Novel Cultivation-Based Approach To Understanding the Miscellaneous Crenarchaeotic Group (MCG) Archaea from Sedimentary Ecosystems

The uncultured miscellaneous crenarchaeotic group (MCG) archaea comprise one of the most abundant microbial groups in the Earth''s subsurface environment. However, very little information is available regarding the lifestyle, physiology, and factors controlling the distribution of members of this group. We established a novel method using both cultivation and molecular techniques, including a pre-PCR propidium monoazide treatment, to investigate viable members of the MCG in vitro. Enrichment cultures prepared from estuarine sediment were provided with one of a variety of carbon substrates or cultivation conditions and incubated for 3 weeks. Compared with the samples from time zero, there was an order-of-magnitude increase in the number of MCG 16S rRNA genes in almost all cultures, indicating that MCG archaea are amenable to in vitro cultivation. None of the tested substrates or conditions significantly stimulated growth of MCG archaea more than the basal medium alone; however, glycerol (0.02%) had a significantly inhibitory effect (P < 0.05). Diversity analysis of populations resulting from four culture treatments (basal medium, addition of amino acids, H₂-CO₂ as the gas phase, or initial aerobic conditions) revealed that the majority of viable MCG archaea were affiliated with the MCG-8 and MCG-4 clusters. There were no significant differences in MCG diversity between these treatments, also indicating that some members of MCG-4 and MCG-8 are tolerant of initially oxic conditions. The methods outlined here will be useful for further investigation of MCG archaea and comparison of substrates and cultivation conditions that influence their growth in vitro.     

Biological control of cereal stem borers in Kenya: A cost benefit approach

Lepidopteran stem borers are the key pests of maize in Sub-Saharan Africa. In the lowland tropics, dry mid-altitude, dry transitional and the moist mid-altitude zones of Kenya, the invasive crambid Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) causes up to 73% yield loss. The International Centre of Insect Physiology and Ecology (ICIPE) started a biological control (BC) program in 1991 to control stem borers in subsistence agriculture in Africa with emphasis on classical BC of C. partellus. The project released the braconid larval parasitoid Cotesia flavipes Cameron (Hymenoptera: Braconidae) in 1993 in coastal Kenya, where it got established and spread to other regions. This study assesses the economic impact of the introduced parasitoid. Temporal data on percentage parasitism by the introduced parasitoid and on stem borer density were collected between 1995 and 2004. Socio-economic data was collected through administration of questionnaires to 300 farmers. Economic impact of the project was calculated as the value of the yield loss abated by the parasitoid based on a model of expected stem borer density and parasitism level. Average annual parasitism increased linearly from the time of introduction to reach 20% parasitism by 2004. The net reduction in total stem borer density over the last 10 years was 33.7%, thus abating 47.3% of yield loss. The region will accumulate a net present value of US $ 183 million in economic benefits in 20 years since release of the parasitoid. Introduction of other parasitoid species targeting the egg and pupal stages of the stem borer life cycle stages would be required for biological control to push yield loss by stem borers to an insignificant level.     

Selecting subsets of genotyped experimental populations for phenotyping to maximize genetic diversity

Selective phenotyping is a way of capturing the benefits of large population sizes without the need to carry out large-scale phenotyping and hence is a cost-effective means of capturing information about gene–trait relationships within a population. The diversity within the sample gives an indication of the efficiency of this information capture; less diversity implies greater redundancy of the genetic information. Here, we propose a method to maximize genetic diversity within the selected samples. Our method is applicable to general experimental designs and robust to common problems such as missing data and dominant markers. In particular, we discuss its application to multi-parent advanced generation intercross (MAGIC) populations, where, although thousands of lines may be genotyped as a large population resource, only hundreds may need to be phenotyped for individual studies. Through simulation, we compare our method to simple random sampling and the minimum moment aberration method. While the gain in power over simple random sampling for all tested methods is not large, our method results in a much more diverse sample of genotypes. This diversity can be applied to improve fine mapping resolution once a QTL region has been detected. Further, when applied to two wheat datasets from doubled haploid and MAGIC progeny, our method detects known QTL for small sample sizes where other methods fail.     

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.     

Current methods for molecular typing of Campylobacter species

Campylobacter remains one of the most common bacterial causes of gastroenteritis worldwide. Tracking sources of this organism is challenging due to the large numbers of human cases, and the prevalence of this organism throughout the environment due to growth in a wide range of animal species. Many molecular subtyping methods have been developed to characterize Campylobacter species, but only a few are commonly used in molecular epidemiology studies. This review examines the applicability of these methods, as well as the role that emerging whole genome sequencing technologies will play in tracking sources of Campylobacter spp. infection.     

