Morphological and life‐history responses of anurans to predation by an invasive crayfish: an integrative approach

Predator‐induced phenotypic plasticity has been widely documented in response to native predators, but studies examining the extent to which prey can respond to exotic invasive predators are scarce. As native prey often do not share a long evolutionary history with invasive predators, they may lack defenses against them. This can lead to population declines and even extinctions, making exotic predators a serious threat to biodiversity. Here, in a community‐wide study, we examined the morphological and life‐history responses of anuran larvae reared with the invasive red swamp crayfish, Procambarus clarkii, feeding on conspecific tadpoles. We reared tadpoles of nine species until metamorphosis and examined responses in terms of larval morphology, growth, and development, as well as their degree of phenotypic integration. These responses were compared with the ones developed in the presence of a native predator, the larval dragonfly Aeshna sp., also feeding on tadpoles. Eight of the nine species altered their morphology or life history when reared with the fed dragonfly, but only four when reared with the fed crayfish, suggesting among‐species variation in the ability to respond to a novel predator. While morphological defenses were generally similar across species (deeper tails) and almost exclusively elicited in the presence of the fed dragonfly, life‐history responses were very variable and commonly elicited in the presence of the invasive crayfish. Phenotypes induced in the presence of dragonfly were more integrated than in crayfish presence. The lack of response to the presence of the fed crayfish in five of the study species suggests higher risk of local extinction and ultimately reduced diversity of the invaded amphibian communities. Understanding how native prey species vary in their responses to invasive predators is important in predicting the impacts caused by newly established predator–prey interactions following biological invasions.     

Novel Loci for metabolic networks and multi-tissue expression studies reveal genes for atherosclerosis

Association testing of multiple correlated phenotypes offers better power than univariate analysis of single traits. We analyzed 6,600 individuals from two population-based cohorts with both genome-wide SNP data and serum metabolomic profiles. From the observed correlation structure of 130 metabolites measured by nuclear magnetic resonance, we identified 11 metabolic networks and performed a multivariate genome-wide association analysis. We identified 34 genomic loci at genome-wide significance, of which 7 are novel. In comparison to univariate tests, multivariate association analysis identified nearly twice as many significant associations in total. Multi-tissue gene expression studies identified variants in our top loci, SERPINA1 and AQP9, as eQTLs and showed that SERPINA1 and AQP9 expression in human blood was associated with metabolites from their corresponding metabolic networks. Finally, liver expression of AQP9 was associated with atherosclerotic lesion area in mice, and in human arterial tissue both SERPINA1 and AQP9 were shown to be upregulated (6.3-fold and 4.6-fold, respectively) in atherosclerotic plaques. Our study illustrates the power of multi-phenotype GWAS and highlights candidate genes for atherosclerosis.     

Microplate screening assay for binding of ligands to bovine or reindeer beta-lactoglobulins

Several analytical methods have been used to determine whether ligands bind to bovine beta-lactoglobulin (betaLG). The most common methods are based on fluorescence quenching. We have miniaturised this method from a quartz cell to a 96-well plate. The miniaturisation was evaluated using retinol. The binding constants between the two methods demonstrated a good correlation. The 96-well plate method is much faster and allows many references to be used in the same analysis. The miniaturised method was used to study the binding of three different ligands (4-HPR, arotinoid, warfarinyl palmitate) modelled to bind to betaLG. The binding data showed that all of these ligands bound to betaLG. The method was further used to demonstrate that reindeer betaLG could also bind the four ligands in the same way as bovine betaLG. Because one aim is to use bovine and reindeer betaLG as a binder molecule for aliments in e.g. functional food or for drugs, the influence of pH was also studied and demonstrated that short-term acidic conditions had only a slight effect on the binding properties.     

