Utility of field-based artificial streams for assessing effluent effects on riverine ecosystems

Experimentation using field-based artificial streams provides a promising, complimentary approach to biomonitoring assessments because artificial streams provide control over relevant environmental variables and true replication of treatments. We have used large and small artificial stream systems, based in the field, to examine the effect of treated bleached kraft pulp mill effluent (BKME) on the benthos of three large rivers in western Canada. Under natural regimes of temperature, water chemistry, and insolation, these artificial streams provide current velocities and substrata to food chains or food webs that are representative of those in the study river. With these tools we have shown that BKME stimulated mayfly growth in the Thompson River above that which could be accounted for by fertilization of their algal food supply. In contrast, moulting frequency was inhibited at high BKME concentrations. Results from artificial streams also indicate that increased algal biomass and abundances of benthic communities downstream of BKME outfalls were induced by nutrient enrichment from the effluent. BKME treatments did not change diatom species richness in the Fraser River, or diatom species diversity in either the Athabasca or Fraser Rivers. Artificial streams provide a means of understanding the mechanisms of stressor effects over a continuum ranging from single stressor effects on specific taxa to the effects of multiple stressors on communities and ecosystems. Because riverside deployment provides environmental realism within a replicated experimental design, this approach can (i) address questions that cannot be examined using laboratory tests or field observations, (ii) improve our mechanistic understanding of stressor effects on riverine ecosystems, and (iii) can contribute directly to the development, parameterization, and testing of models for predicting ecosystem-level responses.     

Impacts of cloud immersion on microclimate, photosynthesis and water relations of Abies fraseri (Pursh.) Poiret in a temperate mountain cloud forest

The red spruce-Fraser fir ecosystem [Picea rubens Sarg.-Abies fraseri (Pursh) Poir.] of the southern Appalachian mountains, USA, is a temperate zone cloud forest immersed in clouds for 30-40% of a typical summer day, and experiencing immersion on about 65% of all days annually. We compared the microclimate, photosynthetic gas exchange, and water relations of Fraser fir trees in open areas during cloud-immersed, low-cloud, or sunny periods. In contrast to sunny periods, cloud immersion reduced instantaneous sunlight irradiance by 10-50%, and midday atmospheric vapor pressure deficit (VPD) was 85% lower. Needle surfaces were wet for up to 16 h per day during cloud-immersed days compared to <1 h for clear days. Shoot-level light-saturated photosynthesis (A (sat)) on both cloud-immersed (16.0 micromol m(-2) s(-1)) and low-cloud (17.9 micromol m(-2) s(-1)) days was greater than A (sat) on sunny days (14.4 micromol m(-2) s(-1)). Daily mean A was lowest on cloud-immersed days due to reduced sunlight levels, while leaf conductance (g) was significantly higher, with a mean value of 0.30 mol m(-2) s(-1). These g values were greater than commonly reported for conifer tree species with needle-like leaves, and declined exponentially with increasing leaf-to-air VPD. Daily mean transpiration (E) on immersed days was 43 and 20% lower compared to sunny and low-cloud days, respectively. As a result, daily mean water use efficiency (A/E) was lowest on cloud-immersed days due to light limitation of A, and high humidity resulted in greater uncoupling of A from g. Thus, substantial differences in photosynthetic CO2 uptake, and corresponding water relations, were strongly associated with cloud conditions that occur over substantial periods of the summer growth season.     

Phylogeography of the Australian freshwater turtle Chelodina expansa reveals complex relationships among inland and coastal bioregions

We examined range‐wide mitochondrial phylogeographical structure in the riverine freshwater turtle Chelodina expansa to determine whether this species exhibits deep genetic divergence between coastal and inland hydrological provinces, as seen in co‐distributed freshwater taxa. We sequenced two mitochondrial loci, genealogical relationships were assessed using a network approach, and relationships among biogeographical regions were tested using analyses of molecular variance. Population history was evaluated using neutrality tests, indices of demographic expansion, and mismatch analyses. Twenty‐one haplotypes were recovered across two mitochondrial haplogroups separated by approximately 4% nucleotide divergence. The haplogroups have discrete geographical boundaries but only partially support a hypothesis of deep divergence between coastal and inland bioregions. The first haplogroup comprises populations from the inland Murray‐Darling Basin and from coastal catchments south of the Mary River in south‐east Queensland. The second haplogroup comprises populations from coastal catchments north of the Mary River. Cryptic phylogeographical barriers separating adjacent coastal populations are congruent with those demonstrated for other freshwater taxa and may result from the combined influences of the Conondale Range and alluvial deposits at the mouth of the Mary River. The findings of the present study demonstrate that freshwater taxa commonly display genetic differentiation within a biogeographical region where no boundaries have been recognized, highlighting the need to uncover cryptic microbiogeographical regions to aid conservation of freshwater biota. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 789–805.     

Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development

Frem1 belongs to a family of structurally related extracellular matrix proteins of which Fras1 is the founding member. Mutations in Fras1 and Frem1 have been identified in mouse models for Fraser syndrome, which display a strikingly similar embryonic skin blistering phenotype due to impaired dermal-epidermal adhesion. Here we show that Frem1 originates from both epithelial and mesenchymal cells, in contrast to Fras1 that is exclusively derived from epithelia. However, both proteins are localized in an absolutely overlapping fashion in diverse epithelial basement membranes. At the ultrastructural level, Frem1 exhibits a clustered arrangement in the sublamina densa coinciding with fibrillar structures reminiscent of anchoring fibrils. Furthermore, in addition to its extracellular deposition, around E16, Frem1 displays an intracellular distribution in distinct epidermal cell types such as the periderm layer and basal keratinocytes. Since periderm cells are known to participate in temporary epithelial fusions like embryonic eyelid closure, defective function of Frem1 in these cells could provide a molecular explanation for the "eyes open at birth" phenotype, a feature unique for Frem1 deficient mouse mutants. Finally, we demonstrate loss of Frem1 localization in the basement membrane but not in periderm cells in the skin of Fras1(-/-) embryos. Taken together, our findings indicate that besides a cooperative function with Fras1 in embryonic basement membranes, Frem1 can also act independently in processes related to epidermal differentiation.     

Transforming growth factor-beta3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk-1, and CD31 gene expression during CL/Fr mouse lip fusion

BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation. CONCLUSIONS: TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.     

Feasibility of using constructed wetland treatment for molasses wastewater treatment

The surface flow constructed wetland (SFCW) with Cyperus involucratus, Typha augustifolia and Thalia dealbata J. Fraser was applied to treat anaerobic treated-molasses wastewater (An-MWW) under the organic loading rates (OLRs) of 612, 696, 806, 929 and 1,213 kg BOD(5)ha(-1)day(-1). The results showed that both removal efficiency and plant growth rate were increased with the decrease of organic loading rate (OLR). All tested-plant species could not grow under OLR of higher than 696 kg BOD(5)mg l(-1) (p>0.05). Also, the plant-biomass of the systems was reduced by 10.4%, 26.5%, and 64.7% of initial plant-biomass under the OLR of 806, 929 and 1,213 kg BOD(5)ha(-1)day(-1), respectively. However, all tested-plant species showed the same pattern on the plant-biomass production yield and removal efficiency. The highest SS, BOD, COD, total phosphorus, NH(4)(+), NO(3)(-) and molasses pigments (MP) removal efficiencies of 90-93%, 88-89%, 67%, 70-76%, 77-82%, 94-95% and 72-77%, respectively were detected under the OLR of 612 kg BOD(5)ha(-1)day.     

Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos

Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.     

Selective decrease of type I collagen synthesis in Fraser mice skin

Quantification and biosynthesis of type I and type III collagens were determined in skin of control and Fraser mice (Cat^Fraser mutation), which exhibit a genetically determined cataract. Skin organ cultures were labelled with [³H]proline. Pepsin-solubilized collagens were studied using three different approaches: (a) differential salt precipitation at neutral pH, followed by SDS-polyacrylamide gel electrophoresis; (b) differential salt precipitation at acid pH followed by SDS-polyacrylamide gel electrophoresis. (c) CNBr peptide analysis. These methods gave consistent and reproducible results, indicating a selective decrease of type I collagen in Fraser mouse skin as compared to control mouse skin. Metabolic labelling of skin organ cultures showed a decreased specific radioactivity of hydroxy[³H]proline in type I collagen of Fraser mouse skin. The concordant results of these experiments suggest a genetically determined alteration of interstitial collagen metabolism in the Fraser mutation apparently specifically concerning the expression of type I collagen gene(s).     

Genetic discontinuity revealed by chloroplast microsatellites in eastern North American Abies (Pinaceae)

Development of conservation strategies for Fraser fir (Abies fraseri) in the southern Appalachian Mountains depends in part on recognition of the extent to which Fraser fir is genetically distinct from the closely related balsam (A. balsamea) and intermediate (A. balsamea var. phanerolepis) fir. These sibling species have exhibited intergrading, clinal variation in morphological, chemical, and genetic characteristics in prior research. Chloroplast microsatellite markers were polymerase chain reaction amplified from genomic DNA samples of 78 individuals representing the geographic ranges of Fraser, balsam, and intermediate fir. Gene diversity levels at two loci ranged among taxa from 0.65 to 0.84. Allele frequencies demonstrated significant differentiation among taxa, with R(ST) values of 0.36 and 0.10. Haplotype diversity and D(SH) were highest for balsam fir and lowest for intermediate fir. A haplotype network analysis based on allele size distribution for the two loci revealed two distinct clusters of haplotypes and population-specific haplotypes. Ninety-two percent of the haplotypes in one cluster were from balsam fir and intermediate fir, and 84% of the haplotypes in the other cluster were from Fraser fir and intermediate fir. The genetic differentiation of chloroplast DNA markers provides justification for the recognition of Fraser fir as a distinct Management Unit (MU) for conservation purposes, regardless of its taxonomic classification.