Pre-unfolding resonant oscillations of single green fluorescent protein molecules

Fluorescence spectroscopy of a green fluorescent protein mutant at single-molecule resolution has revealed a remarkable oscillatory behavior that can also be driven by applied fields. We show that immediately before unfolding, several periodic oscillations among the chemical substates of the protein chromophore occur. We also show that applied alternating electric or acoustic fields, when tuned to the protein characteristic frequencies, give rise to strong resonance effects.     

Neurobiological substrates of dread

Given the choice of waiting for an adverse outcome or getting it over with quickly, many people choose the latter. Theoretical models of decision-making have assumed that this occurs because there is a cost to waiting-i.e., dread. Using functional magnetic resonance imaging, we measured the neural responses to waiting for a cutaneous electric shock. Some individuals dreaded the outcome so much that, when given a choice, they preferred to receive more voltage rather than wait. Even when no decision was required, these extreme dreaders were distinguishable from those who dreaded mildly by the rate of increase of neural activity in the posterior elements of the cortical pain matrix. This suggests that dread derives, in part, from the attention devoted to the expected physical response and not simply from fear or anxiety. Although these differences were observed during a passive waiting procedure, they correlated with individual behavior in a subsequent choice paradigm, providing evidence for a neurobiological link between the experienced disutility of dread and subsequent decisions about unpleasant outcomes.     

Looking beyond the horizon: An early warning system to keep marine mammal information relevant for conservation

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Methacarn preserves mucus integrity and improves visualization of amoebae in gills of Atlantic salmon (Salmo salar L.)

Two aqueous fixation methods (modified Davidson's solution and modified Davidson's solution with 2% (w/v) Alcian blue) were compared against two non‐aqueous fixation methods (methacarn solution and methacarn solution with 2% (w/v) Alcian blue) along with the standard buffered formalin fixation method to (a) improve preservation of the mucous coat on Atlantic salmon, Salmo salar L., gills and (b) to examine the interaction between the amoebae and mucus on the gill during an infection with amoebic gill disease. Aqueous fixatives demonstrated excellent cytological preservation but failed to deliver the preservation of the mucus when compared to the non‐aqueous‐based fixatives; qualitative and semi‐quantitative analysis revealed a greater preservation of the gill mucus using the non‐aqueous methacarn solution. A combination of this fixation method and an Alcian blue/Periodic acid–Schiff staining was tested in gills of Atlantic salmon infected with amoebic gill disease; lectin labelling was also used to confirm the mucus preservation in the methacarn‐fixed tissue. Amoebae were observed closely associated with the mucus demonstrating that the techniques employed for preservation of the mucous coat can indeed avoid the loss of potential mucus‐embedded parasites, thus providing a better understanding of the relationship between the mucus and parasite.     

The effects of starving and feeding on Dover sole (Solea solea,Soleidae, Linnaeus, 1758) stress response and early larval development

In the view of an urgent necessity to improve the quantity and the quality of farmed fish species, there is a strong need to improve our basic knowledge on the effects of first feeding during the developmental stages of fish larvae. High mortality, mainly due to food deprivation or inappropriate food quality, has been observed in many larval fish species, but knowledge about the morphological, biochemical and molecular processes related to this topic is still poorly understood. The understanding of the early larval ontogeny as well as the larval nutritional requirements and the molecular and cellular mechanisms elicited by fish larvae during food deprivation and starvation are thus of primary importance. At this regard, this study investigates, in Dover sole larvae, the effects of starvation and starving/re‐feeding procedures at a morphological, histological, biochemical and molecular level. The results evidenced that starved larvae progressively decrease in growth, lipid content, affected their gastrointestinal tract and muscle development and increased cortisol and heat shock protein 70 levels. On the contrary, starved and re‐fed larvae showed, after the restoration of a favourable feeding condition, a compensatory growth. In conclusion, this is the first study analysing through a multidisciplinary approach the effects of food deprivation on the development of an important economic species, the Dover sole.     

A comparison of the use of different swab materials for optimal diagnosis of amoebic gill disease (AGD) in Atlantic salmon (Salmo salar L.)

