Chitin determination on marine seston in a shallow temperate estuary (Argentina)

Biancalana, Florencia; Kopprio, Gérmán A.; Dutto, M. Sofía; Berasategui, Anabela A.; Fricke, Anna; Garzón-Cardona, John E.; Peterke, Dieter; Lara, Rubén

doi:10.1590/s1679-87592017129806502

Cited by 6 publications

(5 citation statements)

References 36 publications

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“…The appropriate dye concentration may, however, depend on the chitin concentration in the water. We thus recommend 5 µg dye mL −1 , as chitin concentrations in natural waters are expected to be in the lower µg mL −1 range [ 69 ]. CFW-stained chytrids were difficult to distinguish from the background fluorescence on PC filters after staining with 1 µg CFW mL −1 , and even indistinguishable from the background at 5 and 25 µg CFW mL −1 .…”

Section: Discussionmentioning

confidence: 99%

Intercomparison of Two Fluorescent Dyes to Visualize Parasitic Fungi (Chytridiomycota) on Phytoplankton

et al. 2021

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Fungal microparasites (here chytrids) are widely distributed and yet, they are often overlooked in aquatic environments. To facilitate the detection of microparasites, we revisited the applicability of two fungal cell wall markers, Calcofluor White (CFW) and wheat germ agglutinin (WGA), for the direct visualization of chytrid infections on phytoplankton in laboratory-maintained isolates and field-sampled communities. Using a comprehensive set of chytrid–phytoplankton model pathosystems, we verified the staining pattern on diverse morphological structures of chytrids via fluorescence microscopy. Empty sporangia were stained most effectively, followed by encysted zoospores and im-/mature sporangia, while the staining success was more variable for rhizoids, stalks, and resting spores. In a few instances, the staining was unsuccessful (mostly with WGA), presumably due to insufficient cell fixation, gelatinous cell coatings, and multilayered cell walls. CFW and WGA staining could be done in Utermöhl chambers or on polycarbonate filters, but CFW staining on filters seemed less advisable due to high background fluorescence. To visualize chytrids, 1 µg dye mL−1 was sufficient (but 5 µg mL−1 are recommended). Using a dual CFW–WGA staining protocol, we detected multiple, mostly undescribed chytrids in two natural systems (freshwater and coastal), while falsely positive or negative stained cells were well detectable. As a proof-of-concept, we moreover conducted imaging flow cytometry, as a potential high-throughput technology for quantifying chytrid infections. Our guidelines and recommendations are expected to facilitate the detection of chytrid epidemics and to unveil their ecological and economical imprint in natural and engineered aquatic systems.

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Section: Discussionmentioning

confidence: 99%

Intercomparison of Two Fluorescent Dyes to Visualize Parasitic Fungi (Chytridiomycota) on Phytoplankton

et al. 2021

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“…The zooplankton chitin supply from the BBE was mainly presented in the form of degraded particles contributing to a micro-detritus pool in the seston size fraction ≤20 μm. (Biancalana et al, 2017a;2019). It is known that the carcasses and exuviae of zooplankton represent a concentrated reservoir of chitin for the water column bacteria and fungi (Tang et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…m -3 ) during the warm season (Sabatini, 1989;Hoffmeyer, 2004;Dutto et al, 2012;Berasategui et al, 2016). Previous studies on chitin quantification on seston samples from the BBE suggested that most the chitin was micro-detritus from exuviae and pellets of A. tonsa during the warm season (Biancalana et al, 2017a;Biancalana et al, 2019). However, little is known about the production and stock of chitin by this copepod and its contribution to the seston in this ecosystem.…”

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confidence: 99%

Size-fractionated chitin contribution to seston, with linkages to the copepod Acartia

Biancalana

Berasategui

2020

Ocean Coast. Res.

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Size-fractionated chitin contribution to seston, with linkages to the copepod Acartia The main purpose of the present work was to determine chitin from experimental size-fractioned incubations of the copepod Acartia tonsa, considered one of the primary sources of chitin in the Bahía Blanca Estuary. Sampling was performed during the austral warm season at one station in 2014 and 2015. Field-collected females (200 individuals) and males (100 individuals) were incubated in 8 L containers in the laboratory simulating in situ environmental conditions of temperature and salinity for 72 hours. Chitin content was measured in different size fractions (20-60, 60-135 and ≥135 μm). Particulate organic matter and dry weight were also determined in the different size fractions. Highly agglutinated debris, pellets, and natural food (diatoms and tintinnids) were observed in the 20-60 μm size fraction, with a maximum of 0.68±0.21mg L-1 chitin, and the highest contribution of chitin to POC (34.62±18.50 %). Eggs, nauplii and natural food (diatoms and tintinnids) were observed in the 60-135 μm size fraction, with a maximum of 0.20±0.12 mg L-1 chitin and a contribution to POC of 9.80±5.00 %. Acartia adults and their exuviae were observed in the ≥135 μm size fraction, with a maximum concentration of chitin of 0.67±0.40, and a maximum contribution of chitin to POC TS of 30.47±27.23 %. Differences were detected between the different fractions in chitin, POC and DW, indicating that the 20-60 and ≥135 µm size fractions were both an important contribution of chitin in the experiment. Our results suggest that pellets, carcasses and exuviae along with natural food and organic aggregates were the main source of chitin in this system.

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“…A set of samples from Bahia Blanca Estuary (BBE, Argentina) was used for developing the protocol. Water was pre-filtered with a 20 m net, in order to obtain the size fraction ≤20 m that previous studies had shown contains the major bulk fraction of chitin in certain marine environments, including BBE [11,22,30].…”

Section: Methodsmentioning

confidence: 99%

A protocol for the simultaneous identification of chitin-containing particles and their associated bacteria

Biancalana

Kopprio

Lara

et al. 2017

Systematic and Applied Microbiology

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Chitin is the second most abundant polymer on Earth, playing a crucial role in the biogeochemical cycles. A core issue for studying its processing in aquatic systems is the identification and enumeration of chitin-containing particles and organisms, ideally in a manner that can be directly linked to bulk chitin quantification. The aim of this study was the development of such a technique. We successfully combined the methodology of bulk chitin determination using wheat germ agglutinin (FITC-WGA) for staining chitin-containing particles and organisms along with CARD-FISH staining of either chitin-containing eukaryotic cells or bacteria associated with them. Environmental chitin staining was successfully applied to natural water samples. Fungal hyphae, diatoms, and dinoflagellates, sestonic aggregates and chitin-containing structures derived from metazoa were observed. Also, hybridized bacteria attached to chitinaceous debris were clearly visualized. Finally, as proof of principle, cultured yeast cells were simultaneously-targeted by FITC-WGA and the EUK516 probe without exhibiting any interference between both stains. The presented approach appears as a powerful tool to evaluate the contribution of different size classes and organisms to chitin production and consumption, opening the possibility for application of single-cell approaches targeting the ecophysiology of chitin transformations in aquatic systems.

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Chitin determination on marine seston in a shallow temperate estuary (Argentina)

Cited by 6 publications

References 36 publications

Intercomparison of Two Fluorescent Dyes to Visualize Parasitic Fungi (Chytridiomycota) on Phytoplankton

Intercomparison of Two Fluorescent Dyes to Visualize Parasitic Fungi (Chytridiomycota) on Phytoplankton

Size-fractionated chitin contribution to seston, with linkages to the copepod Acartia

A protocol for the simultaneous identification of chitin-containing particles and their associated bacteria

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