Reversibility of predator‐induced plasticity and its effect at a life‐history switch point

In natural systems, organisms are frequently exposed to spatial and temporal variation in predation risk. Prey organisms are known to develop a wide array of plastic defences to avoid being eaten. If inducible plastic defences are costly, prey living under fluctuating predation risk should be strongly selected to develop reversible plastic traits and adjust their defences to the current predation risk. Here, we studied the induction and reversibility of antipredator defences in common frog Rana temporaria tadpoles when confronted with a temporal switch in predation risk by dragonfly larvae. We examined the behaviour and morphology of tadpoles in experimental treatments where predators were added or withdrawn at mid larval development, and compared these to treatments with constant absence or presence of predators. As previous studies have overlooked the effects that developing reversible anti‐predator responses could have later in life (e.g. at life history switch points), we also estimated the impact that changes in antipredator responses had on the timing of and size at metamorphosis. In the presence of predators, tadpoles reduced their activity and developed wider bodies, and shorter and wider tails. When predators were removed tadpoles switched their behaviour within one hour to match that found in the constant environments. The morphology matched that in the constant environments in one week after treatment reversal. All these responses were highly symmetrical. Short time lags and symmetrical responses for the induction/reversal of defences suggest that a strategy with fast switches between phenotypes could be favoured in order to maximise growth opportunities even at the potential cost of phenotypic mismatches. We found no costs of developing reversible responses to predators in terms of life‐history traits, but a general cost of the induction of the defences for all the individuals experiencing predation risk during some part of the larval development (delayed metamorphosis). More studies examining the reversibility of plastic defences, including other type of costs (e.g. physiological), are needed to better understand the adaptive value of these flexible strategies.     

Flat bed scanner in the quantitative assay of 35SO4-incorporation by X-ray film autoradiography of intervertebral disc sections

A rapid quantitation of proteoglycan synthesis distribution in intervertebral disc and endplates is described. Tissue blocks of disc (C7-Th1) in the midsagittal plane from ten female beagles were incubated in the presence of ³⁵SO₄ and prepared as histological slides. For comparison, sulphate incorporation rates in the C5–C6 discs were assayed by liquid scintillation. Autoradiographic film exposed against the labelled sections was developed and digitized for image analysis using a 256 grey level flat bed table scanner connected to a microcomputer. The film density versus dpm (disintegrations per minute) calibration was performed using a set of ³⁵SO₄-labelled glycosaminoglycan standards applied on the same film. Since section thickness, dpm calibration of the film density and the specific activity of sulphate in the medium were known, the incorporations per tissue volume could be calculated. The average incorporation rates of the anterior and posterior annulus fibrosus, nucleus pulposus and vertebral endplates were 5.2±0.9, 5.2±0.8, 4.5±0.6 and 4.1±0.8 pmol/mm³ per h (±SE, n=10), respectively and closely corresponded to those obtained by liquid scintillation. This method offers a convenient and reproducible way to measure the rate of proteoglycan synthesis in large tissue sections but also in thin cartilaginous tissues such as the vertebral endplate.     

Inhibition of the mitochondrial calcium uniporter by antibodies against a 40-kDa glycorproteinT

Polyclonal rabbit antibodies against a Ca²⁺-binding mitochondrial glycoprotein were found to inhibit the uniporter-mediated transport of Ca²⁺ in mitoplasts prepared from rat liver mitochondria. Spermine, a modulator of the uniporter, decreased the inhibition. This glycoprotein ofM _r 40,000, isolated from beef heart mitochondria and earlier shown to form Ca²⁺-conducting channels in black-lipid membranes, thus is a good candidate for being a component of the uniporter. Antibody-IgG was found to specifically bind to mitochondria in human fibroblasts.     

The cytochrome P450-containing monooxygenase of Trichosporon cutaneum: Occurrence and properties

Trichosporon cutaneum metabolizes glucose purely oxidatively and cytochrome P450 was not detected in the reduced CO-difference spectrum of whole cells. However, in the isolated microsomal fraction the corresponding monooxygenase was present as shown by the appearence of cytochrome P450, NADPH-cytochrome c (P450) reductase and cytochrome b₅. The absorption maximum of the terminal oxidase in the reduced CO-difference spectrum shifted between 447 and 448 nm. Derepression of biosynthesis of all components was achieved by transition of the cells from carbon- to oxygen-limited growth in continuous culture. The monooxygenase exhibited aminopyrine demethylation activity but not -hydroxylation activity of lauric acid. With respect to the growth limiting nutrient (carbon and oxygen respectively), mitochondrial cytochrome content showed an analogous behavior as cytochrome P450 and cytochrome b₅.     

