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.     

5-Hydroxytryptamine-Stimulated Inositol Phospholipid Hydrolysis in the Mouse Cortex Has Pharmacological Characteristics Compatible with Mediation via 5-HT2 Receptors but This Response Does Not Reflect Altered 5-HT2 Function After 5,7-Dihydroxytryptamine Lesioning or Repeated Antidepressant Treatments

5-Hydroxytryptamine (5-HT; 3 x 10(-8)-1 x 10(-5)M) produced a dose-dependent increase in phosphatidylinositol/polyphosphoinositide (PI) turnover in mouse cortical slices, as measured by following production of 3H-labelled inositol phosphates (IPs) in the presence of 10 mM LiCl. Analysis of individual IPs, in slices stimulated for 45 min, indicated substantial increases in inositol monophosphate (IP1; 140%) and inositol bisphosphate (IP2; 95%) contents with smaller increases in inositol trisphosphate (IP3; 51%) and inositol tetrakisphosphate (IP4; 48%) contents. The increase in IP3 level was solely in the 1,3,4-isomer. This response was inhibited by the nonselective 5-HT antagonists methysergide, metergoline, and spiperone. It was also inhibited by the selective 5-HT2 antagonists ketanserin and ritanserin but not by the 5-HT1 antagonists isapirone, (-)-propranolol, or pindolol. 5-HT-stimulated IP formation was also unaltered by atropine, prazosin, and mepyramine. Lesioning brain 5-HT neurones using 5,7-dihydroxytryptamine (5,7-DHT; 50 micrograms i.c.v.) produced a 210% (p less than 0.01) increase in the number of 5-HT2-mediated head-twitches induced by 5-methoxy-N,N-dimethyltryptamine (2 mg/kg). However, 5,7-DHT lesioning had no effect on 5-HT-stimulated PI turnover in these mice. Similarly, an electroconvulsive shock (90 V, 1 s) given five times over a 10-day period caused an 85% (p less than 0.01) increase in head-twitch responses but no change in 5-HT-stimulated PI turnover. Decreasing 5-HT2 function by twice-a-day injection of 5 mg/kg of zimeldine or desipramine (DMI) produced 50% (p less than 0.01) and 56% (p less than 0.01), respectively, reductions in head-twitch behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of hematozoa in blood smears

Ten thin blood smears from mourning doves (Zenaida macroura) infected with Haemoproteus maccallumi were examined by each of two observers using identical techniques and microscopy in an attempt to delineate the factors necessary to provide an accurate estimate of the number of parasites/n erythrocytes. The number of erythrocytes examined must be actually counted, not estimated from extrapolated partial counts or from the number of fields of view examined. Doubling the number of erythrocytes counted (1) decreased the overdispersed frequency distribution patterns in only 25% of the replicate counts for numbers of H. maccallumi/100 erythrocytes for a series of 2,000 versus 4,000 erythrocytes counted; and (2) did not significantly increase the accuracy for determining parasite intensities. Thus, the number of erythrocytes that must be counted to determine parasite intensities could be considerably reduced from the 10,000 or 20,000 estimated for most studies, and still provide an accurate determination of the number of parasites/n erythrocytes in datasets collected from hosts with moderate to high levels of parasitemia. This resulted in a decreased amount of time expended by the observer on each blood smear examined. With two equivalently trained individuals, differences between observers examining the same blood smears were minimal. This study suggests an approach by which a more standardized methodology for quantifying blood parasite intensities could be developed.     

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.     

Distribution of activities of aspartate aminotransferase isoenzymes and malate dehydrogenase in guinea pig retinal layers

Distributions of activity of the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase and of malate dehydrogenase (MDH) were determined in guinea pig retinal layers. The distribution of total AAT activity (tAAT = cAAT + mAAT) and of mAAT activity correlated well (r = 0.88-0.91) with the distribution of MDH activity. mAAT activity was highest in the inner segments of the photoreceptors; there was a greater than twelve-fold difference between activity in that layer and in the inner retinal layers. cAAT activity was also highest in the inner segments, but the difference between the activity in the inner segments and the other layers was not nearly as great as with mAAT. cAAT activity was also relatively high in the outer nuclear layer, outer plexiform layer, and part of the inner plexiform layer. The high activity of cAAT, mAAT, and MDH in the inner segments indicates that all of these enzymes are involved in metabolic reactions related to energy production and/or to photoreceptive processes in the outer segments and, therefore, that the enzymes are probably involved in energy-related metabolism at synapses. However, other functions, including those related to neurotransmission, are not excluded.     

Production of a fibronectin-associated lymphokine by cloned mouse T cells

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.