Morphological and free amino acid profile variability,as a tool for stock identification among farmed rainbow trout Oncorhynchus mykiss of different origin

As a tool for stock identification the body morphology and the free amino acid (FAA) profile variability were determined among farmed rainbow trout Oncorhynchus mykiss (Walbaum 1792) from different geographical areas throughout Greece, using geometric morphometrics and Reversed Phase‐High‐Performance Liquid Chromatography. Significant intraspecific body shape variation was detected among the fish stocks of different origin. Canonical Variate Analysis distinguished populations into two groups being consistent to a great extent with a previous population genetics study. General linear models did not support an association of haplotypes and diet with shape. However, shape was significantly correlated with origin, water temperature, geological‐chemical‐climatic zones, histidine and arginine. Therefore, morphological variation was primarily driven by environmentally induced differences among these zones being the result of phenotypic plasticity. Environmental cues and rearing conditions played the pivotal role in comparison to broodstock origin. Specimens presented different FAA profiles depending on their geographical origin. Principal Component Analysis showed a division between eastern and western Greece which might be owed to the existence of climatic fluctuations affecting the abiotic factors. This combined approach offers an important tool for stock identification thus assisting environmental risk assessments in evaluation of potential ecological effects and fostering research excellence in sustainable aquaculture management.     

Phytopathogenic, morphological, genetic and molecular characterization of a Verticillium dahliae population from Crete, Greece

A population of 84?V. dahliae isolates mainly originating from Crete, Greece, was characterized in terms of pathogenicity and virulence on different hosts, in parallel with morphological/physiological characterization, vegetative compatibility grouping and mating type determination. Tomato race 2 was found to have supplanted race 1 and was more virulent on a tomato-susceptible cultivar than race 1. Using a differential host classification system which tests pathogenicity to tomato, eggplant, sweet pepper and turnip, 59 isolates were assigned to tomato, 19 to eggplant, one to sweet pepper and five to tomato-sweet pepper pathogenicity groups. All isolates from Crete fell into VCG subgroups 2A, 2B and 4B, while a remarkably high incidence of bridging isolates (compatible with two or more VCGs) was recorded. The tomato-sweet pepper pathogenicity group was morphologically quite distinct from the others, while conidial length and pigment intensity were discriminatory parameters among VCGs 2A, 2B and 4B. PCR-based molecular marker Tr1/Tr2 was reliable in race prediction among tomato-pathogenic isolates, except for members of VCG 4B, while the application of markers Tm5/Tm7 and 35-1/35-2 was highly successful for tomato-pathogenic isolates. E10 marker was related to VCG 2B, rather than to pathogenicity groups. A single nucleotide polymorphism in the ITS2 region, and two novel molecular markers, M1 and M2, proved useful for the fast and accurate determination of major VCGs 2A, 2B and 4B, and can be used for high-throughput population analyses in future studies. The mating type was unrelated to VCG classification and probably does not control heterokaryon incompatibility in V. dahliae.     

The potential use of carvacrol for the control of <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

The protein kinase HOG1 in high osmolarity glycerol pathway is one of mitogen-activated protein kinases and plays an important role in fungi cellular responses to external stress and stimuli. In this study, AlHOG1, a yeast HOG1 homolog, was isolated from tobacco pathogenic fungus Alternaria longipes and its disrupted mutants were generated using homologous recombination. Compared with the wild-type strain, the mutants grew slower under Minimal-Media, produced less conidia on Potato-Dextrose-Agar and showed a retardation phenotype in conidial germination rate. Colony color observation indicated that AlHOG1 most likely negatively regulated melanin production under nutrients-deficient or osmotic stress. The same action mode of AlHOG1 on hyphal growth in response to certain carbon or nitrogen sources was speculated. Additionally, the disrupted strains were sensitive to osmotic and oxidative stress, and showed increased resistance to dicarboximide, phenylpyrrole and carbendazim fungicides. Meanwhile, results from the comparative analysis between the AlHOG1- and the AlHK1 (encoding a group III histidine kinase)-disrupted mutants showed that AlHOG1 mediated-pathway was partly regulated by the upstream AlHK1 kinase for cellular resistance to salts osmotic stress and that AlHK1 was a primary regulator for cellular sugar and fungicides stress adaptation and likely modulated both AlHOG1-mediated pathway and other unknown mechanisms. Noteworthily, AlHOG1 but not AlHK1 was confirmed to be associated with high concentration glycerol adaptation, which is the first report to our knowledge. Overall, our results indicated that AlHOG1 was involved in multiple physiological processes in A. longipes, including mycelial and conidial development, melanin production, nutrients sensing and multi-stress responses.     