Isoelectric focusing of myelin membrane proteins

Human myelin was isolated from the white matter of autopsy brains. Myelin proteins were characterized by isoelectric focusing in ultrathin slab gels in a pH range from 3.5 to 10 after solubilization with urea and Nonidet P 40. The protein profile in the acidic region (pH below 6.2) revealed at least twelve faint bands which comprised only a few percent of the total myelin proteins. Most of the myelin proteins were focused in the neutral range (pH 6.2–7.8) which showed two sharper and three broader major bands, the total number of bands in this region being about twenty. The basic pH range (pH above 7.8) contained about 30% of the proteins, and revealed a very intense band near the cathode with seven to nine weaker bands below pH 9.0. When the myelin was partially delipidated prior to solubilization, an additional broad band was observed at the area pH 8.0–8.5.     

Theoretical investigations of prostatic acid phosphatase

The phosphotyrosyl protein phosphatase activity of prostatic acid phosphatase (PAP) has been well established. It has also been suggested that PAP partly regulates the activity of growth factor receptors by dephosphorylating the autophosphorylysable tyrosines in them. We studied the binding of the peptides from epidermal growth factor receptor (EGFR) and its homolog (ErbB-2), corresponding to their autophosphorylation sites, to PAP using theoretical modeling and molecular dynamics (MD) simulation methods. Nine different peptides, each with a phosphotyrosine residue, were docked on human PAP. The binding energies of these peptide-PAP complexes were calculated theoretically and compared to experimentally obtained affinities. The peptide Ace--DNLpYYWD--NH2 from ErbB-2(1197-1203) showed the most favorable free energy of binding when estimated theoretically. The results demonstrate that the presence of another tyrosine residue proximate to C-terminal of autophosphorylysable Tyr enhances the binding affinity considerably. The presence of a bulky group instead prevents the binding, as is observed in case of peptide Ace--NLYpYWDQ--NH2 which failed to bind, both in theoretical calculations and experiments. Thus we demonstarted that PAP could potentially bind to EGFR and Erbb-2 and dephosphorylate them. Thus it could be involved in the regulation of the function of such receptors. In addition, complexes of a peptide from AngiotensinII and phosphotyrosine(pY) with human PAP were also modeled. The effects of different protonation states of the titratable active site residues on ligand (pY) binding have also been investigated. For a favorable binding His12 and Asp258 should be neutral, His257 should be positively charged and the phosphate group of the ligand should be in PO(4) (3-) state. Furthermore, the analysis of protein motion as observed during simulations suggests the loop-loop contact in the PAP dimer to be of importance in cooperativity.     

Quantitative genetics of larval life-history traits in Rana temporaria in different environmental conditions

The degree to which genetic variation in a given trait varies among different populations of the same species and across different environments has seldom been quantified in wild vertebrate species. We investigated the expression of genetic variability and maternal effects in three larval life-history traits of the amphibian Rana temporaria. In a factorial laboratory experiment, five widely separated populations (max. 1600 km) were subjected to two different environmental treatments. Animal model analyses revealed that all traits were heritable (h(2) approximately 0.20) in all populations and under most treatment combinations. Although the cross-food treatment genetic correlations were close to unity, heritabilities under a restricted food regime tended to be lower than those under an ad libitum food regime. Likewise, maternal effects (m(2) approximately 0.05) were detected in most traits, and they tended to be most pronounced under restricted food conditions. We detected several cross-temperature genetic and maternal effects correlations that were lower than unity, suggesting that genotype-environment interactions and maternal effect-environment interactions are a significant source of phenotypic variation. The results reinforce the perspective that although the expression of genetic and maternal effects may be relatively homogeneous across different populations of the same species, local variation in environmental conditions can lead to significant variation in phenotypic expression of quantitative traits through genotype-environment and maternal effect-environment interactions.