Growth and foraging consequences of facultative paedomorphosis in the tiger salamander,Ambystoma tigrinum nebulosum

Summary Facultative paedomorphosis in salamanders occurs when larvae respond to varying environmental conditions by either metamorphosing into terrestrial metamorphic adults or retaining their larval morphology to become sexually mature paedomorphic adults. Several hypotheses have been proposed for the evolutionary maintenance of this environmentally induced dimorphism, but few data are available to assess them adequately. We studied a montane population of the tiger salamander,Ambystoma tigrinum nebulosum, and measured the adult growth rate and body condition across three growing seasons to assess the relative costs and benefits of each morph. Metamorphic adults grew more than paedomorphic adults in terms of snout—vent length across years and in weight within years. Dietary analyses and foraging experiments revealed some of the proximate factors that may underlie these differential growth patterns. Across all prey, metamorphs had significantly higher biomass and calories per stomach sample than paedomorphs. Metamorphic diets primarily consisted of the fairy shrimpBranchinecta coloradensis, whereas paedomorphic diets contained a variety of benthic and terrestrial invertebrates. Foraging experiments revealed that both morphs are more successful at capturing fairy shrimp relative to other prey types and both show high electivity toward this prey. However, fairy shrimp occurred only in non-permanent ponds and thus are inaccessible to paedomorphs, which can survive only in permanent ponds. Paedomorphs also experience higher levels of intraspecific competition with large larvae in permanent ponds than metamorphs do in non-permanent ponds. Thus, metamorphs obtain a growth advantage over paedomorphs by foraging in non-permanent ponds that contain fairy shrimp and have reduced intraspecific competition. These results suggest that paedomorphs should have decreased fitness relative to metamorphs, primarily because metamorphs can move into the best habitats for growth. The net fitness effect of morph-specific differences in dispersal depend on whether there are trade-offs with other life history traits. Nonetheless, because the relative benefit of metamorph dispersal will change with environmental conditions in permanent ponds and the surrounding habitat, the relative fitness payoff to each morph should track changes in the environment. Thus, facultative paedomorphosis may be maintained in part by variable, environmentally-specific fitness payoffs to each morph.     

SEXUAL SELECTION IN AMERICAN TOADS: A TEST OF A GOOD-GENES HYPOTHESIS

Adaptive mate choice in species lacking male resource control and/or paternal care might be maintained by selection because preferred males sire genetically superior offspring. For such a process to occur, some male phenotypic trait(s) must both reliably indicate male genetic quality and influence the pattern of mate choice by females. In American toads, Bufo americanus, male body length has been documented to influence female mating patterns: females usually mate with males that are larger than average. However, the relationship between male size and male genetic quality is unknown. We conducted a controlled breeding experiment using 48 sires and 19 dams to determine if larger males sire offspring with superior larval performance characteristics (greater survival to metamorphosis, larger mass at metamorphosis, and earlier metamorphosis). We also aged each sire to test the hypothesis that older males are, on average, genetically superior to younger males. We crossed each female with three sires representing three body size categories (mean and 1 SD ± mean snout-ischium length). Hatchlings (500 from each cross) were reared to metamorphosis in seminatural ponds in the field. Metamorph weight (log transformed) and age at metamorphosis showed significant heritability and were genetically correlated with each other. Hence, sires differed in genetic quality. However, none of the three measures of offspring performance was correlated with sire body size or age. Thus, we obtained no support for the prediction that sire body size or age is related to genetic quality.     

Association of activated properdin with complexes of properdin with C3

An ELISA using antibody to properdin (P), followed by antibody to C3 to detect complexes of P with C3 (P-C3), detected low levels of P-C3 complexes in human serum and plasma samples. Incubating serum for 1 h at 37 degrees C increased the amount of P-C3 and diminished factor B hemolytic activity without altering total alternative pathway activity or C3 activity in serum. When P and C3 in incubated serum were analyzed by size exclusion HPLC, complexes of P-C3 were detected at retention times corresponding to molecular mass measuring in excess of 2 x 10(6) Da. Activation of serum with zymosan or cobra venom factor greatly increased the level of P-C3 and decreased alternative pathway hemolytic activity. Chromatography of proteins eluted from serum-treated zymosan detected a peak of P at 9.7 x 10(5) Da and a peak of P-C3 at 1.5 x 10(6) Da. Functional assays for activated properdin also revealed a peak of activity at 1.5 x 10(6) Da, congruent with the peak of P-C3. Native properdin was detected at 3.9 x 10(5) Da. When native properdin was added to properdin-depleted serum and incubated for 1 h at 37 degrees C, activated properdin was detected at the same position in the chromatograph as were P-C3 complexes. We conclude that incubation of serum at 37 degrees C produces complexes of P with C3, that exposure of serum to alternative pathway activators increases the amount of P-C3, and that generation of P-C3 complexes is associated with the presence of activated P.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

The gastrointestinal tract: A major site of antioxidant action?