Optimization and validation of the reversed-phase high-performance liquid chromatography with fluorescence detection method for the separation of tocopherol and tocotrienol isomers in cereals, employing a novel sorbent material

The separation and determination of tocopherols (Ts) and tocotrienols (T3s) by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed and validated after optimization of various chromatographic conditions and other experimental parameters. Analytes were separated on a PerfectSil Target ODS-3 (250 × 4.6 mm, 3 μm) column filled with a novel sorbent material of ultrapure silica gel. The separation of Ts and T3s was optimized in terms of mobile-phase composition and column temperature on the basis of the best compromise among efficiency, resolution, and analysis time. Using a gradient elution of mobile phase composed of isopropanol/water and 7 °C column temperature, a satisfactory resolution was achieved within 62 min. For the quantitative determination, α-T acetate (50 μg/mL) was used as the internal standard. Detection limits ranged from 0.27 μg/mL (γ-T) to 0.76 μg/mL (γ-T3). The validation of the method was examined performing intraday (n = 5) and interday (n = 3) assays and was found to be satisfactory, with high accuracy and precision results. Solid-phase extraction provided high relative extraction recoveries from cereal samples: 87.0% for γ-T3 and 115.5% for δ-T. The method was successfully applied to cereals, such as durum wheat, bread wheat, rice, barley, oat, rye, and corn.     

Efficacy of different treatment regimens of marbofloxacin in canine visceral leishmaniosis: a pilot study

This phase II, randomized, open-label field trial was designed to evaluate and compare the safety and efficacy of four treatment durations (10, 20, 28 or 40 days) with marbofloxacin administered orally at the dosage of 2mg/kg once a day for canine visceral leishmaniosis. Twenty-four dogs naturally infected with visceral leishmaniosis and without biochemical disorder evidences of renal insufficiency, were recruited by two Greek veterinarian clinics. They were also randomly assigned to one of the four treatment duration groups, and have been clinically, haematologically, biochemically and parasitologically followed-up regularly for 9 months. Efficacy was achieved for 5/6 dogs treated for 28 days, 4/6 dogs treated for 10 or 20 days and for 3/6 dogs treated for 40 days. Moreover, efficacy was reached more quickly (58.4 days) in dogs treated for 28 days. Improvement of clinical signs tended to be better and faster in the 28 days treatment group too. After 9 months of follow-up, a total of three cases could be considered as relapsing (two dogs treated for 40 days and one dog treated for 28 days). There was a significant reduction in amastigotes density in macrophages after 3 months in the four groups when compared with the parasite density at inclusion. No adverse effects were noticed during this 9 months study. Results obtained with marbofloxacin at the dosage of 2mg/kg once a day for 28 days seem encouraging and may offer a safe alternative for treating canine visceral leishmaniosis.     

Genetic diversity and thermotolerance in Holstein cows: Pathway analysis and marker development using whole-genome sequencing

Heat stress causes extensive losses in the dairy sector, due to negative effects on milk production and reproduction. Cows have evolved a series of protective mechanisms, (physiological, biochemical, behavioural) to cope with the thermostressing environments, which have allowed the preservation of productive and reproductive potential of specific animals during summer; these animals are considered thermotolerant and could be used to design programs of selective breeding. These programs, targeting the generations of a population of heat-resistant animals, would increase the frequency of the desired phenotypes, tackling the financial losses on one hand and reducing the carbon footprints of the dairy sector on the other. The development of genomics techniques has enabled genome wide variant calling, to detect SNPs associated with the desired phenotypes. In this study, we used a comparative genomics approach to detect genetic variation associated with thermotolerance and to design molecular markers for characterizing the animals as tolerant/sensitive. A total of 40 cows from each group were split in four sequencing pools and a whole-genome sequencing approach was used. Results and conclusion: Genome-wide genetic variation between groups was characterized and enrichment analysis revealed specific pathways which participate in the adaptive mechanisms of thermotolerance, implicated into systemic and cellular responses, including the immune system functionality, Heat Stress and Unfolded Protein Response. The markers made a promising set of results, as specific SNPs in five genes encoding for Heat Shock Proteins were significantly associated with thermotolerance.