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. 2022 May;16(5):1409-1419.
doi: 10.1038/s41396-022-01192-0. Epub 2022 Jan 18.

Trophic flexibility of marine diplonemids - switching from osmotrophy to bacterivory

Galina Prokopchuk #  1   2 , Tomáš Korytář #  3   4 , Valéria Juricová  3   5 , Jovana Majstorović  3   5 , Aleš Horák  3   5 , Karel Šimek  5   6 , Julius Lukeš  7   8
Affiliations

Trophic flexibility of marine diplonemids - switching from osmotrophy to bacterivory

Galina Prokopchuk et al. ISME J. 2022 May.

Abstract

Diplonemids are one of the most abundant groups of heterotrophic planktonic microeukaryotes in the world ocean and, thus, are likely to play an essential role in marine ecosystems. So far, only few species have been introduced into a culture, allowing basic studies of diplonemid genetics, morphology, ultrastructure, metabolism, as well as endosymbionts. However, it remains unclear whether these heterotrophic flagellates are parasitic or free-living and what are their predominant dietary patterns and preferred food items. Here we show that cultured diplonemids, maintained in an organic-rich medium as osmotrophs, can gradually switch to bacterivory as a sole food resource, supporting positive growth of their population, even when fed with a low biovolume of bacteria. We further observed remarkable differences in species-specific feeding patterns, size-selective grazing preferences, and distinct feeding strategies. Diplonemids can discriminate between low-quality food items and inedible particles, such as latex beads, even after their ingestion, by discharging them in the form of large waste vacuoles. We also detected digestion-related endogenous autofluorescence emitted by lysosomes and the activity of a melanin-like material. We present the first evidence that these omnipresent protists possess an opportunistic lifestyle that provides a considerable advantage in the generally food resource-limited marine environments.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Dynamics of bacterial uptake and growth of diplonemids.
A Growth curves of Diplonema japonicum and Rhynchopus humris cultured in either the nutrient-rich Hemi medium (turquoise), artificial seawater (ASW) supplemented with FLB [7–9 × 106 cells/ml] (maroon), or nutrient-free ASW (bright green). Values are means for triplicates, and the error bars show SDs. Open symbols in FLB and Hemi curves indicate significantly different values (T-test, P < 0.05). The insets show the exponential decay of FLB. B Growth rates of D. japonicum (blue) and R. humris (orange) represented as the best-fit lines.
Fig. 2
Fig. 2. Kinetics of consumption and digestion of bacteria.
A Time-course changes in green fluorescence signal emitted by FLB in food vacuoles of D. japonicum (blue) and R. humris (orange). Upper panel: each individual histogram represents the intensity of the green fluorescence (corresponding to the amount of ingested FLB) on the x-axis and the number of events on the y-axis. The composite of histograms shows the extent of changes in the relative fluorescence signal over time intervals of 24 h, which are color-coded. Lower panel: column chart showing comparison of mean fluorescence intensity (MFI) of both diplonemids. B Changes in abundance of D. japonicum (blue) and R. humris (orange) with ingested pHrodo Green dye-labeled bacteria over time and MFI emitted by these bacteria inside the food vacuoles of the respective diplonemids.
Fig. 3
Fig. 3. Characteristics of diplonemids under different conditions.
General morphology of D. japonicum (A, C) and R. humris (D, F) at 72 h and 120 h time points, respectively, by differential interference contrast (DIC) microscopy. Double arrowheads point to numerous food vacuoles filled with FLB (A, D), and arrowheads indicate storage vacuoles in the Hemi medium-grown cells (C, F). Epifluorescence microscopy of D. japonicum (B) and R. humris (E) showing DAPI-stained nucleus (blue) and food vacuoles (double arrowheads), containing FLB (green) at different stages of digestion. Scale bar: 2.5 µm. G A comparison of major morphometric characteristics (shown as the mean value ± SD and range of cell length, width, and cell volume) of the D. japonicum and R. humris from both culture conditions.
Fig. 4
Fig. 4. Characteristics of FLB-fed diplonemids.
Transmission electron micrographs (TEM) of D. japonicum (A–D) and R. humris (E–G) revealing bacterial prey (double arrowheads) inside the food vacuoles at indicated time points. D Arrows point to the coat enveloping two sessile D. japonicum cells, and the arrowhead shows an excreted vacuole with bacterial residues. H Time-course changes in sizes of D. japonicum and R. humris fed with FLB, represented by forward side scatter (FSC). Time intervals as in Fig. 2A. I DIC micrographs of Sulcionema specki showing successive events of excretion of undigested bacterial residues and released vacuole (J). K TEM shows the content of vacuoles filled with bacteria (double arrowheads) at different stages of digestion. Scale bars: 1 µm (A–G), 5 µm (I–J) and 2 µm (K).
Fig. 5
Fig. 5. Characterization of red autofluorescence of diplonemids.
A Panels showing emergence and dynamics of red fluorescence signal emitted by D. japonicum and R. humris. Time intervals as in Fig. 2A. B Time-course changes in mean red autofluorescence intensity of D. japonicum and R. humris freshly subjected to either the Hemi medium (HEMI; solid line) or artificial seawater (ASW; dashed line). C Confocal microscopy images of live D. japonicum showing the presence of red autofluorescence (af) and its colocalization with lysosomes stained with green LysoTracker (lyso); the signal is absent in the ASW-starved cells. Nucleus was stained with NucBlue (nb). Ph, phase contrast. Scale bar: 1 µm. D Time-course changes in abundance of reactive oxygen species produced by D. japonicum grown on bacteria or medium.
Fig. 6
Fig. 6. Differentially expressed genes of osmotrophic, phagotrophic, and starved diplonemids.
Overall relative levels of the gene expression for the most expressed transcripts (TPM > 400) in D. japonicum (A) and R. humris (B) maintained on a bacterial diet (Bact) and medium (HEMI) or starved in ASW broken down to the respective functional categories (COG). For each COG category, the sample with the highest expression (TPM) was assigned 100% (red), and the expression of the remaining samples was related to this value. The horizontal bars show the cumulative TPM values of all abundant transcripts belonging to the respective COG category. Numbers count the most expressed transcripts included in a given COG.
Fig. 7
Fig. 7. Characterization of melanin-like pigment in trophic and starved D. japonicum.
A Western blot analysis showing the time course (0–96 h) of melanin expression using anti-melanin antibody 6D2 in the diplonemids freshly inoculated into either the Hemi medium (HEMI) or artificial seawater (ASW). Leishmania mexicana (Lm) and synthetic melanin (SM) served as negative and positive controls, respectively. UM – stain-free membrane. B Confocal microscopy images of melanin detected in 72-h-old cultures by immunofluorescence using 6D2 and Alexa Fluor 647 goat anti-mouse antibody. DAPI stained the nucleus. Ph, phase contrast. Scale bar: 1 µm. Optical absorption spectra (C) and fluorescence (D) emission spectra of pigment-containing fraction extracted from 72-h-old cultures and compared with synthetic melanin (SM).
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