Advertisement

Protoplasma

, Volume 253, Issue 6, pp 1421–1431 | Cite as

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

  • Eleonora Scalco
  • Alberto Amato
  • Maria Immacolata Ferrante
  • Marina Montresor
Original Article

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.

Keywords

Chloroplasts Diatoms Life cycle Pseudo-nitzschia multistriata Sexual reproduction Time-lapse cinematography 

Notes

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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

709_2015_891_MOESM1_ESM.doc (30 kb)
ESM 1 (DOC 30 kb)
709_2015_891_MOESM2_ESM.mpg (8.1 mb)
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)
709_2015_891_MOESM3_ESM.mpg (4.5 mb)
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)
709_2015_891_MOESM4_ESM.mpg (2.2 mb)
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)
709_2015_891_MOESM5_ESM.mpg (1.6 mb)
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)
709_2015_891_MOESM6_ESM.mpg (160 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)
709_2015_891_MOESM7_ESM.mpg (1.5 mb)
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)
709_2015_891_MOESM8_ESM.mpg (786 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)
709_2015_891_MOESM9_ESM.mpg (3.5 mb)
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)
709_2015_891_MOESM10_ESM.eps (58 mb)
Supplementary Fig. 1 Schematic drawings of the life cycle of Pseudo-nitzschia multistriata showing the different life stages and their nuclei (EPS 59389 kb)
709_2015_891_Fig6_ESM.gif (21 kb)
High resolution image (GIF 20 kb)
709_2015_891_MOESM11_ESM.eps (57.6 mb)
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)
709_2015_891_Fig7_ESM.gif (215 kb)
High resolution image (GIF 215 kb)

