Wastewater Treatment Effluent Reduces the Abundance and Diversity of Benthic Bacterial Communities in Urban and Suburban Rivers

In highly urbanized areas, wastewater treatment plant (WWTP) effluent can represent a significant component of freshwater ecosystems. As it is impossible for the composition of WWTP effluent to match the composition of the receiving system, the potential exists for effluent to significantly impact the chemical and biological characteristics of the receiving ecosystem. We assessed the impacts of WWTP effluent on the size, activity, and composition of benthic microbial communities by comparing two distinct field sites in the Chicago metropolitan region: a highly urbanized river receiving effluent from a large WWTP and a suburban river receiving effluent from a much smaller WWTP. At sites upstream of effluent input, the urban and suburban rivers differed significantly in chemical characteristics and in the composition of their sediment bacterial communities. Although effluent resulted in significant increases in inorganic nutrients in both rivers, surprisingly, it also resulted in significant decreases in the population size and diversity of sediment bacterial communities. Tag pyrosequencing of bacterial 16S rRNA genes revealed significant effects of effluent on sediment bacterial community composition in both rivers, including decreases in abundances of Deltaproteobacteria, Desulfococcus, Dechloromonas, and Chloroflexi sequences and increases in abundances of Nitrospirae and Sphingobacteriales sequences. The overall effect of the WWTP inputs was that the two rivers, which were distinct in chemical and biological properties upstream of the WWTPs, were almost indistinguishable downstream. These results suggest that WWTP effluent has the potential to reduce the natural variability that exists among river ecosystems and indicate that WWTP effluent may contribute to biotic homogenization.     

Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.     

Display of active beta-glucosidase on the surface of Schizosaccharomyces pombe cells using novel anchor proteins

Here, we demonstrate display of beta-glucosidase (BGL) on the surface of Schizosaccharomyces pombe cells using novel anchor proteins. A total of four candidate anchor proteins (SPBC21D10.06c, SPBC947.04, SPBC19C7.05, and SPBC359.04c) were selected from among almost all of S. pombe membrane proteins. The C-terminus of each anchor protein was genetically fused to the N-terminus of BGL, and the fusion protein was expressed using S. pombe as a host. The highest cell surface-associated BGL activity (107 U/10⁵ cells was achieved with SPBC359.04c serving as the anchor, followed by SPBC947.04 (44 U/10⁵ cells) and SPBC21D10.06c (38 U/10⁵ cells). S. pombe displaying BGL with SPBC359.04c as an anchor showed the highest growth on 2 % cellobiose (10.7?×?10⁷ cells/mL after 41 h of cultivation from an initial density of 0.1?×?10⁷ cells/mL). Additionally, culturing BGL-displaying S. pombe in medium containing cellobiose as the sole carbon source did not affect protein expression, and ethanol fermentation from cellobiose was successfully demonstrated using BGL-displaying S. pombe. This is the first report describing a cell surface display system for the functionalization of S. pombe.     

Catch-up growth in Japanese quail (Coturnix Japonica): relationships with food intake, metabolic rate and sex

The effects of early environmental conditions can profoundly affect individual development and adult phenotype. In birds, limiting resources can affect growth as nestlings, but also fitness and survival as adults. Following periods of food restriction, individuals may accelerate development, undergoing a period of rapid “catch-up” growth, in an attempt to reach the appropriate size at adulthood. Previous studies of altricial birds have shown that catch-up growth can have negative consequences in adulthood, although this has not been explored in species with different developmental strategies. Here, we investigated the effects of resource limitation and the subsequent period of catch-up growth, on the morphological and metabolic phenotype of adult Japanese quail (Coturnix japonica), a species with a precocial developmental strategy. Because males and females differ in adult body size, we also test whether food restriction had sex-specific effects. Birds that underwent food restriction early in development had muscles of similar size and functional maturity, but lower adult body mass than controls. There was no evidence of sex-specific sensitivity of food restriction on adult body mass; however, there was evidence for body size. Females fed ad lib were larger than males fed ad lib, while females subjected to food restriction were of similar size to males. Adults that had previously experienced food restriction did not have an elevated metabolic rate, suggesting that in contrast to altricial nestlings, there was no metabolic carry-over effect of catch-up growth into adulthood. While Japanese quail can undergo accelerated growth after re-feeding, timing of food restriction may be important to adult size, particularly in females. However, greater developmental flexibility compared to altricial birds may contribute to the lack of metabolic carryover effects at adulthood.     

