Diversity in Mediterranean farm ponds: trade‐offs and synergies between irrigation modernisation and biodiversity conservation

1. Agricultural intensification has caused dramatic biodiversity loss in many agricultural landscapes over the last century. Here, we investigated whether new types of farm ponds (made of artificial substrata) in intensive systems and natural‐substratum ponds in traditional farming systems differ in their value for aquatic biodiversity conservation. 2. We analysed the main patterns of environmental variation, compared α‐, β‐ and γ‐diversity of macroinvertebrates between ponds types and evaluated the role of submerged aquatic vegetation (SAV). Generalised additive models (GAM) were used to analyse the relationships of α‐ and β‐diversity with environmental predictors, and variation partitioning to separate the effect of environmental and spatial characteristics on the variation in macroinvertebrate assemblages. Moran’s eigenvector maps (MEMs) were used to define spatial variables. 3. A principal coordinate analysis (PCoA) detected a primary environmental gradient that separated nutrient‐rich ponds from those dominated by SAV; a secondary morphometric gradient distinguished natural‐substratum ponds, with large surface area and structural complexity, from artificial‐substratum ponds with steeper slopes. Natural‐substratum ponds had almost twice the α‐ and γ‐diversity of artificial‐substratum ponds, and diversity significantly increased when SAV was present, particularly in artificial‐substratum ponds. Total phosphorus (TP) strongly contributed to explain the patterns in diversity, while SAV was a significant predictor of assemblage composition and diversity. GAMs revealed optima of both α‐diversity at intermediate SAV covers and β‐diversity at intermediate–high TP concentrations. 4. These findings have important implications for conservation planning. Adaptation of artificial‐substratum ponds by adding natural substratum and smoothing the gradient of pond margins would improve their conservation value. Development of SAV with occasional harvests and certain cautionary measures to control nutrient levels may also improve both the agronomical and environmental function of ponds.     

Characterization of a G Protein -Coupled Guanylyl Cyclase -B Receptor from Bovine Tracheal Smooth Muscle

A G protein-coupled natriuretic peptide-guanylyl cyclase receptor-B (NPR-B) located in plasma membranes from bovine tracheal smooth muscle shows complex kinetics and regulation. NPR-B was activated by natriuretic peptides (CNP-53 > ANP-28) at the ligand extracellular domain, stimulated by Gq-protein activators, such as mastoparan, and inhibited by Gi-sensitive chloride, interacting at the juxtamembrane domain. The kinase homology domain was evaluated by the ATP inhibition of Mn²⁺-activated NPR-B, which was partially reversed by mastoparan. The catalytic domain was studied by kinetics of Mn²⁺/Mg²⁺ and GTP, and the catalytic effect with GTP analogues with modifications of the /γ phosphates and ribose moieties. Most NPR-B biochemical properties remained after detergent solubilization but the mastoparan activation and chloride inhibition of NPR-B disappeared. Our results indicate that NPR-B is a highly regulated nano-machinery with domains acting at cross-talk points with other signal transducing cascades initiated by G protein-coupled receptors and affected by intracellular ligands such as chloride, Mn²⁺, Mg²⁺, ATP, and GTP.     

Elevated non-esterified fatty acid concentrations during bovine oocyte maturation compromise early embryo physiology

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism.     

Seed germination temperatures of eight Mexican Agave species with economic importance

The genetic diversity of Agave plants is threatened by clonal commercial reproduction and climatic change. Sexual reproduction is uncommon and research on seed germination is scarce. The present study evaluated the seed germination of Agave lechuguilla, Agave striata, Agave americana var. marginata, Agave asperrima, Agave cupreata, Agave duranguesis, Agave angustifolia ssp. tequilana and Agave salmiana at constant temperatures (10, 15, 20, 25, 30, 35 and 40°C). Initial imbibition (after the first 12 h) was significantly variable among species, positively correlated with seed weight (r = 0.6560, P < 0.001) and increased with temperature (from 35% at 10°C to 66% at 40°C). Temperature affected maximum imbibition (83–150%) for A. asperrima, A. lechuguilla, A. salmiana and A. striata; other species averaged 110%. Most germination kinetics best fitted a logistic model, whereas only a few treatments fit a Weibull model. The time to germination onset diminished (P < 0.05) from 125–173 h at 15°C to 68–84 h at 25°C, and then ascended to 84–196 h at 35°C. The mean germination rate and seed germination percentage after 312 h peaked at 25°C (0.50–0.95% seeds/h and 85–99%, respectively) and fell (P < 0.05) to near zero at 10 and 40°C. Temperatures of 10, 35 and 40°C were partially lethal to A. asperrima, A. duranguensis and A. salmiana seeds. The time to germination onset, seed germination percentage after 312 h and mean germination rate are best described by a Gaussian distribution, with its optimum at approximately 25°C. Thus, optimum temperatures are related to the ecological characteristics of each species area.     

