Reply to Hussey et al.: The requirement for accurate diet-tissue discrimination factors for interpreting stable isotopes in sharks

Carbon and nitrogen stable isotope analyses have improved our understanding of food webs and movement patterns of aquatic organisms. These techniques have recently been applied to diet studies of elasmobranch fishes, but isotope turnover rates and isotope diet–tissue discrimination are still poorly understood for this group. We performed a diet switch experiment on captive sandbar sharks (Carcharhinus plumbeus) as a model shark species to determine tissue turnover rates for liver, whole blood, and white muscle. In a second experiment, we subjected captive coastal skates (Leucoraja spp.) to serial salinity reductions to measure possible impacts of tissue urea content on nitrogen stable isotope values. We extracted urea from spiny dogfish (Squalus acanthias) white muscle to test for effects on nitrogen stable isotopes. Isotope turnover was slow for shark tissues and similar to previously published estimates for stingrays and teleost fishes with low growth rates. Muscle isotope data would likely fail to capture seasonal migrations or diet switches in sharks, while liver and whole blood would more closely reflect shorter term movement or shifts in diet. Nitrogen stable isotope values of skate blood and skate and dogfish white muscle were not affected by tissue urea content, suggesting that available diet–tissue discrimination estimates for teleost fishes with similar physiologies would provide accurate estimates for elasmobranchs.     

What paths do advantageous alleles take during short‐term evolutionary change?

Combining experimental evolution with whole‐genome resequencing is a promising new strategy for investigating the dynamics of evolutionary change. Published studies that have resequenced laboratory‐selected populations of sexual organisms have typically focused on populations sampled at the end of an evolution experiment. These studies have attempted to associate particular alleles with phenotypic change and attempted to distinguish between different theoretical models of adaptation. However, neither the population used to initiate the experiment nor multiple time points sampled during the evolutionary trajectory are generally available for examination. In this issue of Molecular Ecology, Orozco‐terWengel et al. (2012) take a significant step forward by estimating genome‐wide allele frequencies at the start, 15 generations into and at the end of a 37‐generation Drosophila experimental evolution study. The authors identify regions of the genome that have responded to laboratory selection and describe the temporal dynamics of allele frequency change. They identify two common trajectories for putatively adaptive alleles: alleles either gradually increase in frequency throughout the entire 37 generations or alleles plateau at a new frequency by generation 15. The identification of complex trajectories of alleles under selection contributes to a growing body of literature suggesting that simple models of adaptation, whereby beneficial alleles arise and increase in frequency unimpeded until they become fixed, may not adequately describe short‐term response to selection.     

Measurement by radioimmunoassay of casein content in rabbit mammary gland during pregnancy and after prolactin stimulation in organ culture

A specific homologous radioimmunoassay was developed to measure rabbit beta-casein in rabbit mammary gland with a sensitivity of 0.5 ng/ml protein. It was used to measure casein concentration during pregnancy and in organ culture of mammary gland explants. Casein was detectable in virgin mammary glands, showed a small increase during the first half of pregnancy, increased more than 20-fold between Days 21 and 27, and diminished somewhat on the first days of lactation. After 24 hr of culture, mammary gland explants had no detectable casein, but the addition of increasing concentrations of prolactin to a culture medium which contained insulin (5 micrograms/ml) and cortisol (0.5 microgram/ml) induced a regular increase in the casein content of the tissue. Casein started to increase when 10 ng/ml of prolactin was present and maximal values were achieved for 100 ng/ml of the hormone.     

Peroxidative membrane damage in human erythrocytes induced by a concerted action of iodoacetate, vanadate and ferricyanide

Human erythrocytes incubated without substrate in the presence of iodoacetate (0.2 mM), vanadate (0.5 mM) and ferricyanide (5 mM) form aqueous membrane leaks of equivalent radii of 0.5-0.8 nm leading to complete colloid-osmotic lysis within 180 min. All three components are indispensable for the effect. Inosine but not glucose markedly enhances the rate of hemolysis. These effects are due to oxidative damage, as indicated by concomitant destruction of polyunsaturated fatty acids and suppression of both effects by radical scavengers. Hemoglobin is not oxidized under these conditions. GSH and membrane SH levels remain almost normal, and no crosslinking or irreversible aggregation of membrane proteins is observed. In the absence of O2 no membrane damage can be observed. It is proposed that radical formation originates from reduction of O2 by NADPH, analogous to processes described in microsomal membranes. NADH seems not to be involved, since leak formation occurs in spite of the blockage of NADH formation by iodoacetate. Vanadate and ferricyanide are probably required to amplify the peroxidative reaction sufficiently to overcome the cellular antioxidative capacity.     

Random sample planning in morphometry

This report tries to explain principles and presuppositions of planning sample sizes. The aim is estimation of the optimal, that means minimally necessary sample size, justifying an investigation ethically and economically. The problems are complicated because of the necessity of nested sampling or samples within samples. We describe the most important presuppositions using an example of mean value estimation. These are: establishing of demands for exactness, of error probability, and knowledge of variance of the parameters. It is explained in detail by means of binomially distributed variables as we find them in the point counting method.     

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.