Characterization of humus-protease complexes extracted from soil

Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A_I with nominal molecular weight (nmw) > 100 kD, A_II with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A_II > A_I. The lowest amino acid N/organic C was found in the R fraction, whereas A_II fraction was rich in humic acids, carbohydrates and amino acid N and A_I fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A_I, A_II and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity.     

First attempts towards the restoration of gorgonian populations on the Mediterranean continental shelf

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Leishmania-specific isotype levels and their relationship with specific cell-mediated immunity parameters in canine leishmaniasis

In the current retrospective study, Leishmania infantum-specific IgG, IgA and IgM levels were determined by ELISA in 106 untreated dogs with clinically-patent leishmaniasis (Sx) and in 171 clinically healthy dogs (Asx) from Spain to investigate the relationship between these Ig isotypes and clinical status. In addition, we studied if different Leishmania-specific humoral pattern exists between Asx dogs with and without cellular mediated immunity (CMI). Fifty-six dogs were assessed by means of lymphoproliferation assay (LPA), interferon production (IFN) and leishmanin skin test (LST), 71 dogs by means of both LPA and IFN and 44 only by LST. Both Sx and Asx dogs produced Leishmania-specific IgG, IgA and IgM antibodies, however the levels and proportion of positive dogs for each Ig isotype were significantly higher in Sx than in Asx ones (P<0.001). Analysis of immunological profiles determined for each cellular technique (positive and negative cellular response for each technique combined with positive or negative specific humoral response) showed that Asx dogs constituted a high heterogeneous group. No correlations were observed between CMI tests and specific IgG or IgM levels. However, a significant inverse correlation was demonstrated between specific IgA levels and LPA response (Spearman's r=-0.220; P=0.035). In general, the low correlation detected between CMI tests and isotype levels might indicate that the immune response is not strongly polarized in the majority of Asx dogs. Additionally, this study suggests that dogs developing T-cell response are probably able to avoid the dissemination of the parasite at least to mucosal surfaces and, as a consequence, to produce low or background specific IgA levels. Further studies are needed to investigate the relationship between specific IgA and parasite load, especially at mucosal site.     

Effect of drought on diameter increment of Quercus ilex, Phillyrea latifolia, and Arbutus unedo in a holm oak forest of NE Spain

The present study was carried out to elucidate the drought growth responses of Quercus ilex L., Phillyrea latifolia L., Arbutus unedo L., and other accompanying woody species of the Mediterranean holm oak forest. We submitted holm oak forest stands in Prades mountains (NE Spain) to a 2-year experimental drought. We reduced soil water availability about 15% by plastic strips and funnels that partially excluded rain throughfall and by ditch interception of water runoff. Mean stem diameter increment showed a great variation depending on the species. A. unedo had larger growth rates than Q. ilex and P. latifolia, but it was also the species that experimented the highest growth reduction in the drought plots (77%), suggesting a higher drought sensitivity than Q. ilex (55%) and P. latifolia (no drought effect). The growth reduction was specially marked in the larger trees. Aboveground stand biomass increment, estimated from stem diameter by allometric relationships, was 1.9 Mg ha⁻¹ per year in the control plots. The 15% reduction in the upper soil moisture produced 42% reduction in this biomass increment. In the drier conditions predicted in this Mediterranean area in the frame of climate change, an important reduction of growth rates can be hence expected, accompanied by a gain of dominance of drought-tolerant species such as P. latifolia in detriment of more mesic species such as Q. ilex.     

Effects of Rapid Cooling Prior to Freezing on the Quality of Canine Cryopreserved Spermatozoa

The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality. In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsala procedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid, cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionated or unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same cooling rates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments was replicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability (spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM, PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the cooling rate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significant differences among the males (P<0.01) in the sperm quality parameters evaluated after thawing. The interaction between the males and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsala method at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionated glycerol.     

DNA‐analysis to monitor fisheries and aquaculture: Too costly?

Evidence from DNA‐analysis is commonplace in human criminal investigations, and while it is increasingly being used in wildlife crime, to date, its application to control and enforcement activities in fisheries and aquaculture has only been sporadic. Contemporary DNA‐analysis tools are capable of addressing a broad range of compliance issues, species identification, mislabelling of fish products, determining the origin of catches and the farm of origin of aquaculture escapees. Such applications have the potential to ensure traceability along the fish product supply chain and to combat consumer fraud and Illegal, Unreported and Unregulated fishing. Nevertheless, DNA‐analysis is not yet used routinely in investigations into compliance with fisheries and aquaculture legislation. One potential reason for this is that DNA‐analysis techniques may have been regarded as too expensive. However, costs have plummeted over the past decade prompting us to objectively assess whether the costs associated with routine use of DNA‐analysis techniques for fisheries and aquaculture control and enforcement activities do constitute an impediment. Based on a number of recent fisheries and aquaculture compliance investigations that incorporated DNA‐analysis, our results indicate that the use of genetic analysis was justified and worthwhile in all cases examined. We therefore conclude that the costs associated with DNA‐analysis do not represent a barrier to the routine adoption of DNA‐analysis techniques in fisheries and aquaculture compliance investigations. Thus, control and enforcement agencies should be encouraged to use such techniques routinely.     

