Combining Flow Cytometry and Real-Time PCR Methodology to Demonstrate Consumption by Prymnesium parvum1

Bowers, Holly A., Andreas Brutemark, Wanderson F. Carvalho, and Edna Granéli, 2010. Combining Flow Cytometry and Real-Time PCR Methodology to Demonstrate Consumption by Prymnesium parvum. Journal of the American Water Resources Association (JAWRA) 46(1):133-143. DOI: 10.1111/j.1752-1688.2009.00397.x Abstract: Harmful algal bloom species can persist in the environment, impacting aquatic life and human health. One of the mechanisms by which some harmful algal bloom species are able to persist is by consumption of organic particles. Methods to demonstrate and measure consumption can yield insight into how populations thrive. Here, we combine flow cytometry and real-time PCR to demonstrate consumption of a cryptophyte species (Rhodomonas sp.) by a toxic mixotrophic haptophyte (Prymnesium parvum). Using flow cytometry, the feeding frequency of a population of P. parvum cells was calculated using the phycoerythrin (PE) fluorescence signal from Rhodomonas sp. and the fluorescence of an acidotropic probe labeling the food vacuoles. Feeding frequency increased in the beginning of the experiment and then began to decline, reaching a maximum of 47.5% of the whole P. parvum population after 212 min. The maximum number of consumed Rhodomonas sp. cells was 0.8 per P. parvum cell, and occurred after 114 min corresponding to an ingestion rate of 0.4 Rhodomonas sp. cells/P. parvum/h. Cells from the feeding P. parvum population were sorted, washed, and subjected to a real-time PCR assay targeting the cryptophyte 18S locus. There was a correlation between cycle threshold (Ct) values and number of consumed prey cells calculated by fluorescence. Overall, this study shows that flow cytometric analysis, of the acidotropic probe and prey pigments, is an efficient and rapid tool in enumerating food vacuoles and the number of prey cells consumed. Furthermore, we suggest that real-time PCR can be applied to cells sorted by flow cytometry, thus allowing for the detection and potential quantification of the targeted prey cells.     

Postnatal management and long-term outcome for survivors with congenital diaphragmatic hernia

Significant advances in the postnatal management of patients with congenital diaphragmatic hernia (CDH) have resulted in a remarkable improvement in survival rates over the past two decades. The success of current postnatal management of CDH patients has rendered fetal intervention to be limited to the most severe cases, and the role for prenatal treatment of CDH patients remains unclear. The adoption of lung-preserving strategies including high-frequency oscillatory ventilation (HFOV) and extracorporeal membrane oxygenation (ECMO) have improved CDH outcomes especially in those patients with significant ventilatory or circulatory compromise. Survival rates of up to 90% are being reported in some high-volume centers. However, the increased survival in CDH patients has been accompanied by an increase in neurological, nutritional and musculoskeletal morbidity among the long-term survivors. This has resulted in the need to provide resources for the long-term follow-up and support of this patient population. In this article, the postnatal management strategies and primary and secondary outcomes of high-volume international pediatric surgical centers will be reviewed. Finally, the role of a multidisciplinary management team for the follow-up of long-term CDH survivors will be discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Sulfamethazine uptake by plants from manure-amended soil

Animal manure is applied to agricultural land as a means to provide crop nutrients. However, animal manure often contains antibiotics as a result of extensive therapeutic and subtherapeutic use in livestock production. The objective of this study was to evaluate plant uptake of a sulfonamide-class antibiotic, sulfamethazine, in corn (Zea mays L.), lettuce (Lactuca sativa L.), and potato (Solanum tuberosum L.) grown in a manure-amended soil. The treatments were 0, 50, and 100 microg sulfamethazine mL(-1) manure applied at a rate of 56 000 L ha(-1). Results from the 45-d greenhouse experiment showed that sulfamethazine was taken up by all three crops, with concentrations in plant tissue ranging from 0.1 to 1.2 mg kg(-1) dry weight. Sulfamethazine concentrations in plant tissue increased with corresponding increase of sulfamethazine in manure. Highest plant tissue concentrations were found in corn and lettuce, followed by potato. Total accumulation of sulfamethazine in plant tissue after 45 d of growth was less than 0.1% of the amount applied to soil in manure. These results raise potential human health concerns of consuming low levels of antibiotics from produce grown on manure-amended soils.     

