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A double staining method using SYTOX green and calcofluor white for studying fungal parasites of phytoplankton.

ABSTRACT: We propose a double staining method based on the combination of two fluorochromes, calcofluor white (CFW; specific chitinous fluorochrome) and SYTOX green (nucleic acid stain), coupled to epifluorescence microscopy for counting, identifying, and investigating the fecundity of parasitic fungi of phytoplankton and the putative relationships established between hosts and their chytrid parasites. The method was applied to freshwater samples collected over two successive years during the terminal period of autumnal cyanobacterial blooms in a eutrophic lake. The study focused on the uncultured host-parasite couple Anabaena macrospora (cyanobacterium) and Rhizosiphon akinetum (Chytridiomycota). Our results showed that up to 36.6% of cyanobacterial akinetes could be parasitized by fungi. Simultaneously, we directly investigated the zoosporic content inside the sporangia and found that both the host size and intensity of infection conditioned the final size and hence fecundity of the chytrids. We found that relationships linking host size, final parasite size, and chytrid fecundity were conserved from year to year and seemed to be host-chytrid couple specific. We concluded that our double staining method was a valid procedure for improving our knowledge of uncultured freshwater phytoplankton-chytrid couples and so of the quantitative ecology of chytrids in freshwater ecosystems.

SUBMITTER: Gerphagnon M

PROVIDER: S-EPMC3697587 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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A double staining method using SYTOX green and calcofluor white for studying fungal parasites of phytoplankton.

Gerphagnon Mélanie M Latour Delphine D Colombet Jonathan J Sime-Ngando Télesphore T

Applied and environmental microbiology 20130419 13

We propose a double staining method based on the combination of two fluorochromes, calcofluor white (CFW; specific chitinous fluorochrome) and SYTOX green (nucleic acid stain), coupled to epifluorescence microscopy for counting, identifying, and investigating the fecundity of parasitic fungi of phytoplankton and the putative relationships established between hosts and their chytrid parasites. The method was applied to freshwater samples collected over two successive years during the terminal per ...[more]

PMID: 23603679

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Project description:BACKGROUND:Blood parasites belonging to the Apicomplexa, Trypanosomatidae and Filarioidea are widespread in birds and have been studied extensively. Microscopical examination (ME) of stained blood films remains the gold standard method for the detection of these infections in birds, particularly because co-infections predominate in wildlife. None of the available molecular tools can detect all co-infections at the same time, but ME provides opportunities for this to be achieved. However, fixation, drying and staining of blood films as well as their ME are relatively time-consuming. This limits the detection of infected hosts during fieldwork when captured animals should be released soon after sampling. It is an obstacle for quick selection of donor hosts for parasite experimental, histological and other investigations in the field. This study modified, tested and described the buffy coat method (BCM) for quick diagnostics (~?20 min/sample) of avian blood parasites. METHODS:Blood of 345 birds belonging to 42 species was collected, and each sample was examined using ME of stained blood films and the buffy coat, which was examined after centrifugation in capillary tubes and after being transferred to objective glass slides. Parasite detection using these methods was compared using sensitivity, specificity, positive and negative predictive values and Cohen's kappa index. RESULTS:Haemoproteus, Leucocytozoon, Plasmodium, microfilariae, Trypanosoma and Lankesterella parasites were detected. BCM had a high sensitivity (>?90%) and specificity (>?90%) for detection of Haemoproteus and microfilariae infections. It was of moderate sensitivity (57%) and high specificity (>?90%) for Lankesterella infections, but of low sensitivity (20%) and high specificity (>?90%) for Leucocytozoon infections. Trypanosoma and Plasmodium parasites were detected only by BCM and ME, respectively. According to Cohen's kappa index, the agreement between two diagnostic tools was substantial for Haemoproteus (0.80), moderate for Lankesterella (0.46) and fair for microfilariae and Leucocytozoon (0.28) infections. CONCLUSIONS:BCM is sensitive and recommended as a quick and reliable tool to detect Haemoproteus, Trypanosoma and microfilariae parasites during fieldwork. However, it is not suitable for detection of species of Leucocytozoon and Plasmodium. BCM is a useful tool for diagnostics of blood parasite co-infections. Its application might be extended to studies of blood parasites in other vertebrates during field studies.

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A double staining method using SYTOX green and calcofluor white for studying fungal parasites of phytoplankton.

Publications

A double staining method using SYTOX green and calcofluor white for studying fungal parasites of phytoplankton.

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