Diets rich in fruits and vegetables delay the onset of many age-related diseases, and contain a complex mixture of antioxidants (including ascorbate, carotenoids, vitamin E and other phenolics such as the flavonoids). However, diet also contains pro-oxidants, including iron, copper, H₂O₂, haem, lipid peroxides and aldehydes. Nitrite is frequently present in diet, leading to generation of reactive nitrogen species in the stomach. In considering the biological importance of dietary antioxidants, attention has usually focussed on those that are absorbed through the gastrointestinal tract into the rest of the body. In the present paper we develop the argument that the high levels of antioxidants present in certain foods (fruits, vegetables, grains) and beverages (e.g. green tea) play an important role in protecting the gastrointestinal tract itself from oxidative damage, and in delaying the development of stomach, colon and rectal cancer. Indeed, carotenoids and flavonoids do not seem to be as well absorbed as vitamins C and E. Hence their concentrations can be much higher in the lumen of the GI tract than are ever achieved in plasma or other body tissues, making an antioxidant action in the GI tract more likely. Additional protective mechanisms of these dietary constituents (e.g. effects on intercellular communication, apoptosis, cyclooxygenases and telomerase) may also be important.     

Nitric oxide and peroxynitrite. The ugly, the uglier and the not so good: a personal view of recent controversies

Nitric oxide, a gaseous free radical, is poorly reactive with most biomolecules but highly reactive with other free radicals. Its ability to scavenge peroxyl and other damaging radicals may make it an important antioxidant in vivo, particular in the cardiovascular system, although this ability has been somewhat eclipsed in the literature by a focus on the toxicity of peroxynitrite, generated by reaction of O^·-₂ with NO^· (or of NO^- with O₂). On balance, experimental and theoretical data support the view that ONOO^- can lead to hydroxyl radical (OH^·) generation at pH 7.4, but it seems unlikely that OH^· contributes much to the cytotoxicity of ONOO^-. The cytotoxicity of ONOO^- may have been over-emphasized: its formation and rapid reaction with antioxidants may provide a mechanism of using NO^· to dispose of excess O^·-₂, or even of using O^·-₂ to dispose of excess NO^·, in order to maintain the correct balance between these radicals in vivo. Injection or instillation of “bolus” ONOO^- into animals has produced tissue injury, however, although more experiments generating ONOO^- at steady rates in vivo are required. The presence of 3-nitrotyrosine in tissues is still frequently taken as evidence of ONOO^- generation in vivo, but abundant evidence now exists to support the view that it is a biomarker of several “reactive nitrogen species”. Another under-addressed problem is the reliability of assays used to detect and measure 3-nitrotyrosine in tissues and body fluids: immunostaining results vary between laboratories and simple HPLC methods are susceptible to artefacts. Exposure of biological material to low pH (e.g. during acidic hydrolysis to liberate nitrotyrosine from proteins) or to H₂O₂ might cause artefactual generation of nitrotyrosine from NO^-₂ in the samples. This may be the origin of some of the very large values for tissue nitrotyrosine levels quoted in the literature. Nitrous acid causes not only tyrosine nitration but also DNA base deamination at low pH: these events are relevant to the human stomach since saliva and many foods are rich in nitrite. Several plant phenolics inhibit nitration and deamination in vitro, an effect that could conceivably contribute to their protective effects against gastric cancer development.     

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins.We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.     

Monogamy in marine fishes

The formation of long-term pair bonds in marine fish has elicited much empirical study. However, the evolutionary mechanisms involved remain contested and previous theoretical frameworks developed to explain monogamy in birds and mammals are not applicable to many cases of monogamy in marine fish. In this review, we summarise all reported occurrences of social monogamy in marine fish, which has so far been observed in 18 fish families. We test quantitatively the role of ecological and behavioural traits previously suggested to be important for the evolution of monogamy and show that monogamous species occur primarily in the tropics and are associated with coral reef environments in which territory defence and site attachment is facilitated. However, there is little evidence that obligately monogamous species are smaller in body size than species that can adopt a polygynous mating system. We review the evidence pertaining to six hypotheses suggested for the evolution of monogamous pair bonds: (1) biparental care, (2) habitat limitation, (3) low population density/low mate availability/low mobility, (4) increased reproductive efficiency, (5) territory defence, and (6) net benefit of single mate sequestration. We outline predictions and associated empirical tests that can distinguish between these hypotheses, and assess how generally each hypothesis explains monogamy within and between breeding periods for species with different types of territories (i.e. feeding only or feeding and breeding). Hypotheses (1) and (2) have limited applicability to marine fishes, while hypotheses (3)-(5) have little empirical support beyond the species for which they were designed. However, the role of paternal care in promoting monogamous pair bonds is not explicit in these hypotheses, yet paternal care has been reported in more than 70 monogamous marine fish. We show that paternal care may act to increase the likelihood of monogamy in combination with each of the proposed hypotheses through decreased benefits to males from searching for additional mates or increased advantages to females from sequestering a single high-quality mate. Among species defending breeding and feeding territories, the benefits, both within and between reproductive periods, of sequestering a single high-quality mate (hypothesis 6) appear to be the best explanation for socially monogamous pairs. For species without parental care (i.e. holding only feeding territories), territory defence (hypothesis 5) in combination with the benefits of guarding a large mate (hypothesis 6) could potentially explain most instances of monogamy. Empirical studies of marine fishes over the past two decades are therefore slowly changing the view of monogamy from a mating system imposed upon species by environmental constraints to one with direct benefits to both sexes.