doi: 10.1371/journal.pone.0181098. eCollection 2017.
Morphological and transcriptomic evidence for ammonium induction of sexual reproduction in Thalassiosira pseudonana and other centric diatoms
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- PMID:28686696
- PMCID: PMC5501676
- DOI: 10.1371/journal.pone.0181098
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Morphological and transcriptomic evidence for ammonium induction of sexual reproduction in Thalassiosira pseudonana and other centric diatoms
Eric R Moore et al. PLoS One..
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Abstract
The reproductive strategy of diatoms includes asexual and sexual phases, but in many species, including the model centric diatom Thalassiosira pseudonana, sexual reproduction has never been observed. Furthermore, the environmental factors that trigger sexual reproduction in diatoms are not understood. Although genome sequences of a few diatoms are available, little is known about the molecular basis for sexual reproduction. Here we show that ammonium reliably induces the key sexual morphologies, including oogonia, auxospores, and spermatogonia, in two strains of T. pseudonana, T. weissflogii, and Cyclotella cryptica. RNA sequencing revealed 1,274 genes whose expression patterns changed when T. pseudonana was induced into sexual reproduction by ammonium. Some of the induced genes are linked to meiosis or encode flagellar structures of heterokont and cryptophyte algae. The identification of ammonium as an environmental trigger suggests an unexpected link between diatom bloom dynamics and strategies for enhancing population genetic diversity.
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Figures
The average cell size of a population of asexually dividing diatoms decreases as a result of differential thecae inheritance. At a critically small size, cells can initiate sexual reproduction and differentiate into male and female cells. Meiosis in the male spermatogonangium produces multinucleate spermatogonia that divide into individual haploid spermatocytes. Meiosis in the female oogonia produces a single functional haploid nucleus that is fertilized by a flagellated spermatocyte through an opening in the oogonia thecae. Fertilized oogonia expand into a large auxospore where new, large thecae are formed for the new initial cell.
(A) Proportion of sexual cells (oogonia and auxospores) relative to the total population in cultures ofT.pseudonana grown in the presence of NH4Cl or NaNO3; n = 3 independent cultures, average of 300 cells counted per replicate. Oogonia and auxospores were only observed beginning in stationary phase, data are mean values, error bars are s.d.. Inset: corresponding growth curve linking the onset of stationary phase with first appearance of sexual cells on day six. (B) Sexual cells were observed in cultures with NH4Cl present at inoculation (blue hatched and solid blue bars) or following NH4Cl addition at the onset of stationary phase (yellow bars). Legend shows concentration of nitrogen source provided at inoculation and concentration of nitrogen source added at the time of the second dosing. Two control treatments were supplied 200 μM nitrogen source at inoculation only. Inset: corresponding growth curve showing the onset stationary phase and timing of 2nd nitrogen addition; n = 3 independent cultures, average of 281 cells counted per replicate, data are mean values, error bars are s.d..
The life cycle stages ofT.pseudonana (A-K), T. weissflogii (L) and C. cryptica (M-O) imaged using scanning electron microscopy (SEM), light (LM), and confocal microscopy (CFM). A: Two vegetative cells (LM, CCMP1335).B: Oogonium displaying separation of thecae (arrowhead) and putative pycnotic nucleus indicated by the arrow (CFM, CCMP1015).C: Oogonium sharply bending at the thecae junction. Arrowhead indicates protrusion of the plasma membrane (CFM, CCMP1335). Oogonia images are representative of 38 total images.D: Spermatogonium containing multiple spermatocytes seen as individual red (DNA stained) clusters (CFM, CCMP1335); representative of 8 images.E: Motile spermatocytes (in red, arrow) with moving flagella (arrowheads, CFM, CCMP1335, representative of 10 images).F-G: SEM images of spermatocytes (arrowhead) attached to early oogonia (SEM, CCMP1335, representative of 20 images).H,I: Auxospores; representative of 60 images in CCMP1015 (H, LM) and CCMP1335 (I, CFM) showing bulging where mother valve was attached (arrowhead). Two nuclei are visible in red following non-cytokinetic mitosis.J: Small parental cell (arrow) with initial cells produced by sexual reproduction to the left (partial valve view) and right (girdle view) indicated by the arrowheads (SEM, CCMP1335).K: 7 x 12 μm initial cell (LM); j and k representative of 12 images of CCMP1335.L:T.weissflogii auxospore (LM); representative of 12 similar images.M:C.cryptica spermatogonium (upper left) and vegetative cell (lower right). CFM shows stained DNA (red, arrow) and multiple nuclei in the spermatogonium. Arrowheads indicate chlorophyll autofluorescence (green). Oogonium (N, representative of 6 images) and auxospore (O, representative of 4 similar images) ofC.cryptica (LM). Confocal microscopy images (b-e, i, m) show chlorophyll autofluorescence (green) and Hoescht 33342 stained DNA (red). Scale bars:A: 5 μm;B-E: 5 μm;F: 2 μm;G: 1 μm;H-O: 10 μm.
A: 18s rRNA phylogeny of diatoms including pennates (pink rectangle), centrics (blue rectangle). Highlighted in red are the four strains induced into sexual reproduction in this study. Species for which some evidence already exists for sexual reproduction are starred [9, 13, 16, 28, 33, 38].B-D: Changes in DNA and chlorophyll fluorescence in exponential (EXP), stationary (STA) and late stationary (L-STA) growth phases ofT.pseudonana induced by ammonium; 30,000 events recorded, representative of two biological replicates.E: Coulter Counter distributions of cell diameter forT.pseudonana cultures in exponential phase of growth and maintained in NaNO3 (red) and after six successive 25% transfers to medium with ammonium (blue). Each new culture was allowed to remain in stationary phase for three days before the next 25% transfer was made. Single replicates of cultures with cell densities of 2.4 x 106 ml-1 (NaNO3) and 2.3 x 106 ml-1 (ammonium). Dashed lines are the mode for each peak.
A: Heat map of 89 genes having annotated functions that were differentially expressed during differentiation and sexual reproduction inT.pseudonana CCMP1335. Color indicates normalized expression value (FPKM) for each nitrogen treatment (control = 100 μM NO3-; 100NH4 = 100 μM NH4+; 800NH4 = 800 μM NH4+) and growth phase (EXP, STA, L-STA).B: FPKM values of select genes across growth phases for each nitrogen treatment.
References
- Nelson DM, Tréguer P, Brzezinski MA, Leynaert A, Quéguiner B. Production and dissolution of biogenic silica in the ocean: Revised global estimates, comparison with regional data and relationship to biogenic sedimentation Biogeochem Cycles. 1995;9(3):359–72.
- Mann DG. The species concept in diatoms. Phycologia. 1999;38:437–95.
- Simonsen R. The diatom system: Ideas on phylogeny. Bacillaria. 1979;2:9–71.
- Lewis WM. The diatom sex clock and its evolutionary importance. Amer Naturalist. 1984;123:73–80.
- Chepurnov VA, Mann DG, Sabbe K, Vyverman W. Experimental studies on sexual reproduction in diatoms In: Jeon KW, editor. A Survey of Cell Biology. International Review of Cytology. 237 London, UK: Elsevier Academic Press; 2004. p. 91–154. - PubMed
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