The floral transition is not the developmental switch that confers competence for the Arabidopsis age-related resistance response to Pseudomonas syringae pv. tomato

Age-related resistance (ARR) is a plant defense response characterized by enhanced resistance to certain pathogens in mature plants relative to young plants. In Arabidopsis thaliana the transition to flowering is associated with ARR competence, suggesting that this developmental event is the switch that initiates ARR competence in mature plants (Rusterucci et al. in Physiol Mol Plant Pathol 66:222–231, 2005). The association of ARR and the floral transition was examined using flowering-time mutants and photoperiod-induced flowering to separate flowering from other developmental events that occur as plants age. Under short-day conditions, late-flowering plant lines ld-1 (luminidependens-1), soc1-2 (suppressor of overexpression of co 1-2), and FRI ⁺ (FRIGIDA) displayed ARR before the transition to flowering occurred. Early-flowering svp-31, svp-32 (short vegetative phase), and Ws-2 were ARR-defective, whereas early-flowering tfl1-14 (terminal flower 1-14) displayed ARR at the same time as Col-0. While svp-31, svp-32 and Ws-2 produced few rosette leaves, tfl1-14 produced a rosette leaf number similar to Col-0, suggesting that the development of a minimum number of rosette leaves is necessary to initiate ARR competence under short-day conditions. Photoperiod-induced transient expression of FT (FLOWERING LOCUS T) caused precocious flowering in short-day-grown Col-0 but this was not associated with ARR competence. Under long-day conditions co-9 (constans-9) mutants did not flower but displayed an ARR response at the same time as Col-0. This study suggests that SVP is required for the ARR response and that the floral transition is not the developmental event that regulates ARR competence.     

Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants

Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli–maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP₁) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri.     

Linking phospholipase C isoforms with differentiation function in human vascular smooth muscle cells

The phosphoinositol-phospholipase C (PLC) family of enzymes consists of a number of isoforms, each of which has different cellular functions. PLCγ₁ is primarily linked to tyrosine kinase transduction pathways, whereas PLCδ₁ has been associated with a number of regulatory proteins, including those controlling the cell cycle. Recent studies have shown a central role of PLC in cell organisation and in regulating a wide array of cellular responses. It is of importance to define the precise role of each isoform, and how this changes the functional outcome of the cell. Here we investigated differences in PLC isoform levels and activity in relation to differentiation of human and rat vascular smooth muscle cells. Using Western blotting and PLC activity assay, we show that PLCδ₁ and PLCγ₁ are the predominant isoforms in randomly cycling human vascular smooth muscle cells (HVSMCs). Growth arrest of HVSMCs for seven days of serum deprivation was consistently associated with increases in PLCδ₁ and SM α-actin, whereas there were no changes in PLCγ₁ immuno-reactivity. Organ culture of rat mesenteric arteries in serum free media (SFM), a model of de-differentiation, led to a loss of contractility as well as a loss of contractile proteins (SM α-actin and calponin) and PLCδ₁, and no change in PLCγ₁ immuno-reactivity. Taken together, these data indicate that PLCδ₁ is the predominant PLC isoform in vascular smooth muscle, and confirm that PLCδ₁ expression is affected by conditions that affect the cell cycle, differentiation status and contractile function.     

Effect of age and environmental conditions on the movement characteristics of Pacific oyster (Crassostrea gigas) trochophores

The movement characteristics of Pacific oyster trochophores have received very little coverage in the scientific literature. Described here are not only changes in the swimming characteristics of Crassostrea gigas trochophores (size: 53–77 μm) in relation to time after fertilization and to inter‐female variation but also the effects of salinity and pH on movement characteristics. The percentage of motile trochophores was measured on images obtained through a dissecting microscope and the Velocity Average Path (VAP) was assessed using a Computer Assisted Sperm Analysis (CASA) system. At 20°C, the first movements of the trochophores were observed at 6.5 h post‐fertilization. The mean (± SD) percentage of swimming trochophores and the VAP observed in seawater at 11.5 h post‐fertilization were 85 ± 10% and 146 ± 75 μm s^?1, respectively (n = 8 females). Significant inter‐female variation in the percentage of motile trochophores (range: 66 ± 16–93 ± 3%) and in the VAP (55 ± 47–180 ± 90 μm s^?1) was detected. Most of the trochophores were motile in a 9‰ salinity solution. Adjusting the pH of the seawater to values from 5.10 to 9.08 had no effect on swimming characteristics. The present study shows that the movement of oyster trochophores exhibits high plasticity in relation to environmental conditions because the highest percentages of swimming trochophores and optimal velocity values were recorded within large ranges of salinity and pH. Further research is required to determine whether the swimming performances of trochophores can be used to assess embryo quality in commercial hatcheries.