Land Use,Land-Use Change,Forestry, and Agricultural Activities in the Clean Development Mechanism: Estimates of Greenhouse Gas Offset Potential

Activities involving land use, land-use change,forestry, and agriculture (LUCF) can help reducegreenhouse gas (GHG) concentrations in the atmosphereby increasing biotic carbon storage, by decreasing GHGemissions, and by producing biomass as a substitutefor fossil fuels. Potential activities includereducing rates of deforestation, increasing landdevoted to forest plantations, regenerating secondaryforest, agroforestry, improving the management offorests and agricultural areas; and producing energycrops.Policymakers debating the inclusion of a variety ofLUCF activities in the Clean Development Mechanism(CDM) of the Kyoto Protocol need to consider themagnitude of the carbon contribution these activitiescould make. Existing estimates of the cumulative GHGoffset potential of LUCF activities often take aglobal or regional approach. In contrast, land-usedecisions are usually made at the local level anddepend on many factors including productive capacityof the land, financial considerations of thelandowner, and environmental concerns. Estimates ofGHG offset potential made at a local, or at mostcountry, level that incorporate these factors may belower, as well as more useful for policy analyses,than global or large regional estimates. Whilecountry-level estimates exist for forestry activities,similar estimates utilizing local information need tobe generated for agricultural activities and biofuels,as well as for the cumulative potential of all LUCFactivities in a particular location.     

The Fate of Murine Norovirus and Hepatitis A Virus During Preparation of Fresh Produce by Cutting and Grating

Human noroviruses and hepatitis A virus (HAV) are commonly associated with outbreaks occurring in restaurant establishments and catered events. Food handlers are major contributing factors to foodborne illnesses initiated in the kitchen setting. In this study, transfer of HAV and murine norovirus (MNV-1), a human norovirus surrogate, between produce (cucumbers, strawberries, tomatoes, cantaloupes, carrots, and honeydew melons) and common kitchen utensils (graters and knives) was investigated. The extent of virus transfer to produce during utensil application, in the presence and the absence of food residue, and the impact of knife surface properties (sharp, dull, serrated) was also investigated. Transfer of MNV-1 and HAV from produce items, initially contaminated with ~5.5 log PFU, to knives and graters during application ranged from 0.9 to 5.1 log PFU. MNV-1 transfer to knives was the greatest for cucumbers, strawberries, and tomatoes, and the least for honeydew melons, while transfer of HAV to knives was greater for tomatoes and honeydew melons than strawberries, cantaloupes, and cucumbers. After preparation of a contaminated produce item, knife cross-contamination easily occurred as viruses were detected on almost all of the seven produce items successively prepared. Produce residues on utensils often resulted in less virus transfer when compared to utensils without residue accumulation. Knife surface properties did not impact virus transfer. The ease of virus transfer between produce and utensils demonstrated by the current study highlights the importance of efforts aimed toward preventing cross-contamination in the kitchen environment.     

Ecosystem ecology meets adaptive management: food web response to a controlled flood on the Colorado River, Glen Canyon

Large dams have been constructed on rivers to meet human demands for water, electricity, navigation, and recreation. As a consequence, flow and temperature regimes have been altered, strongly affecting river food webs and ecosystem processes. Experimental high-flow dam releases, i.e., controlled floods, have been implemented on the Colorado River, U.S.A., in an effort to reestablish pulsed flood events, redistribute sediments, improve conditions for native fishes, and increase understanding of how dam operations affect physical and biological processes. We quantified secondary production and organic matter flows in the food web below Glen Canyon dam for two years prior and one year after an experimental controlled flood in March 2008. Invertebrate biomass and secondary production declined significantly following the flood (total biomass, 55% decline; total production, 56% decline), with most of the decline driven by reductions in two nonnative invertebrate taxa, Potamopyrgus antipodarum and Gammarus lacustris. Diatoms dominated the trophic basis of invertebrate production before and after the controlled flood, and the largest organic matter flows were from diatoms to the three most productive invertebrate taxa (P. antipodarum, G. lacustris, and Tubificida). In contrast to invertebrates, production of rainbow trout (Oncorhynchus mykiss) increased substantially (194%) following the flood, despite the large decline in total secondary production of the invertebrate assemblage. This counterintuitive result is reconciled by a post-flood increase in production and drift concentrations of select invertebrate prey (i.e., Chironomidae and Simuliidae) that supported a large proportion of trout production but had relatively low secondary production. In addition, interaction strengths, measured as species impact values, were strongest between rainbow trout and these two taxa before and after the flood, demonstrating that the dominant consumer-resource interactions were not necessarily congruent with the dominant organic matter flows. Our study illustrates the value of detailed food web analysis for elucidating pathways by which dam management may alter production and strengths of species interactions in river food webs. We suggest that controlled floods may increase production of nonnative rainbow trout, and this information can be used to help guide future dam management decisions.