References

  1. Amato A, Kooistra WHCF, Levialdi Ghiron JH, Mann DG, Pröschold T, Montresor M (2007) Reproductive isolation among sympatric cryptic species in marine diatoms. Protist 158:193–207PubMedCrossRefGoogle Scholar
  2. Amato A, Luedeking A, Kooistra WHCF (2010) Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy). Toxicon 55:157–161PubMedCrossRefGoogle Scholar
  3. Amato A, Montresor M (2008) Morphology, phylogeny, and sexual cycle of Pseudo-nitzschia mannii sp. nov. (Bacillariophyceae): a pseudo-cryptic species within the P. pseudodelicatissima complex. Phycologia 47:487–497CrossRefGoogle Scholar
  4. Amato A, Orsini L (2015) Rare interspecific breeding in Pseudo-nitzschia. Phytotaxa 217:145–154CrossRefGoogle Scholar
  5. Amato A, Orsini L, D’Alelio D, Montresor M (2005) Life cycle, size reduction patterns, and ultrastructure of the pennate planktonic diatom Pseudo-nitzschia delicatissima (Bacillariophyceae). J Phycol 41:542–556CrossRefGoogle Scholar
  6. Botte V, Ribera d’Alcalà M, Montresor M (2013) Hydrodynamic interactions at low Reynolds number: an overlooked mechanism favouring diatom encounters. J Plankton Res 35:914–918CrossRefGoogle Scholar
  7. Casteleyn G, Chepurnov VA, Leliaert F, Mann DG, Bates SS, Lundholm N, Rhodes L, Sabbe K, Vyverman W (2008) Pseudo-nitzschia pungens (Bacillariophyceae): a cosmopolitan diatom species? Harmful Algae 7:241–257CrossRefGoogle Scholar
  8. Chepurnov VA, Mann DG, Sabbe K, Vannerum K, Casteleyn G, Verleyen E, Peperzak L, Vyverman W (2005) Sexual reproduction, mating system, chloroplast dynamics and abrupt cell size reduction in Pseudo-nitzschia pungens from the North Sea (Bacillariophyta). Eur J Phycol 40:379–395CrossRefGoogle Scholar
  9. Chepurnov VA, Mann DG, Sabbe K, Vyverman W (2004) Experimental studies on sexual reproduction in diatoms. Int Rev Cytol 237:91–154PubMedCrossRefGoogle Scholar
  10. Chepurnov VA, Mann DG, Vyverman W, Sabbe K, Danielidis DB (2002) Sexual reproduction, mating system, and protoplast dynamics of Seminavis (Bacillariophyceae). J Phycol 38:1004–1019CrossRefGoogle Scholar
  11. Cote P, Whiteway M (2008) The role of Candida albicans FAR1 in regulation of pheromone-mediated mating, gene expression and cell cycle arrest. Mol Microbiol 68:392–404PubMedCrossRefGoogle Scholar
  12. D’Alelio D, Amato A, Luedeking A, Montresor M (2009) Sexual and vegetative phases in the planktonic diatom Pseudo-nitzschia multistriata. Harmful Algae 8:225–232CrossRefGoogle Scholar
  13. D’Alelio D, Ribera d’Alcalà M, Dubroca L, Sarno D, Zingone A, Montresor M (2010) The time for sex: a biennial life cycle in a marine planktonic diatom. Limnol Oceanogr 55:106–114CrossRefGoogle Scholar
  14. Davidovich NA, Bates SS (1998) Sexual reproduction in the pennate diatoms Pseudo-nitzschia multiseries and P. pseudodelicatissima (Bacillariophyceae). J Phycol 34:126–137CrossRefGoogle Scholar
  15. Edgar R, Drolet D, Ehrman JM, Kaczmarska I (2014) Motile male gametes of the araphid diatom Tabularia fasciculata search randomly for mates. PLoS ONE 9:e101767PubMedPubMedCentralCrossRefGoogle Scholar
  16. Edler L, Elbrächter M (2010) The Utermöhl method for quantitative phytoplankton analysis. In: Karlson B, Cusack CK, Bresnan E (ed.) Microscopic and molecular methods for quantitative phytoplankton analysis. IOC Manuals and Guides n. 55, 13-20.Google Scholar
  17. Garcia-Muse T, Steinberg G, Perez-Martin J (2003) Pheromone-induced G(2) arrest in the phytopathogenic fungus Ustilago maydis. Eukaryot Cell 2:494–500PubMedPubMedCentralCrossRefGoogle Scholar
  18. Geitler L (1973) Auxosporenbidung und systematik bei penaten diatomeen und die zytologie von Cocconeis-sippen. Österr Botsch Zagreb 122:299–321CrossRefGoogle Scholar
  19. Gillard J, Frenkel J, Devos V, Sabbe K, Paul C, Rempt M, Inze D, Pohnert G, Vuylsteke M, Vyverman W (2013) Metabolomics enables the structure elucidation of a diatom sex pheromone. Angew Chem Int Ed 52:854–857CrossRefGoogle Scholar
  20. Greiner S, Sobanski J, Bock R (2015) Why are most organelle genomes transmitted maternally? BioEssays 37:80–94PubMedCrossRefGoogle Scholar
  21. Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrate animals. Plenum, New York, pp 29–60CrossRefGoogle Scholar
  22. Hiltz M, Bates SS, Kaczmarska I (2000) Effect of light:dark cycles and cell apical length on the sexual reproduction of the pennate diatom Pseudo-nitzschia multiseries (Bacillariophyceae) in culture. Phycologia 39:59–66CrossRefGoogle Scholar
  23. Holtermann KE, Bates SS, Trainer VL, Odell A, Armbrust EV (2010) Mass sexual reproduction in the toxigenic diatoms Pseudo-nitzschia australis and P. pungens (Bacillariophyceae) on the Washington coast. J Phycol 46:41–52CrossRefGoogle Scholar
  24. Jensen KG, Moestrup Ø, Schmid A-MM (2003) Ultrastructure of the male gametes from two centric diatoms, Chaetoceros laciniosus and Coscinodiscus wailesii (Bacillariophyceae). Phycologia 42:98–105CrossRefGoogle Scholar
  25. Kociolek JP, Stoermer EF (1989) Chromosome numbers in diatoms: a review. Diatom Res 4:47–54CrossRefGoogle Scholar
