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The sexual phase of the diatom Pseudo-nitzschia multistriata: cytological and time-lapse cinematography characterization

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Abstract

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.

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Acknowledgments

The authors would like to thank Dr. Giovanna Benvenuto (Unit Morpho-Functional Analyses and Bioimaging, Stazione Zoologica Anton Dohrn) for her assistance with CLSM and time-lapse microscopy. E.S. has been supported by a PhD fellowship from Stazione Zoologica Anton Dohrn. A.A. was funded by the EC FP7-People COFUND (GA n. 600407) and RITMARE (Ricerca ITaliana per il MARE) Flagship Project. This work was partially supported by the Marie Curie FP7-PEOPLE-2011-CIG 293887 (GyPSy) grant to M.I.F.

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  1. Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy

    Eleonora Scalco, Alberto Amato, Maria Immacolata Ferrante & Marina Montresor

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  1. Eleonora Scalco
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  2. Alberto Amato
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  3. Maria Immacolata Ferrante
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  4. Marina Montresor
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Correspondence to Eleonora Scalco.

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Handling Editor: Tsuneyoshi Kuroiwa

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Supplementary Movie 1

‘Courtship’ behaviour between a small Pm+ and large Pm− cell of Pseudo-nitzschia multistriata before pairing. Real-time movie. Large Pm− cells move randomly around one small Pm+ cell until one large Pm− cell pairs with the small Pm+. Scale bar = 25 μm; on the top relative time course (MPG 8294 kb)

Supplementary Movie 2

Sexual reproduction in Pseudo-nitzschia multistriata. Real-time movie. The paired gametangia (small Pm− on the top and the large Pm+ below) underwent gametogenesis. The cytoplasm rearranged and later segregated in two distinct protoplasts, which contracted into two rounded gametes. The two gametes produced in the Pm+ gametangium conjugated with the corresponding two Pm− gametes and produced two zygotes attached to the Pm− gametangium. The two zygotes expanded in a bi-polar way, originating two auxospores. The four chloroplasts inside the auxospore are visible. Scale bar = 50 μm; on the top relative time course (AVI) (MPG 4618 kb)

Supplementary Movie 3

Chloroplast movement during gametogenesis. Real-time movie. The arrowed gametangium on the right side (Pm+) underwent gametogenesis. The two chloroplasts divided and slid along the valves: the two daughter chloroplasts on the left side slid downwards and the other two on the right side, upwards. Plasmokinesis occurred, visible in the middle portion of the gametangial cell. The two protoplasts, each containing two daughter chloroplasts, contracted forming two rounded gametes. The gametangium on the left side (Pm−) had just started gametogenesis and four chloroplasts are visible. Scale bar = 50 μm; on the top relative time course (AVI) (MPG 2270 kb)

Supplementary Movie 4

Chloroplast movement during gametogenesis. Real-time movie. The gametangium on the right side (Pm+) underwent gametogenesis. The two chloroplasts divided and slid along the valves: the two daughter chloroplasts on the left side slid downwards and the other two on the right side, upwards. Plasmokinesis occurred, visible in the middle portion of the gametangial cell. The two protoplasts, each containing two daughter chloroplasts, contracted forming two rounded gametes. The gametangium on the left side (Pm−) had just started gametogenesis and four chloroplasts are visible. Scale bar = 50 μm; on the top relative time course (AVI) (MPG 1670 kb)

Supplementary Movie 5

Gamete conjugation in Pseudo-nitzschia multistriata. Real-time movie. Two paired gametangia (small Pm− on the top and large Pm+ below, arrowed) at the end of gametogenesis, each of them with two rounded gametes. The conjugation between two active Pm+ and two passive Pm− gametes was recorded. Scale bar = 50 μm; on the top relative time course (AVI) (MPG 160 kb)

Supplementary Movie 6

Incorrect chloroplast movement during gametogenesis. Real-time movie. Two paired gametangia (small Pm− on the left side, and large Pm+ on the right side). The large gametangium (Pm+) underwent gametogenesis, while the cytoplasm rearranged the two chloroplasts divided and slid along the valves: the two daughter chloroplasts on the left side slid to the centre of the gametangium while the other two on the right side, slid one upwards and one downwards. Plasmokinesis occurred, visible in three portion of the gametangial cell contracted into three rounded anomalous gametes. The upper and the lower anomalous gametes contained one chloroplast each and the middle anomalous gamete contained two daughter chloroplasts. The gametangium on the left side (Pm−) produced two rounded gametes. Scale bar = 50 μm; on the top relative time course (AVI) (MPG 1532 kb)

Supplementary Movie 7

Incorrect chloroplast movement during gametogenesis. Real-time movie. Two paired gametangia (small Pm−, arrowed, and large Pm+). The Pm− gametangium underwent gametogenesis, while the cytoplasm rearranged the two chloroplasts divided and slid along the valves: the two daughter chloroplasts on the left side slid to the centre of the gametangium while the other two on the right side, slid one upwards and one downwards. Plasmokinesis occurred, visible in three portion of the gametangial cell contracted into three rounded anomalous gametes. The upper and the lower anomalous gametes contained one chloroplast each and the middle anomalous gamete contained two daughter chloroplasts. The larger gametangium (Pm+) produced two rounded gametes. Scale bar = 50 μm; on the top relative time course (AVI) (MPG 786 kb)

Supplementary Movie 8

Automixis event in Pseudo-nitzschia multistriata. Real-time movie. The arrowed gametangium underwent gametogenesis, plasmokinesis occurred and the two protoplasms contracted into two rounded gametes that conjugated between them. Eventually the anomalous zygote degenerated. Scale bar = 50 μm; on the top relative time course (AVI) (MPG 3558 kb)

Supplementary Fig. 1

Schematic drawings of the life cycle of Pseudo-nitzschia multistriata showing the different life stages and their nuclei (EPS 59389 kb)

High resolution image (GIF 20 kb)

Supplementary Fig. 2

Confocal Z-stack projections (a-b) and LM micrographs (c-d) of anomalous sexual stages: (a, b) triploid zygotes attached to the Pm− gametangium, a residual body on the lower side in a; (c-d) tetraploid auxospore with eight chloroplasts. Green, nuclei stained with SYBR Green I, red, chlorophyll autofluorescence. Scale bars = 10 μm (EPS 58954 kb)

High resolution image (GIF 215 kb)

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Scalco, E., Amato, A., Ferrante, M.I. et al. The sexual phase of the diatom Pseudo-nitzschia multistriata: cytological and time-lapse cinematography characterization. Protoplasma 253, 1421–1431 (2016). https://doi.org/10.1007/s00709-015-0891-5

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