrally located extradural spinal cord compressive lesions possibly due to intervertebral disc disease. Compression at the level of Th3-Th4 intervertebral disc space was considered responsible for the paraparesis. The lesion was approached via a right-sided lateral partial corpectomy as described for dogs. Complete spinal decompression was achieved, as documented intraoperatively by visual inspection and palpation of the spinal canal. No surgery related complications were encountered and the cat improved gradually within 8 weeks after the procedure. At 1 year follow-up only a slight proprioceptive deficit in the right hind limb could be noted. This is the first report of partial lateral corpectomy in a cat and should encourage the use of this technique even in small patients.