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Species-specific effects and the ecological role of programmed cell death in the microalgaeAnkistrodesmus (Sphaeropleales, Selenastraceae)

Marcelo M Barreto Filho1,2,4,Helena H Vieira1,3,J Jeffrey Morris4,Inessa L Bagatini1,✉
1Laboratory of Phycology, Department of Botany, Federal University of São Carlos (UFSCar), Brazil
2Programa de Pós-Graduação em Ecologia e Recursos Naturais (PPGERN), UFSCar, São Carlos, São Paulo, Brazil
3Hydrobiologický Ústav, Biologické centre AV ČR, v.v.i, České Budějovice, Czechia
4Department of Biology, University of Alabama at Birmingham, Birmingham, AL, USA

Electronic supplementary material is available online athttps://doi.org/10.6084/m9.figshare.c.6238476.

✉

Corresponding author.

Roles

Marcelo M Barreto Filho:Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing
Helena H Vieira:Conceptualization, Methodology, Writing – review & editing
J Jeffrey Morris:Formal analysis, Investigation, Visualization, Writing – review & editing
Inessa L Bagatini:Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing

Received 2022 Jun 6; Accepted 2022 Sep 27; Collection date 2022 Oct.

© 2022 The Author(s)

Published by the Royal Society. All rights reserved.

PMCID: PMC9579752  PMID:36259168

Abstract

Reports of programmed cell death (PCD) in phytoplankton raise questions about the ecological evolutionary role of cell death in these organisms. We induced PCD by nitrogen deprivation and unregulated cell death (non-PCD) in one strain of the green microalgaAnkistrodesmus densus and investigated the effects of the cell death supernatants on phylogenetically related co-occurring organisms using growth rates and maximum biomass as proxies of fitness. PCD-released materials fromA. densus CCMA-UFSCar-3 significantly increased growth rates of two conspecific strains compared to healthy culture (HC) supernatants and improved the maximum biomass of allA. densus strains compared to related species. Although growth rates of non-A. densus with PCD supernatants were not statistically different from HC treatment, biomass gain was significantly reduced. Thus, the organic substances released by PCD, possibly nitrogenous compounds, could promote conspecific growth. These results support the argument that PCD may differentiate species or subtypes and increases inclusive fitness in this model unicellular chlorophyte. Further research, however, is needed to identify the responsible molecules and how they interact with cells to provide the PCD benefits.

Keywords:Ankistrodesmus, programmed cell death, inclusive fitness, kin selection, phytoplankton

1. Introduction

In contrast to non-PCD death (e.g. predation, physico-chemical damage), where death is an unintended side-effect of other processes, programmed cell death (PCD) is an active, genetically encoded process [1,2]. In multicellular organisms, PCD may have evolved to prevent individual cells from replicating indefinitely [3–5]. However, PCD occurrence in single-celled microorganisms [6–8] raises intriguing questions about its origin and maintenance [9] since the cell is the individual, and PCD brings no obvious benefits.

The potential species-specific benefits of PCD in unicells were demonstrated in the chlorophyteChlamydomonas reinhardtii [10,11], possibly by nutrient recycling. Previously, we reported typical PCD markers (i.e. ultrastructural alterations and phosphatidylserine (PS) externalization) in another model, green algaAnkistrodesmus densus CCMA-UFSCar-3 under nitrogen deprivation [12]. Here, we investigated whether the PCD and non-PCD modes of death inA. densus have differential survival effects on related co-occurring strains [13]. PCD increased the inclusive fitness ofA. densus but significantly reduced maximum biomass in related species. Our data support the argument that the fitness effects of PCD can depend on genetic relatedness between individuals and may differentiate species [11], and further we provide some evidence even for ecotype differentiation. Our results also suggest that the method of death may differently affect the nutrient flow in the cell and in aquatic ecosystems [14].

2. Material and methods

(a) . Organisms and culture conditions

All organisms (electronic supplementary material, table S1) were axenic and maintained at the Culture Collection of Freshwater Microalgae in the Federal University of São Carlos (CCMA-UFSCar; World Data Centre for Microorganisms 835).