The consequences of metabolic changes in high-yielding dairy cows on oocyte and embryo quality

Unsatisfactory reproductive performance in dairy cows, such as reduced conception rates, in addition to an increased incidence of early embryonic mortality, is reported worldwide and has been associated with a period of negative energy balance (NEB) early post partum. Typically, NEB is associated with biochemical changes such as high non-esterified fatty acid (NEFA), high β-hydroxybutyrate (β-OHB) and low glucose concentrations. The concentrations of these and other metabolites in the follicular fluid (FF) of high-yielding dairy cows during NEB were determined and extensively analyzed, and then were replicated in in vitro maturation models to investigate their effect on oocyte quality. The results showed that typical metabolic changes during NEB are well reflected in the FF of the dominant follicle. However, the oocyte seems to be relatively isolated from extremely elevated NEFA or very low glucose concentrations in the blood. Nevertheless, the in vitro maturation models revealed that NEB-associated high NEFA and low glucose levels in the FF are indeed toxic to the oocyte, resulting in deficient oocyte maturation and developmental competence. Induced apoptosis and necrosis in the cumulus cells was particularly obvious. Furthermore, maturation in saturated free fatty acid-rich media had a carry-over effect on embryo quality, leading to reduced cryotolerance of day 7 embryos. Only β-OHB showed an additive toxic effect in moderately hypoglycemic maturation conditions. These in vitro maturation models, based on in vivo observations, suggest that a period of NEB may hamper the fertility of high-yielding dairy cows through increased NEFA and decreased glucose concentrations in the FF directly affecting oocyte quality. In addition to oocyte quality, these results also demonstrate that embryo quality is reduced following an NEB episode. This important observation may be linked to the typical diet provided to stimulate milk yield, or to physiological adaptations sustaining the high milk production. Research into this phenomenon is ongoing.     

Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus

A cell line, PHL, has been successfully established from newly hatched herring larvae. The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing. These cells retain a diploid karotype after 65 population doublings. PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant. Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills. In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products. This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring. In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species.     

Genomic cloning of novel isotypes of the rainbow trout interleukin-8

A cDNA clone, designated IL-8nL, was obtained by suppression subtractive hybridisation between lipopolysaccharide-stimulated and non-stimulated populations of the rainbow trout macrophage-like cell line, RTS11. IL-8nL was similar but not identical to a recently published sequence of the gene encoding rainbow trout interleukin-8 (IL-8). Amplification of genomic DNA by the polymerase chain reaction (genomic PCR) using a single outbred trout with common primers in the 5' and 3' untranslated regions gave six distinct genomic sequences, including one ( IL-8A) almost identical to that of the published IL-8 gene and another identical to IL-8nL. The other four clones were termed IL-8B, IL-8C, IL-8D and IL-8E. The deduced amino acid sequences of IL-8A through IL-8E are all identical to the published IL-8, while the IL-8nL protein has a substitution of Arg87 to Lys. Analysis of ten outbred trout by genomic PCR of a repeat region in exon 4, which has three different sizes in the above alleles, revealed a shorter, fourth fragment termed IL-8X and another of the same size as IL-8nL, but with a different single nucleotide replacement, called IL-8nL2. These results, together with a Southern blot of the same ten individuals showing up to five bands, indicate that rainbow trout has at least four copies of the IL-8 gene. Like IL-8nL, IL-8X lacks the repeat sequence in exon 4 and encodes a protein identical to IL-8nL protein. Polymerase chain reaction of the repeat region was useful for typing rainbow trout into four categories, and the type III and IV fish have a new allele, IL-8F, which lacks one repeat unit compared with IL-8A.     

Evaluation of a porcine lens and fluorescence assay approach for in vitro ocular toxicological investigations

Cell biology, as monitored with the fluorescent indicator dyes Alamar Blue and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), and lens optical quality, as measured with an in vitro scanning laser system, have been used to evaluate in vitro the condition of porcine lenses after being placed in a culture medium. The measurements, beginning from week one of culture, were compared statistically. Optical quality and cellular viability, as measured with either dye, were unchanged in lenses that had been maintained for 6 weeks in modified M199 medium. Some lenses were treated with 0.152J/cm(2) UVB radiation, and a decline was observed after 48 hours in both optical and metabolic capabilities, as indicated by a decreased capacity of the lenses to reduce Alamar Blue. The measurements with CFDA-AM did not show complete concordance with the other indicators of lens health after UV treatment, making this dye less reliable as applied currently to lens cultures. Overall, the findings suggest that porcine lenses can be maintained for weeks in culture, and that their condition can be evaluated quantitatively by assays that probe cellular functions and optical properties. Such a system should prove valuable for in vitro ocular pharmacotoxicological research.