Intrinsic Properties of So-Called Dormant Probiotic Bacteria, Determined by Flow Cytometric Viability Assays

Plate counting and four culture-independent flow cytometric assays were used to determine the viability and intrinsic properties of three probiotic strains during storage. The strains showed reduction in plate counts but were able to maintain esterase activity, intact cytoplasmic membrane, and pH gradient. The apparently uncultivable probiotic cells were active and stress resistant.     

REACTION NORMS OF MORPHOLOGICAL AND LIFE-HISTORY TRAITS TO LIGHT AVAILABILITY IN IMPATIENS CAPENSIS

For plants, light availability is an important environmental factor that varies both within and between populations. Although the existence of sun and shade “ecotypes” is controversial, it is often assumed that trade-offs may exist between performance in sun and in shade. This study therefore investigated variation in reaction norms to light availability within and between two neighboring natural populations of the annual Impatiens capensis, one in full sun and the other in a forest understory. Seedlings were collected randomly from both populations and grown to maturity in a greenhouse under two light conditions: full light and 18% of full light. Selfed full-sib seed families were collected from plants from both populations grown in both parental light environments. To characterize family reaction norms, seedlings from each family were divided into the same two light treatments and individuals were scored for a variety of morphological and life-history traits. The maternal light environment had little impact on progeny reaction norms. However, the two study populations differed both qualitatively and quantitatively in plastic response to light availability (indicated by significant population x environment interactions in mixed-model ANCOVA). Much of this difference was attributable to population differences in light sensitivity of axillary meristem allocation patterns, which produced concurrent differences in reaction norms for a suite of developmentally linked traits. Within each population, different sets of traits displayed significant variation in plasticity (indicated by significant family x environment interactions). Thus, the genetic potential for evolutionary response to selection in heterogeneous light environments may differ dramatically between neighboring plant populations. Between-environment genetic correlations were largely positive in the woods population and positive or nonsignificant in the sun population; there was no evidence for performance trade-offs across environments or sun or shade “specialist” genotypes within either population. There was little evidence that population differences represented adaptive differentiation for sun or shade; rather, the results suggested the hypothesis of differential selection on patterns of meristem allocation caused by population differences in timing of mortality and intensity of competition.     

Large scale geographic clines of parasite damage to Populus tremula L.

According to the geographic mosaic theory of coevolution (GMTC), clines of traits reflecting local co‐adaptation (including resistance genes) should be common between a host and its parasite and should persist across time. To test the GMTC‐assumption of persistent clinal patterns we compared the natural prevalence of two parasites on aspen Populus tremula trees: mining moths of the genus Phyllocnistis and leaf rust Melampsora spp. Damage data were collated from the Swedish National Forest Damage Inventory (2004–2006). In addition, occurrence of the parasites was scored in field conditions in two common gardens in the north and south of Sweden over five growing seasons (2004–2008), then related to biomass (stem height and diameter) and to concentrations of eleven leaf phenolics. Phyllocnistis mainly occurred in the northern garden, a distribution range which was confirmed by the countrywide inventory, although Phyllocnistis was more abundant on southern clones, providing evidence for possible local maladaptation. Melampsora occurred all over the country and in both gardens, but built up more quickly on northern clones, which suggests a centre of local clone maladaptation in the north. Stem growth also followed a clinal pattern as did the concentration of three phenolic compounds: benzoic acid, catechin and cinnamic acid. However, only benzoic acid was related to parasite presence: negatively to Phyllocnistis and positively to Melampsora and it could thus be a potential trait under selection. In conclusion, clines of Phyllocnistis were stronger and more persistent compared to Melampsora, which showed contrasting clines of varying strength. Our data thus support the assumption of the GMTC model that clines exist in the border between hot and cold spots and that they may be less persistent for parasites with an elevated gene flow, and/or for parasites which cover relatively larger hot spots surrounded by fewer cold spots.     

When can we measure stress noninvasively? Postdeposition effects on a fecal stress metric confound a multiregional assessment

