Subtilase cytotoxin encoding genes are present in human, sheep and deer intimin-negative, Shiga toxin-producing Escherichia coli O128:H2

Shiga toxin-producing Escherichia coli (STEC) O128:H2 is recognised worldwide to be an important non-O157 STEC associated with human illness and in particular with causing haemolytic uraemic syndrome. This serotype is commonly isolated from sheep and is being increasingly isolated from deer. We determined the virulence profile and genetic relationships of one human, six sheep and five deer intimin-negative STEC O128:H2 strains isolated in Spain over a 7-year period. Our goals were to establish the presence of other virulence-associated factors, such as SubAB, in intimin-negative STEC O128:H2 strains involved in human disease and in that case, to determine if sheep and/or deer represent a reservoir of SubAB-positive STEC O128:H2. All the strains lacked the eae gene and carried subtilase cytotoxin (SubAB) encoding genes (subAB) and tia genes, but not saa gene, suggesting the presence of the recently identified new variant of SubAB, encoded on a putative pathogenicity island together with tia. We report for the first time the presence of subtilase cytotoxin encoding genes in intimin-negative STEC O128:H2 strains pathogenic for humans and how this finding might explain their clinical relevance despite neither carrying eae nor stx subtypes associated with severe clinical outcomes, but only stx1c and stx2b. Multilocus sequence typing analysis revealed that STEC O128:H2 strains from sheep and deer belong to the clonal lineage of STEC O128:H2 strains involved in diarrhoeal and haemorrhagic diseases in humans. Our results indicate that sheep and deer represent a reservoir of SubAB-positive STEC O128:H2 strains and thus a potential source of human infection.     

Phenological and water-use patterns underlying maximum growing season length at the highest elevations: implications under climate change

Consequences of climate change on tree phenology are readily observable, but little is known about the variations in phenological sensitivity to drought between populations within a species. In this study, we compare the phenological sensitivity to temperature and water availability in Abies pinsapo Boiss., a drought-sensitive Mediterranean fir, across its altitudinal distribution gradient. Twig growth and needle fall were related to temperature, precipitation and plant water status on a daily scale. Stands located at the top edge of the distributional range showed the most favourable water balance, maximum growth rates and little summer defoliation. Towards higher elevations, the observed delay in budburst date due to lower spring temperatures was overcome by a stronger delay in growth cessation date due to the later onset of strong water-deficit conditions in the summer. This explains an extended growing season and the greatest mean growth at the highest elevation. Conversely, lower predawn xylem water potentials and early partial stomatal closure and growth cessation were found in low-elevation A. pinsapo trees. An earlier and higher summer peak of A. pinsapo litterfall was also observed at these water-limited sites. Our results illustrate the ecophysiological background of the ongoing altitudinal shifts reported for this relict tree species under current climatic conditions.     

Carbon mineralization rates at different soil depths across a network of European forest sites (FORCAST)

Most of the carbon (C) in terrestrial ecosystems is stored in the mineral soil layers. Thus, the response of the mineral soil to potential increases in temperature is crucial for the prediction of the impact of climate change on terrestrial ecosystems. Samples from three mineral soil layers were collected from eight mature forest sites in the European network CARBOEUROFLUX and were incubated at four temperatures (4, 10, 20 and 30°C) for c. 270 days. Carbon mineralization rates were related to soil and site characteristics. Soil water holding capacity, C content, nitrogen (N) content and organic matter all decreased with soil depth at all sites, with significantly larger amounts of organic matter, C and N in the top 0–5 cm of mineral soil than in the deeper layers. The conifer forest soils had significantly lower pH, higher C/N ratios and carbon contents in the top 5 cm than the broadleaf forest soils. Carbon mineralization rates decreased with soil depth and time at all sites but increased with temperature, with the highest rates measured at 30°C for all sites. Between 50 and 70% of the total C respired after 270 days of incubation came from the top 5 cm. The percentage C loss was small in all cases, ranging from 1 to 10%. A two‐compartment model was fitted to all data to derive the labile/active and slow/recalcitrant fractions, as well as their decomposition constants. Although the labile fraction was small in all cases, we found significantly larger amounts of labile C in the broadleaf forest soils than in the conifer forest soils. No statistically significant differences were found in the temperature sensitivity parameter Q₁₀ among sites, soil layers or between conifer and broadleaf soils. The average Q₁₀ for all soils was 2.98 (± 0.10). We found that despite large differences among sites, C mineralization can be successfully predicted as a combined function of site leaf area index, mean annual temperature and content of labile carbon in the soil (R² = 0.93).     

Alternatives in Donkey semen cryopreservation: Mare vs. Jenny Colostrum

The aim of this study was to test and compare two new components in extenders for freezing donkey semen: mare colostrum and jenny colostrum. Colostrum was obtained from four mares and four jennies right after the foal's birth. Ejaculates were collected from five fertile donkeys. Sperm samples were pooled, diluted and cryopreserved in three different experimental extender groups: lactose supplemented with egg yolk extender (20%) as the control group, lactose supplemented with jenny colostrum extender (20%), and lactose supplemented with mare colostrum extender (20%). After thawing, we evaluated the sperm motility by means of computer‐assisted analysis, viability by SYBR‐14 and propidium iodide (PI), membrane functional by HOS test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC‐PNA) and PI. The results demonstrated that lactose–jenny colostrum extender displayed significantly higher values (p < .05) in nearly all parameters evaluated – Total Motility, Viability, HOS test, VCL, VSL, VAP, LIN, STR and WOB –, compared with mare colostrum and egg yolk extenders after thawing. In conclusion, the extender containing jenny colostrum used for donkey semen cryopreservation improved the donkey sperm quality after the freezing–thawing process.     

