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.     

Embryonic heart mesenchymal cell migration in laminin

Laminin, a large glycoprotein and major component of basement membranes, influences cell adhesion, migration, morphology, and differentiation. A peptide sequence, YIGSR, from the B1 chain of laminin has been found to correspond to an active site for cell adhesion. We report here that cardiac mesenchymal cells migrate vigorously within three-dimensional gels of laminin and that the YIGSR peptide will completely abolish this migratory activity. In contrast, migration of the mesenchymal cells into three-dimensional gels composed of collagen or collagen + laminin is not effected by YIGSR or other peptides (GRGDS, GRGDTP) reported to mediate cellular adhesion.     

The effects of in vivo administration of endotoxin on the functions and interaction of hepatocytes and Kupffer cells

It was the purpose of this study to determine the effects of the in vivo administration of endotoxin on certain in vitro hepatocyte and Kupffer cell functions. An Alzet osmotic pump that contained endotoxin (LPS, 2.5 mg/100g) was implanted into the peritoneal cavity of 300g guinea pigs and delivered the endotoxin over a period of four days. In vivo administration of LPS did not cause a change in the in vitro release of albumin by isolated hepatocytes. However, when hepatocytes were co-cultured with Kupffer cells there was a significant decrease in albumin release for both control and LPS-treated animals. There was no difference between control and LPS-treated animals in the release of C3 by hepatocytes. However, there was a significant increase over the control group in C3 release by Kupffer cells from LPS-treated animals. When hepatocytes and Kupffer cells were cultured together, their release of C3 was not additive. Kupffer cells from LPS-treated animals released significantly greater amounts of PGE2 than control animals when stimulated in vitro with LPS. Thus, these Kupffer cells appeared to be primed to respond to an in vitro challenge of LPS. Kupffer cells from LPS-treated animals had significantly depressed antibody dependent cellular cytotoxicity (ADCC). This endotoxin model is useful for determining the in vivo effects of endotoxin on cellular function and gives some indirect evidence for the detrimental effects of LPS on the immune system and host defense.     

Effects of various experimental manipulations on neostriatal acetylcholine and dopamine release

The release of endogenous acetylcholine and dopamine and the appearance of their metabolites, choline and dihydroxyphenylacetic acid (DOPAC), from neostriatal slices prepared from Fischer 344 rats was examined under various experimental conditions. There was a dose-dependent increase in the amount of neurotransmitter or metabolite as the medium potassium concentration was increased from 5 to 50 mM. Over an eight minute period in Krebs Ringer bicarbonate buffer containing 25 mM potassium, the rate of release of acetylcholine was 6 to 13 times greater than that of dopamine. The dopamine endogenous to the slice preparation appeared to have little effect on the release of endogenous acetylcholine since manipulations that significantly altered dopamine release (depletion with 6-hydroxydopamine or uptake inhibition with nomifensine) had minimal effects on the cholinergic neurons. In contrast, increasing the endogenous acetylcholine in the preparation by inhibiting acetylcholinesterase resulted in a 1.2 to 12 fold increase in dopamine release depending upon the incubation time and the potassium concentration. These studies indicate that within the neostriatal slices there is minimal influence of the endogenous dopamine on the cholinergic neurons, whereas the extracellular acetylcholine can influence dopamine release when its concentration is increased by inhibition of acetylcholinesterase.     

Socialization processes in a female lowland gorilla

A low-interactive, captive, female lowland gorilla, Molly, was studied following the introduction into her enclosure of three gorillas, two males and a female, raised from birth in captive gorilla groups. Observations were made 6 mo after the new gorillas were introduced. Throughout the period of observation, Molly interacted in an affiliative manner with one of the males, playing or sitting quietly with him in a tree (where Molly spent most of her time) and occasionally on the ground. Agonistic displays between Molly and the new female decreased after they were released in the enclosure without the males for a series of days. Molly, however, continued to react to the other male, the most dominant, in an agonistic manner, and usually retreated from his reach, climbed the tree, and/or grimaced and piloerected whenever he approached. Although Molly's continued avoidance of the dominant male impeded her complete socialization, we propose that the interventions employed in this study—introduction of new younger gorillas into and an enclosure, and a series of dyadic separations between the noninteractive gorilla and each of the new group members—are possible strategies that can be used to facilitate socialization of captive, noninteractive gorillas.     

Identification of Biochemical Defects in Pancreatic Islets of fa/fa Rats: A Developmental Study

KIBENGE, MOLLY T AND CATHERINE B CHAN. Identification of biochemical defects in pancreatic islets of fa/fa rats: a developmental study. Obes Res. 1995;3:171–178. Adult obese (fa/fa) Zucker rats hypersecrete insulin in response to glucose and other secretagogues. Functional changes in islet ot2-adrenoceptors (8) and glycolytic regulation (9) have been reported. In this study, the development of these biochemical lesions in islets isolated from suckling (3 week old) and weanling (5 week old) lean and fa/fa rats was investigated and compared to results in adult animals. Glucose (15 mM)-induced insulin secretion was inhibited by mannoheptulose (MH) in lean (n=8) but not fa/fa (n=10) adult rats, indicating loss of sensitivity of glucokinase to competitive inhibition. Sensitivity to MH was somewhat reduced in the islets of 3- and 5-week-old fa/fa (n=7 and 12) compared to lean (n=15 and 9) rats, requiring 30–100 fold higher concentrations to achieve significant inhibition. At 3 weeks of age fa/fa rats did not differ from lean controls in either islet insulin content or body weight, but both parameters were increased in fa/fa rats by 5 weeks. The presence of altered α₂-adrenoceptor function in fa/fa rats could not be confirmed in this study. Unlike the previous report, prazosin did not antagonize α₂-agonist mediated inhibition of insulin secretion. The presence of defective regulation of the glycolytic pathway by mannoheptulose in suckling and weanling rats may contribute to development of hyperinsulinemia in fa/fa rats.     

Vertical bars on male Xiphophorus multilineatus: a signal that deters rival males and attracts females

We examined the function of the vertical bar pattern on maleswordtails (Xiphophorus multilinneatus) as a signal in bothmale-male competition and female choice. This pattern had previouslybeen described as an aggressive signal because males intensifiedthe bars during male-male encounters in the laboratory. Ourfield observations supported this observation and also showedthat bars intensified when males courted females. The intensityof bars was correlated with access to females in the field.Within the size range of males that have bars, however, neitherbar number nor male size appeared to influence access to females.We used freeze-branding to remove the bars from males in thelaboratory so that we could control for characters correlatedwith bar intensity, and tested males and females separatelyso that we could separate the influence of these two componentsof sexual selection. We compared the responses of males andfemales to males that had their bars removed and control malesfreeze-branded between the bars. Test males responded more aggressivelyto males without bars as compared to control males. In addition,females showed a preference for control males over males thathad their bars removed. These results suggest that the barsmay function as a signal that deters rival males and attractsfemales.     

Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor “processing” within 4 h of administration followed by recovery or “replenishment” of ER levels to the initial level by 20 h. The term “processing” has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor “processing” constitutes disappearance of receptor protein and the later “replenishment” phase represents new ER protein rather than recycling of “processed” receptor. Progesterone-action, on the other hand, influenced only the “replenishment” phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was “processed” and “replenishment” already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.