Three strains ofAnkistrodesmus densus (CCMA-UFSCar-3, CCMA-UFSCar-128 and CCMA-UFSCar-239), twoA. fusiformis (CCMA-UFSCar-605, CB 2009/30) and oneA. stipitatus (CCMA-UFSCar-277) were cultured in WC medium, pH 7.0 [12], at 23 ± 2°C, and 200 µmol photons m−2 s−1. Growth curves used optical density at 750 nm (OD750) as a proxy for biomass which has been shown to be more reliable than pigment concentration for green microalgae [15]. Axenic condition was confirmed as described in [16].

(b) . Released materials from PCD, non-PCD and healthy cultures

The released materials from PCD cultures were obtained fromA. densus CCMA-UFSCar-3. Two controls were used (electronic supplementary material, figure S1): a non-PCD culture, obtained by disrupting cells using a microwave [17], and a healthy culture (HC) which had mostly intact exponentially growing cells (electronic supplementary material, figures S1 and S2).

Mid-late exponential cultures ofA. densus (CCMA-UFSCar-3) were centrifuged at 10g for 10 min and resuspended in culture medium to a final density of 1 × 106 cells ml−1 (electronic supplementary material, figure S1).Ankistrodesmus densus (CCMA-UFSCar-3) was induced to PCD by nitrogen deprivation using a nitrogen-free WC medium [12]. HC and non-PCD cultures were grown in complete WC medium under standard conditions. Prior to supernatant collection, non-PCD cultures were microwaved for 3 min (Electrolux MEF28, 700 W) causing rapid cell lysis and death [17].

Supernatants from PCD, non-PCD and HC were obtained by vacuum filtration through a calcined 2.7 µm pore size glass fibre filter (electronic supplementary material, figures S1 and S2). Prior to filtration, each culture was tested for PS externalization.

(c) . Fitness experiment

Concurrently with supernatant filtration, late exponential target cultures of allAnkistrodesmus strains (electronic supplementary material, table S1) were centrifuged at 10g for 10 min and resuspended to approximately 3–4 × 104 cells ml−1 in new WC medium with lower (0.25 mM) NaNO3, allowing nitrogen-limited growth where we could detect potential growth enhancement by nitrogen supplementation from PCD exudates.

Triplicate experimental cultures were prepared by resuspending cells of target cultures in supernatants of each treatment in a 1 : 2 proportion (supernatant:culture) to a final concentration of approximately 20–30 cells µl−1 [11], and incubating under standard conditions for growth measurements.NO3− concentrations were measured with an ion chromatography system (Thermo Scientific, Waltham, MA, USA). FinalNO3− concentrations in experimental cultures were estimated as follows: (supernatant concentrations * 1/3) + (WC medium concentration * 2/3) (electronic supplementary material, table S2). Growth was measured by OD750 [15].

(d) . PCD detection

The externalization of PS in conjunction with membrane integrity retention is an early sign of PCD in chlorophytes [6,11,18], includingA. densus [19]. Early PCD-positive cells were detected by flow cytometry (FACSCalibur cell analyser, Becton-Dickinson, San Jose, CA, USA) using the Apoptosis Detection Kit, BD Pharmingen, following the manufacturer's protocol, as detailed in [12]. Cytograms were analysed using FlowJo (10.5.3) with a minimum of 1 × 104 cells for each sample. Quadrants were analysed according to [12].

(e) . DNA barcoding and phylogenetic analysis

ThetufA gene sequences ofA. densus were obtained from GenBank (accession nos.KT003380,KT003398 andKT003399) [20]. Sequences of other strains were obtained as described in [21] using primers tufAGF4 [22] and tufAR [23]. Sequences were aligned using Geneious 10.2.6. The phylogenetic tree was constructed using the plugin MrBayes 3.2.6 [24] with the substitution model GTR + I + G [22],Chlorolobion braunii CCMA-UFSCar-137 as outgroup, chain length of 1 100 000 with four heated chains, subsample frequency of 100, and burn-in of 250 000. Sequences were deposited at GenBank (accession nos.OM683275–OM683277).