Acute intrinsic renal failure and blood coagulation disorders after a snakebite in a dog

Acute intrinsic renal failure was diagnosed in a two-year-old, male, German shepherd dog following a Vipera aspis bite. Clinical signs included depression, hypersalivation, vomiting, tachypnoea, abdominal pain, splenomegaly, oliguria with haematuria and haemolysed serum. Leucocytosis with a shift to the left, thrombocytopenia, prolonged coagulation times (activated partial thromboplastin time, prothrombin time and thrombin time), hypofibrinogenaemia, azotaemia and hyposthenuria were the most prominent laboratory abnormalities. Histopathological evaluation of the kidneys showed a discrete glomerular hypercellularity, mesangial lysis and renal tubules filled with many hyaline casts and some necrotic cells.     

Phosphorus content in five representative landscape units of the Lomas de Arequipa (Atacama Desert-Peru)

Phosphorus forms and content were studied in soils of the Lomas de Arequipa (Atacama desert, Peru) using a fractionation method. These Lomas are small hills periodically submitted to the El Niño-Southern Oscillation (ENSO) which causes heavy rainfall. Sample soils were randomly selected in five landscape types characterized by vegetation: cactaceae (Cac), cactaceae and herbaceous (CacHerb), shrubs (Shr), trees with cover < 60% (Tree) and shrubs or trees with cover > 60%) (ShrTree). All the soils were strongly acidic and classified as loamy sand, sandy loam or silt loam. Organic carbon content was under 1% in Cac or CacHerb, then increased strongly in ShrTree (6.50%). Considering phosphorus, all the forms (labile as well resistant forms) increased markedly from Cac soils to ShrTree soils. In all the soils, the labile forms (Resin-P: range 45–105 μg g^− 1; NaHCO3-Pi: 23–123 μg g^− 1; or NaHCO3-Po: 10–122 μg g^− 1) were very high. These high phosphorus contents were attributed to the specific climatic conditions of the Lomas that feature a long period of vegetation dormancy (very dry period) and a short period of growth, following ENSO-associated precipitation. We suggested that during the dry period, plant decay and microbial cells death lead to release and accumulation of labile P in the soil, the rainfall wetting the soil, permitting vegetation growth. In this respect, the Lomas climatic conditions contribute to soil fertility, especially as labile forms of phosphorus are chiefly concerned.     

Seasonal patterns of root-surface phosphatase activities in a Mediterranean shrubland. Responses to experimental warming and drought

Mediterranean ecosystems are water limited and the current general circulation Models (GCM) and ecophysiological models forecast a warming and a further increase of drought in the next decades. A stronger water stress can decrease the capacity for nutrient absorption by plants. We conducted a field experiment to simulate forecasted drought and warming in a Mediterranean calcareous shrubland to assess the performance of root-surface phosphatase activities of the dominant shrub Globularia alypum. These enzyme activities were higher in autumn and spring, when the climate conditions were optimal for plant activity, than in summer or winter, when there was either lack of water or cold temperatures. A decrease in soil moisture in drought plots decreased root-surface phosphatase activity (29% in summer and 25% in autumn). The decrease in root-surface phosphatase activity in drought plots coincided with a decrease in P leaf concentrations and P accumulation in aboveground biomass and loss of photosynthetic capacity of some dominant shrub species of this ecosystem, and with a tendency to increase total soil-P. These results suggest that the expected drier conditions in this Mediterranean shrubland in the next decades will slow down the P uptake by plants, thereby, diminishing the P contents in biomass and increasing total P contents in soil in non-available forms and that this can be, in part, attributable to a result of the decrease in root-surface phosphatase activity.     

Factors involved in alleviating water stress by partial crop removal in pear trees

We studied the relief of water stress associated with fruit thinning in pear (Pyrus communis L.) trees during drought to determine what mechanisms, other than stomatal adjustment, were involved. Combinations of control irrigation (equal to crop water use less effective rainfall) and deficit irrigation (equal to 20% of control irrigation), fruit load (unthinned and thinned to 40 fruits per tree) and root pruning (pruned and unpruned) treatments were applied to pear (cv. 'Conference') trees during Stage II of fruit development. Daily patterns of midday stem water potential (Psi(stem)) and leaf conductance to water vapor (g(l)) of deficit-irrigated trees differed after fruit thinning. In response to fruit thinning, gl progressively declined with water stress until 30 days after fruit thinning and then leveled off, whereas the effects of decreased fruit load on Psi(stem) peaked 30-40 days after fruit thinning and then tended to decline. Soil water depletion was significantly correlated with fruit load during drought. Our results indicate that stomatal adjustment and the resulting soil water conservation were the factors determining the Psi(stem) response to fruit thinning. However, these factors could not explain differences in daily patterns between g(l) and Psi(stem) after fruit thinning. In all cases, effects of root pruning treatments on Psi(stem) in deficit-irrigated trees were transitory (Psi(stem) recovered from root pruning in less than 30 days), but the recovery of Psi(stem) after root pruning was faster in trees with low fruit loads. This behavior is compatible with the concept that the water balance (reflected by Psi(stem) values) was better in trees with low fruit loads compared with unthinned trees, perhaps because more carbon was available for root growth. Thus, a root growth component is hypothesized as a mechanism to explain the bimodal Psi(stem) response to fruit thinning during drought.