Heat Shock Protein 70 and Sex Steroid Receptors in the Follicular Structures of Induced Ovarian Cysts

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERα than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERα in the treated group. An increase in total ERα protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERβ in animals with COD, and the total protein expression of ERβ was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.     

Changes in the spatial structure of oak carbon-based secondary compounds after pine harvesting

In natural plant populations, leaf polyphenols show high intraspecific variation that occurs both temporally and spatially. Leaf phenolics may be induced by diverse ecological factors such as light, nitrogen availability or herbivory attack. Both light and nitrogen availability can show spatial structure in forested stands, meaning that they each have a high degree of autocorrelation, which can determine the appearance of spatial structure in leaf polyphenols. However, the availability of these resources may be drastically changed by forest disturbance, and little is known about the effect of forest disturbance on the spatial pattern and scale of leaf secondary compounds. We hypothesise that the spatial structure of leaf polyphenols in understory vegetation will disappear due to forest harvesting, because these compounds depend on light availability, yet it will remain unaltered for those compounds that either depend on the availability of other resources or are under major genetic control. The study was performed in young pedunculate oak (Quercus robur) populations growing either under a pine canopy (Pinus pinaster) stand or in a pine harvested stand in NW Spain. The spatial structures of green and senescent leaf polyphenols, tannins, non-tannin polyphenols and nitrogen were analysed in both stands using geostatistical analysis. The spatial structures observed for green and senescent leaf polyphenols and tannins in the forested stand disappeared in the harvested stand. However, non-tannin polyphenols, as well as nitrogen, showed spatial structure in both stands. Understanding these changes may be important for the successful recovery of native oak populations growing under pine forests in NW Spain, one of the priorities of the local government. Our results showed that changes in the concentration of leaf secondary compounds after disturbance may be accompanied by differences in their spatial properties, which may have important consequences for ecosystem function.     

Does otolith macrostructure record environmental or biological events? The case of black hake (Merluccius polli and Merluccius senegalensis)

Fish age determination using otoliths requires a prior understanding of growth mark deposition patterns (translucent rings, TR) as well as their connection with internal or external events experienced by the fish. This study analysed the macrostructural seasonal ring deposition pattern observed in transversal sections of black hake otoliths. A total of 793 black hake otoliths were collected in autumn and spring 2007 from research and commercial surveys carried out in continental and shelf waters off Mauritania. Most of the Merluccius polli otoliths presented narrow and wide translucent rings (NTR and WTR, respectively) regardless of fish size, whereas Merluccius senegalensis otoliths only showed NTR. This seemed to be a sign of ontogenetic discrepancy between the two black hake species, whose otoliths confirmed the existence of significant differences in their growth patterns.The frequency distributions of the number of TR counted along the ventral radius (VR) of the otolith from the nucleus (birth date) and from the ventral edge (death date) were analysed to ascertain whether a specific endogenous event (Hypothesis A) or a precise environmental event (Hypothesis B) could restrain growth leading to the formation of TR. The general TR frequency distribution pattern was somewhat similar for both hake species, which showed marked TR at comparable distances. Within each species, TR frequency distributions of their distances from the nucleus along the otolith VR were quite similar between sexes, seasons, and fish sizes. Our results support the idea of a coincident biological event, such as first maturity, slowing down the growth process and thus provoking the formation of TR in otoliths of both species.This study also experience difficulty using the complex and highly variable macrostructural pattern of black hake otoliths to establish age interpretation criteria for these two species.     

Contribution and stability of forest-derived soil organic carbon during woody encroachment in a tropical savanna. A case study in Gabon

In this study, we quantified the contribution of forest-derived carbon (FDC) to the soil organic C (SOC) pool along a natural succession from savanna (S) to mixed Marantaceae forest (MMF) in the Lopè National Park, Gabon. Four 1-ha plots, corresponding to different stages along the natural succession, were used to determine the SOC stock and soil C isotope composition (δ¹³C) to derive the FDC contribution in different soil layers down to 1 m depth. Besides, to investigate changes in SOC stability, we determined the ¹⁴C concentration of SOC to 30 cm depth and derived turnover time (TT). Results indicated that SOC increased only at the end of the succession in the MMF stage, which stored 46% more SOC (41 Mg C ha^?1) in the 0–30 cm depth than the S stage (28.8 Mg C ha^?1). The FDC contribution increased along forest succession affecting mainly the top layers of the initial successional stages to 15 cm depth and reaching 70 cm depth in the MMF stage. The TT suggests a small increase in stability in the 0–5 cm layer from S (146 years) to MMF (157 years) stages. Below 5 cm, the increase in stability was high, suggesting that FDC can remain in soils for a much longer time than savanna-derived C. In conclusion, the natural succession toward Marantaceae forests can positively impact climate change resulting in large SOC stocks, which can be removed from the atmosphere and stored for a much longer time in forest soils compared to savanna soils.