  26. Koester JA, Swalwell JE, von Dassow P, Armbrust EV (2010) Genome size differentiates co-occurring populations of the planktonic diatom Ditylum brightwellii (Bacillariophyta). BMC Evol Biol 10:1–11PubMedPubMedCentralCrossRefGoogle Scholar
  27. Laney SR, Olson RJ, Sosik HM (2012) Diatoms favor their younger daughters. Limnol Oceanogr 57:1572–1578CrossRefGoogle Scholar
  28. Lelong A, Hégaret H, Soudant P, Bates SS (2012) Pseudo-nitzschia (Bacillariophyceae) species, domoic acid and amnesic shellfish poisoning: revisiting previous paradigms. Phycologia 51:168–216CrossRefGoogle Scholar
  29. Levialdi Ghiron JH, Amato A, Montresor M, Kooistra WHCF (2008) Plastid inheritance in the planktonic raphid pennate diatom Pseudo-nitzschia delicatissima (Bacillariophyceae). Protist 159:91–98PubMedCrossRefGoogle Scholar
  30. Lewis WMJ (1983) Interruption of synthesis as a cost of sex in small organisms. Am Nat 121:825–833CrossRefGoogle Scholar
  31. Mann DG (1994) Auxospore formation, reproductive plasticity and cell structure in Navicula ulvacea and resurrection of the genus Dickieia (Bacillariophyta). Eur J Phycol 29:141–157CrossRefGoogle Scholar
  32. Mann DG (1996) Chloroplast morphology, movements and inheritance in diatoms. In: Chaudhary SB and Agrawal SB (eds.) Cytology, genetics and molecular biology of algae. SPB Academic, 249-274.Google Scholar
  33. Miyamura S (2010) Cytoplasmic inheritance in green algae: patterns, mechanisms and relation to sex type. J Plant Res 123:171–184PubMedCrossRefGoogle Scholar
  34. Mos L (2001) Domoic acid: a fascinating marine toxin. Environ Toxicol Pharmacol 9:79–85PubMedCrossRefGoogle Scholar
  35. Motomura T, Nagasato C, Kimura K (2010) Cytoplasmic inheritance of organelles in brown algae. J Plant Res 123:185–192PubMedCrossRefGoogle Scholar
  36. Pickett-Heaps JD, West J (1998) Time-lapse video observations on sexual plasmogamy in the red alga Bostrychia. Eur J Phycol 33:43–56CrossRefGoogle Scholar
  37. Pulido O (2008) Domoic acid toxicologic pathology: a review. Mar Drugs 6:180–219PubMedPubMedCentralCrossRefGoogle Scholar
  38. Quijano-Scheggia S, Garcés E, Andree K, Fortuño JM, Camp J (2009) Homothallic auxosporulation in Pseudo-nitzschia brasiliana (Bacillariophyta). J Phycol 45:100–107PubMedCrossRefGoogle Scholar
  39. Quijano-Scheggia S, Garces E, Sampedro N, van Lenning K, Flo E, Andree K, Fortuno JM, Camp J (2008) Identification and characterisation of the dominant Pseudo-nitzschia species (Bacillariophyceae) along the NE Spanish coast (Catalonia, NW Mediterranean). Sci Mar 72:343–359Google Scholar
  40. Rines JEB, Donaghay PL, Dekshenieks MM, Sullivan JM, Twardowski MS (2002) Thin layers and camouflage: hidden Pseudo-nitzschia spp. (Bacillariophyceae) populations in a fjord in the San Juan Islands, Washington, USA. Mar Ecol Prog Ser 225:123–137CrossRefGoogle Scholar
  41. Round FE, Crawford RM, Mann DG (1990) The diatoms. Biology and morphology of the genera. Cambridge University Press, CambridgeGoogle Scholar
  42. Sabatino V, Russo MT, Patil S, d’Ippolito G, Fontana A, Ferrante MI (2015) Establishment of genetic transformation in the sexually reproducing diatoms Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis and inheritance of the transgene. Mar Biotechnol 17:452–462PubMedCrossRefGoogle Scholar
  43. Sarno D, Zingone A, Montresor M (2010) A massive and simultaneous sex event of two Pseudo-nitzschia species. Deep-Sea Res Pt II 57:248–255CrossRefGoogle Scholar
  44. Sato S, Beakes G, Idei M, Nagumo T, Mann DG (2011) Novel sex cells and evidence for sex pheromones in diatoms. PLoS ONE 6:e26923PubMedPubMedCentralCrossRefGoogle Scholar
  45. Scalco E, Stec K, Iudicone D, Ferrante MI, Montresor M (2014) The dynamics of sexual phase in the marine diatom Pseudo-nitzschia multistriata (Bacillariophyceae). J Phycol 50:817–828PubMedCrossRefGoogle Scholar
  46. Teng ST, Lim HC, Lim PT, Dao VH, Bates SS, Leaw CP (2014) Pseudo-nitzschia kodamae sp. nov. (Bacillariophyceae), a toxigenic species from the Strait of Malacca, Malaysia. Harmful Algae 34:17–28CrossRefGoogle Scholar
  47. Thwaites GHK (1847) On conjugation in the diatomaceae. Ann Mag Nat Hist 20:9–11Google Scholar
  48. Trainer VL, Bates SS, Lundholm N, Thessen AE, Cochlan WP, Adams NG, Trick CG (2012) Pseudo-nitzschia physiological ecology, phylogeny, toxicity, monitoring and impacts on ecosystem health. Harmful Algae 14:271–300CrossRefGoogle Scholar
  49. von Dassow P, Petersen TW, Chepurnov VA, Armbrust EV (2008) Inter- and intraspecific relationships between nuclear DNA content and cell size in selected members of the centric diatom genus Thalassiosira (Bacillariophyceae). J Phycol 44:335–349CrossRefGoogle Scholar
  50. Wichard T, Oertel W (2010) Gametogenesis and gamete release of Ulva mutabilis and Ulva lactuca (Chlorophyta): regulatory effects and chemical characterization of the “swarming inhibitor”. J Phycol 46:248–259CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  1. 1.Integrative Marine EcologyStazione Zoologica Anton DohrnNaplesItaly

Personalised recommendations