Measurement of stress hormone metabolites in fecal samples has become a common method to assess physiological stress in wildlife populations. Glucocorticoid metabolite (GCM) measurements can be collected noninvasively, and studies relating this stress metric to anthropogenic disturbance are increasing. However, environmental characteristics (e.g., temperature) can alter measured GCM concentration when fecal samples cannot be collected immediately after defecation. This effect can confound efforts to separate environmental factors causing predeposition physiological stress in an individual from those acting on a fecal sample postdeposition. We used fecal samples from American pikas (Ochotona princeps) to examine the influence of environmental conditions on GCM concentration by (1) comparing GCM concentration measured in freshly collected control samples to those placed in natural habitats for timed exposure, and (2) relating GCM concentration in samples collected noninvasively throughout the western United States to local environmental characteristics measured before and after deposition. Our timed‐exposure trials clarified the spatial scale at which exposure to environmental factors postdeposition influences GCM concentration in pika feces. Also, fecal samples collected from occupied pika habitats throughout the species' range revealed significant relationships between GCM and metrics of climate during the postdeposition period (maximum temperature, minimum temperature, and precipitation during the month of sample collection). Conversely, we found no such relationships between GCM and metrics of climate during the predeposition period (prior to the month of sample collection). Together, these results indicate that noninvasive measurement of physiological stress in pikas across the western US may be confounded by climatic conditions in the postdeposition environment when samples cannot be collected immediately after defecation. Our results reiterate the importance of considering postdeposition environmental influences on this stress metric, especially in multiregional comparisons. However, measurements of fecal GCM concentration should prove useful for population monitoring within an eco‐region or when postdeposition exposure can be minimized.     

Phylogenetic composition and structure of tree communities shed light on historical processes influencing tropical rainforest diversity

The Neotropics, Afrotropics and Madagascar have different histories which have influenced their respective patterns of diversity. Based on current knowledge of these histories, we developed the following predictions about the phylogenetic structure and composition of rainforest tree communities: (Hypothesis 1) isolation of Gondwanan biotas generated differences in phylogenetic composition among biogeographical regions; (H2) major Cenozoic extinction events led to lack of phylogenetic structure in Afrotropical and Malagasy communities; (H3) greater angiosperm diversification in the Neotropics led to greater phylogenetic clustering there than elsewhere; (H4) phylogenetic overdispersion is expected near the Andes due to the co‐occurrence of magnoliids tracking conserved habitat preferences and recently diversified eudicot lineages. Using abundance data of tropical rainforest tree species from 94 communities in the Neotropics, Afrotropics and Madagascar, we computed net relatedness index (NRI) to assess local phylogenetic structure, i.e. phylogenetic clustering vs. overdispersion relative to regional species pools, and principal coordinates of phylogenetic structure (PCPS) to assess variation in phylogenetic composition across communities. We observed significant differences in phylogenetic composition among biogeographical regions (agreement with H1). Overall phylogenetic structure did not differ among biogeographical regions, but results indicated variation from Andes to Amazon. We found widespread phylogenetic randomness in most Afrotropical and all Malagasy communities (agreement with H2). Most of central Amazonian communities were phylogenetically random, although some communities presented phylogenetic clustering (partial agreement with H3). We observed phylogenetic overdispersion near the Andes (agreement with H4). We were able to identify how differences in lineage composition are related to local phylogenetic co‐occurrences across biogeographical regions that have been undergoing different climatic and orographic histories during the past 100 Myr. We observed imprints of the history following Gondwana breakup on phylobetadiversity and local phylogenetic structure of rainforest tree communities in the Neotropics, Afrotropics and Madagascar.     

C and N allocation in soil under ryegrass and alfalfa estimated by 13C and 15N labelling

Background and Aims

Below-ground translocated carbon (C) released as rhizodeposits is an important driver for microbial mobilization of nitrogen (N) for plants. We investigated how a limited substrate supply due to reduced photoassimilation alters the allocation of recently assimilated C in plant and soil pools under legume and non-legume species.

Methods

A non-legume (Lolium perenne) and a legume (Medicago sativa) were labelled with ¹⁵N before the plants were clipped or shaded, and labelled twice with ¹³CO₂ thereafter. Ten days after clipping and shading, the ¹⁵N and ¹³C in shoots, roots, soil, dissolved organic nitrogen (DON) and carbon (DOC) and in microbial biomass, as well as the ¹³C in soil CO₂ were analyzed.

Results

After clipping, about 50 % more ¹³C was allocated to regrowing shoots, resulting in a lower translocation to roots compared to the unclipped control. Clipping also reduced the total soil CO₂ efflux under both species and the ¹³C recovery of soil CO₂ under L. perenne. The ¹⁵N recovery increased in the shoots of M. sativa after clipping, because storage compounds were remobilized from the roots and/or the N uptake from the soil increased. After shading, the assimilated ¹³C was preferentially retained in the shoots of both species. This caused a decreased ¹³C recovery in the roots of M. sativa. Similarly, the total soil CO₂ efflux under M. sativa decreased more than 50 % after shading. The ¹⁵N recovery in plant and soil pools showed that shading has no effect on the N uptake and N remobilization for L. perenne, but, the ¹⁵N recovery increased in the shoot of M. sativa.

Conclusions

The experiment showed that the dominating effect on C and N allocation after clipping is the need of C and N for shoot regrowth, whereas the dominating effect after shading is the reduced substrate supply for growth and respiration. Only slight differences could be observed between L. perenne and M. sativa in the C and N distribution after clipping or shading.     