Maize chromosomal knobs are located in gene-dense areas and suppress local recombination

Knobs are conspicuous heterochromatic regions found on the chromosomes of maize and its relatives. The number, locations, and sizes of knobs vary dramatically, with most lines containing between four and eight knobs in mid-arm positions. Prior data suggest that some knobs may reduce recombination. However, comprehensive tests have not been carried out, primarily because most knobs have not been placed on the genetic map. We used fluorescent in situ hybridization and two recombinant inbred populations to map seven knobs and to accurately place three knobs from the B73 inbred on the genomic sequence assembly. The data show that knobs lie in gene-dense regions of the maize genome. Comparisons to 23 other recombinant inbred populations segregating for knobs at the same sites confirm that large knobs can locally reduce crossing over by as much as twofold on a cM/Mb scale. These effects do not extend beyond regions ~10 cM to either side of knobs and do not appear to affect linkage disequilibrium among genes within and near knob repeat regions of the B73 RefGen_v2 assembly.     

RECONSTRUCTING THE COMPLEX EVOLUTIONARY ORIGIN OF WILD ALLOPOLYPLOID TOBACCOS (NICOTIANA SECTION SUAVEOLENTES)

Nicotiana (Solanaceae) provides an ideal system for understanding polyploidization, a pervasive and powerful evolutionary force in plants, as this genus contains several groups of allotetraploids that formed at different times from different diploid progenitors. However, the parental lineages of the largest group of allotetraploids, Nicotiana section Suaveolentes, have been problematic to identify. Using data from four regions of three low‐copy nuclear genes, nuclear ribosomal DNA, and regions of the plastid genome, we have reconstructed the evolutionary origin of sect. Suaveolentes and identified the most likely diploid progenitors by using a combination of gene trees and network approaches to uncover the most strongly supported evidence of species relationships. Our analyses best support a scenario where a member of the sect. Sylvestres lineage acted as the paternal progenitor and a member of either sect. Petunioides or sect. Noctiflorae that also contained introgressed DNA from the other, or a hypothetical hybrid species between these two sections, was the maternal progenitor. Nicotiana exemplifies many of the factors that can complicate the reconstruction of polyploid evolutionary history and highlights how reticulate evolution at the diploid level can add even greater complexity to allopolyploid genomes.     

Sensitivity to RNA interference-mediated gene silencing in intact and ligated larvae of the armyworm, Mythimna separata (Lepidoptera: Noctuidae)

RNA interference (RNAi) is a common tool for analysis of gene function in both model and non-model insects, but it is becoming evident that RNAi efficiency varies considerably from species to species. We examined RNAi efficiency in larvae of the armyworm Mythimna separata (Walker) using multiple genes and tissues. First, we showed that five different target genes exhibited distinct tissue distribution patterns by quantitative determination of mRNA in total hemocytes, foregut, midgut, hindgut, Malpighian tubules and fat body: neuroglian mRNA was most abundant in fat body; inhibitor of apoptosis proteins mRNA was found to be ubiquitous; aquaporin 4 mRNA was most enriched in hindgut; cueball and prophenoloxidase 2 were mainly expressed in hemocytes. Second, we assessed sensitivity to gene silencing by double-strand RNA injection of these five genes in the six different tissues. We found that these genes generally showed refractoriness to double-strand RNA-mediated gene knockdown irrespective of the tissue tested. Finally, we demonstrated that appreciable gene knockdown was achieved at least in the adhering hemocyte fraction when larval isolated abdomen was prepared by ligation and subjected to dsRNA injection. Our study thus added detailed information on the refractoriness of larval tissues of a lepidopteran insect to gene silencing through RNAi and provided a new potential approach to improve RNAi efficiency.