Routine gill swabbing is a non-destructive sampling method used for the downstream qPCR detection and quantitation of the pathogen Neoparamoeba perurans, a causative agent of amoebic gill disease (AGD). Three commercially available swabs were compared aiming their application for timelier AGD diagnosis (Calgiswab^® (calcium alginate fibre-tipped), Isohelix^® DNA buccal and cotton wool-tipped). Calcium alginate is soluble in most sodium salts, which potentially allows the total recovery of biological material, hence a better extraction of target organisms’ DNA. Thus, this study consisted of (a) an in vitro assessment involving spiking of the swabs with known amounts of amoebae and additional assessment of retrieval efficiency of amoebae from agar plates; (b) in vivo testing by swabbing of gill arches (second, third and fourth) of AGD-infected fish. Both in vitro and in vivo experiments identified an enhanced amoeba retrieval with Calgiswab® and Isohelix® swabs in comparison with cotton swabs. Additionally, the third and fourth gill arches presented significantly higher amoebic loads compared to the second gill arch. Results suggest that limiting routine gill swabbing to one or two arches, instead of all, could likely lead to reduced stress-related effects incurred by handling and sampling and a timelier diagnosis of AGD.     

The Pelagos Sanctuary for Mediterranean marine mammals

• 1. In February 2002, France, Italy and Monaco agreed to establish an international sanctuary for Mediterranean marine mammals. The resulting Pelagos Sanctuary encompasses over 87500 km² of the north‐western Mediterranean Sea, extending between south‐eastern France, Monaco, north‐western Italy and northern Sardinia, and surrounding Corsica and the Tuscan Archipelago.
• 2. The Pelagos Sanctuary illustrates how the tenets of Marine Protected Area (MPA) design can be reconciled with the dynamic nature of oceanic systems, because its spatial scale was defined by oceanographic and ecological considerations, specifically the location of the Ligurian permanent frontal system.
• 3. By expanding protective measures beyond national waters, the Pelagos Sanctuary also sets a precedent for the implementation of pelagic protected areas in the high seas. The Pelagos Sanctuary will contribute to the conservation of the Mediterranean Sea at two scales: (i) locally, by protecting important cetacean foraging and breeding grounds in the Ligurian Sea, and by providing ‘umbrella’ protection to other marine predators in this area; and (ii) regionally, by empowering other conservation measures, such as the Specially Protected Areas Protocol of the Barcelona Convention and the wider goals of the Agreement on the Conservation of Cetaceans of the Black and Mediterranean Seas (ACCOBAMS).
• 4. However, because few cetacean species are resident within the Sanctuary, their effective long‐term conservation will require large‐scale management and coordinated monitoring throughout the Mediterranean basin.

Copyright © 2007 John Wiley & Sons, Ltd.     

Alternative matrices for cortisol measurement in fish

Plasma cortisol is the most commonly used indicator of stress in fish but, as the blood sampling procedure itself can be a source of stress, it would be helpful to measure cortisol using less invasive matrices. It is also necessary to find alternative matrices as stress indicators in dead fish in which blood sampling is impossible. In the present study, we investigated transport stress in three aquaculture species, European sea bass (Dicentrarchus labrax L.), common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum), by cortisol determination (radioimmunoassay) in plasma and other matrices (skin mucus, gut content, lateral muscle and caudal fin). Cortisol significantly increased after transport in all species and matrices, except in the sea bass gut content, where it remained unchanged. The three species responded to transport stress by producing different cortisol levels. In conclusion, the significant correlation found between plasma cortisol and most of the other matrices opens up the possibility of using them to evaluate stress in fish: mucus sampling is a less invasive method than blood sampling, and in addition to muscle and fin sampling, it can be used in postmortem fish.     

INTRAVENOUS ALFAXALONE ANAESTHESIA IN LEOPARD GECKOS (EUBLEPHARIS MACULARIUS)

Intravenous alfaxalone, administered at a dose of 5 mg/kg in the jugular vein, was evaluated in 20 leopard geckos (Eublepharis macularius) to ascertain its ability to provide anesthesia. The induction time, time to loss of mandibular tone, interval of deep anesthesia, and full recovery time were 27.5 ± 30.7 seconds (10 to 56 seconds), 1.3 ± 1.4 minutes (11 seconds to 4 minutes), 12.5 ± 2.2 minutes (11.11 to 15.39 minutes), and 18.8 ± 12.1 minutes (10.4 to 52.31 minutes), respectively. A significant reduction in heart rate (74 ± 12.9 beats/minute) was recorded between 2 and 24 minutes after alfaxalone administration. A significant decrease in respiratory rate (26.8 ± 10.1 breaths/minute) was recorded 2 minutes after alfaxalone administration, and respiratory rate remained lower than the basal rate (31.4 ± 3.1 breaths/minute) for 24 minutes but without statistical significance. The intravenous administration of alfaxalone in leopard geckos achieved a rapid onset of anesthesia and a suitable recovery time. Based on this investigation, an afaxalone dose of 5 mg/kg intravenously proved to be suitable for sedation before tracheal intubation. Moreover, the administration route via the jugular vein, was acceptable in leopard geckos; a species in which other venipuncture sites can be challenging or inaccessible.