Organ-specific change inDolichos biflorus lectin binding by myocardial endothelial cells during in vitro cultivation

Summary Endothelial cells of the NMRI mouse strain express a cell surface glycoprotein recognized by the lectinDolichos biflorus agglutinin (DBA). This study documents a marked organ-specific increase in DBA-specific lectin binding of myocardium-derived endothelial cells (MEC) of the NMRI/GSF mouse during in vitro cultivation. An up to 20-fold increase in DBA binding sites is observed in long-term culture, an increase not found in other NMRI-derived endothelial cell lines (e.g., brain, aorta). The increase appears restricted to DBA in that binding with other lectins (PNA, WGA) was unaltered. NMRI MEC cultures maintain typical endothelial cell attributes such as cobblestone morphology on confluence, expression of endothelial cell-specific surface markers, and production of angiotensin-converting enzyme. Cultures routinely become aneuploid within 4 passages, several passages before upregulation of the DBA binding site(s). Myocardial endothelial cells sorted to obtain DBA^hi and DBA^lo cell populations generally maintained their sorted phenotype for 3 to 4 passages. Limiting dilution cloning resulted in clones varying in DBA expression. Clones for DBA^hi expression maintained their DBA affinity for at least 10 passages (>30 doublings), whereas DBA^lo clones gave rise to varying numbers of DBA^hi cells within 2 to 4 passages. We hypothesize that the change in DBA affinity accompanies in vitro aging, that the change is independent of alterations in karyotype, and that the increase in DBA affinity may reflect a change in one or more other endothelial cell properties. Additional studies will be necessary to determine whether the in vitro changes are correlated with specific functional alterations and whether they accurately reflect progressive changes of MEC in vivo.     

The length of peptide substrates has a marked effect on hydroxylation by the hypoxia-inducible factor prolyl 4-hydroxylases

Three hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the HIFs by hydroxylating prolines at two separate sites in the oxygen-dependent degradation domain (ODDD) of their alpha subunits. We compared in vitro hydroxylation by purified recombinant human HIF-P4Hs of 19-20- and 35-residue peptides corresponding to the two sites in HIF-alphas and purified recombinant HIF-1alpha and HIF-2alpha ODDDs of 248 and 215 residues. The increase in the length of peptides representing the C-terminal site from 19 to 20 to 35 residues reduced the K(m) values to 90-800 nm, i.e. to 0.7-11% of those for the shorter peptides, whereas those representing the N-terminal site were 10-470 microm, i.e. 10-135%. The K(m) values of HIF-P4H-1 for the recombinant HIF-alpha ODDDs were 10-20 nm, whereas those of HIF-P4H-2 and -3 were 60-140 nm, identical values being found for the wild-type HIF-1alpha ODDD and its N site mutant. The K(m) values for the C site mutant were about 5-10 times higher but only 0.2-3% of those for the 35-residue N site peptides, and this marked difference suggested that the HIF-P4Hs may become bound first to the C-terminal site of an ODDD and that this binding may enhance subsequent binding to the N-terminal site. The K(m) values of HIF-P4H-2 for oxygen determined with the HIF-1alpha ODDD and both its mutants as substrates were all about 100 microm, being 40% of those reported for the three HIF-P4Hs with a 19-residue peptide. Even this value is high compared with tissue O(2) levels, indicating that HIF-P4Hs are effective oxygen sensors.     

Egg and larval load assessment and its influence on oviposition behaviour of the leaf beetle Galerucella nymphaeae

The oviposition behaviour of the water-lily beetle Galerucella nymphaeae was examined. This species is a specialist herbivore on the floating leaves of nymphaeids Nymphaeaceae and especially on the yellow water-lily, Nuphar lutea. Females lay their eggs in clutches on the leaves, and after hatching, the larvae feed on the leaves. The quality of the leaves decreases quickly after the larvae hatch, and eventually the leaves will sink below the water surface, whereupon the eggs, 1st-instar larvae and pupae are killed by drowning. The influence of conspecific eggs, larvae and feeding tracks on the oviposition preferences of the beetles was tested. Females were allowed to choose between fresh leaves and leaves with conspecific eggs and larvae as well as between leaves with larvae and leaves with feeding tracks but no larvae. An attempt was also made to determine whether eggs and larvae affect the oviposition rate of females when they are not given the opportunity to oviposit on untouched leaves. The results indicate that females tended to avoid leaves with conspecific larvae or to exhibit a decreased oviposition rate on such leaves. Females also avoided conspecific eggs, although the oviposition rate was not influenced by the presence of conspecific eggs. When females were allowed to choose between leaves with larvae and leaves with feeding tracks, possible discrimination against leaves with larvae just fails to reach the 5% level.