Identification of Microsatellite Markers for Plasmopara viticola and Establishment of High throughput Method for SSR Analysis

The Oomycete Plasmopara viticola is the causal organism of downy mildew on grapevine (Vitis spp.). In order to set up the techniques for investigating downy mildew disease dynamics and genetic structure, co-dominant, neutral, highly reproducible and polymorphic microsatellite markers for P. viticola were developed. Five markers, two with a (TC)_n repeat (loci BER and ISA), two with a (TC)_n(AC)_n repeat (loci CES and REX) and one with a (CT)_n(CTAT)_n repeat (locus GOB), were selected. Simple sequence repeat (SSR) markers revealed different degrees of polymorphism within 190 oil spots (disease symptoms) collected from an infected Italian vineyard. The most polymorphic SSR marker GOB showed 43 alleles (Nei's expected gene diversity H_e = 0.89) while CES, ISA, BER and REX showed 14 (H_e = 0.71), 4 (H_e = 0.57), 3 (H_e = 0.24) and 1 allele (H_e = 0), respectively. A high throughput DNA extraction method, that allowed molecular analysis of this obligate pathogen directly in the host without any isolation procedure, was developed. The quality and quantity of oil spots did not influence the SSR analysis. Amplified SSR loci were separated by electrophoresis on a Beckman–Coulter 2000XL sequencer and automatically analysed. The objective of this study was to develop molecular biological tools and methods that allow high throughput analysis of the downy mildew populations.     

Calvaria and orbital metastases of pulmonary adenosquamous carcinoma in a cat: a diagnostic challenge

An 11-year-old cat with a 4-month history of lethargy, inappetence, dysphagia, partial mandibular paralysis and weight loss, was euthanized due to the rapid deterioration of his condition. Post-mortem radiographic examination revealed severe bone lysis of the left zygomatic arch, temporal and parietal bones. Magnetic resonance imaging of the head showed a large isointense mass of the left side of the skull associated with extensive lysis of the parietal and temporal bones and destruction of the adjacent tympanic bulla. Gross and histological examinations revealed a pulmonary adenosquamous carcinoma of the left lung, with metastases to the spleen, liver, mesenteric lymph nodes, mesentery, diaphragm, abdominal aorta, left orbit and calvaria. No limb or digit metastases were detected.     

Detection of enzymatic activity and partial sequence of a chitinase gene in <Emphasis Type="Italic">Metschnikowia pulcherrima</Emphasis> strain MACH1 used as post-harvest biocontrol agent

Two antagonistic yeast strains Metschnikowia pulcherrima MACH1 and Rhodotorula sp. PW34 were tested for their efficacy against Botrytis cinerea in vitro and in vivo on apples. Metschnikowia pulcherrima strain MACH1 showed higher inhibition of B. cinerea compared to the strain PW34 in vitro on potato dextrose broth. Further, yeast strain MACH1 showed higher efficacy in reducing grey mould on apples compared to PW34 and the untreated control. In addition, partially purified extracellular proteins from strain MACH1 showed an inhibition to B. cinerea in vitro. The antagonistic yeast strains were tested for their efficacy to produce chitinases in different liquid media, including apple juice, amended with or without cell wall preparations (CWP) of B. cinerea. The study showed a higher production of chitinases from M. pulcherrima strain MACH1 when compared to PW34. Interestingly, the strain MACH1 secreted higher chitinases in the presence of cell wall fractions of B. cinerea. For this reason, the chitinase gene of strain MACH1 was amplified using PCR reactions and the nucleotide sequence data showed high homology to chitinases of other yeast strains. The results of the current study show that M. pulcherrima strain MACH1 has the ability to secrete chitinases in different liquid media including apple juice, and the enzyme could be involved in the post-harvest biological control of B. cinerea.     

Vitamin B6 supplementation increases the docosahexaenoic acid concentration of muscle lipids of rainbow trout (Oncorhynchus mykiss)

Fish fillet quality is influenced by feed quality. In particular, vitamin B₆ availability improves growth rate and nutritional value, because it is related to protein and lipid metabolism. The present study investigates the effect of increasing amounts of vitamin B₆ on growth, fatty acid composition and lipid peroxidation of muscle tissue of rainbow trout (Oncorhynchus mykiss). Quadruplicate groups of trout of a mean initial weight of 110 g were fed commercial diets supplemented with four different quantities of vitamin B₆ (0, 10, 25 and 50 mg kg^?1 diet). Over the experimental period, there were no significant differences in weight gain and feed intake, while vitamin B₆ concentration of muscle was affected by dietary intake, even though its increase was not proportional to the concentrations in the feed. The fatty acid composition of muscle lipid showed differences between the four groups. The percentage of long‐chain unsaturated fatty acids, in particular docosahexaenoic acid, increased significantly in vitamin B₆‐supplemented groups. However, despite the increased fatty acid unsaturation index, lipid peroxidation parameters such as vitamin E content, malondialdehyde production, glutathione reductase and glutathione peroxidase activities did not show significant differences, meaning that the higher level of long‐chain unsaturated fatty acid did not increase the muscle susceptibility to oxidative stress.     

Leiopathes glaberrima millennial forest from SW Sardinia as nursery ground for the small spotted catshark Scyliorhinus canicula

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Effects of population density on seabass (Dicentrarchus labrax, L.) gene expression

The issue of animal welfare in aquaculture is growing of interest and there is an increasing consumer demand for documentation of safe and ethically defendable food production. In this context, we have looked for molecular markers among those genes whose expression results modified by the different farming conditions. We have compared gene expression of seabass farmed at different population densities by differential display. We have obtained six bands differentially expressed whose sequences we have deposited in the public databases; two of them resulted suppressed by high population density, while four were induced by the treatment. Population density has, therefore, an effect at gene level by repressing or enhancing the expression of different genes. These genes can be used as biomarkers to rapidly detect, by polymerase chain reaction (PCR), the occurred exposure of the fish to the stressor.

We are certain that the new molecular techniques will find their place in the everyday management of fish farming; on the other hand, we are also aware that the scarcity of the genomic resources for some fish species, in spite of their economical interest, will retard the beneficial effects that modern biotechnology could bring to aquaculture industry. Therefore, an effort should be made to reduce, as far as it concerns genomics resources, the gap that separates farming species from “model organisms”.     

New sites expanding the “Sardinian cold‐water coral province” extension: A new potential cold‐water coral network?

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