Identification of bacteria associated with Dinoflagellates (Dinophyceae) Alexandrium spp. using tyramide signal amplification fluorescent in situ hybridization and confocal microscopy

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA-FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA-FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.

Reducing social and environmental impacts of urban freight transport: a review of some major cities

This paper reviews the options available to policy makers in their efforts to reduce the negative impacts of urban freight transport. After providing a summary of the categories of negative impacts that can be targeted together with the specific policy initiatives available, it reviews the actions taken by policy makers across in cities within four countries (UK, Japan, the Netherlands and France). In the case of the UK and Japan attention is focused on a single city as an exemplar of some of the developments. In the case of the Netherlands and France the discussion is wider.

Reducing job-irrelevant bias in performance and appraisals: compliance and beyond

Job-irrelevant discrimination seems as ubiquitous as the performance appraisals in which it is commonly detected. This paper explores both compliance-based and more proactive approaches that deal with the various possible sources of discrimination in performance appraisal ratings. The suggestions lead to a code of practice for performance management in firms across cultures and national boundaries.