(f) . Statistical analysis

Statistical analyses were performed with R (v. 4.02) [25]. For growth rates (r) analysis, OD750 datasets were ln transformed and at least three consecutive points, from the first experimental day until the end of exponential growth, were selected for linear regression (electronic supplementary material, table S3), with the resulting slope representingr [26]. The lag phase was included because it could be an experimental response. Maximum biomass yields were obtained by selecting average maximum OD750 values of three replicates (electronic supplementary material, table S4).

Maximal biomass andr were compared between treatments with linear models using thelr function.P-values for the models were computed with thelrtest function [27].Post hoc analyses and contrast comparisons were performed with estimated marginal means [28] using the package/functionemmeans [29].P-values for comparisons were from Bonferroni-corrected Tukey tests. Custom contrasts were performed to test differences between maximum biomass of non-PCD and PCD treatments betweenA. densus CCMA-UFSCar-3 and other strains using the functioncontrast [27] and the interaction between the variables ‘strain' and ‘treatments' as predictors. We further analysed differences between PCD and non-PCD treatments between all strains withinA. densus versus unrelated species with linear mixed-effects models using thelme4 package [30]. Fixed effects were if the strains wereA. densus or not, while random effects were the individual strains tested.

3. Results

(a) . PCD detection

Nitrogen-deprived cultures had 23% of cells positive to FITC-Annexin V, indicating PS externalization (electronic supplementary material, figure S3c), whereas healthy control (HC) and non-PCD (microwaved) cultures had, respectively, only 6.63 and 0.058% of FITC-positive cells (electronic supplementary material, figure S3b,d). This pattern was reversed for propidium iodide (PI), a marker of unregulated cell death: nitrogen-deprived, control and non-PCD cultures presented, respectively, 0.43%, 1.32% and 53.3% of cells positive for PI.

(b) . Intra and interspecific effects of PCD materials fromA. densus

Initial concentrations of dissolvedNO3− in each experimental culture were 10.62, 29.97 and 25.81 mg l−1 for PCD, non-PCD and HC, respectively. In HC, where NO−3 was the sole nitrogen source, growth was low for allA. densus strains, especially CCMA-UFSCar-3, likely because there was insufficient nitrogen to meet their demand as observed in other microalgae [31,32]. There was a statistically significant difference in growth rates (r) between treatments for each strain (lrtest,p < 0.001, adjustedR2 0.81, electronic supplementary material, table S5). The strains CCMA-UFSCar-3 and CCMA-UFSCar-239 had significantly higher growth rates with both PCD and non-PCD supernatant treatments (p < 0.05,n = 3, electronic supplementary material, table S6) compared to HC (figure 1a,b). HC and PCD were not significantly different for all other strains, includingA. densus strain CCMA-UFSCar-128 (p > 0.05,n = 3). Interestingly, despite the lowerNO3− concentration, average growth rates of PCD cultures were greater than non-PCD cultures for allA. densus strains, but the difference was not significant (p > 0.05,n = 3) (figure 1b).

Figure 1.

Figure 1.

(a) Growth curves of the strains CCMA-UFSCar 3, 128, 239, 277, 605 and CB 2009/30 with HC, PCD and non-PCD supernatants (n = 3). OD750 values were ln transformed. (b) Growth rater (n = 3) was computed using the slope of the regression of OD750 versus time [23]. (c) Maximum biomass values (n = 3) were obtained as maximum OD750 from linear growth curves up to the point cultures were still actively growing. Significance levels are from pairwisepost hoc Tukey tests,p < 0.05.

Our linear model using maximum OD750 (electronic supplementary material, table S4) as a response variable (adjustedR2 0.98, lrtest,p = 2.2−16, electronic supplementary material, table S5) revealed that all non-PCD cultures had significantly higher maximum biomass compared to PCD and HC treatments (figure 1c; electronic supplementary material, table S7). However, our linear mixed-effects model showed that the difference in biomass yield between non-PCD and PCD treatments was significantly lower amongA. densus strains compared to non-A. densus strains (p < 0.001,figure 2; electronic supplementary material, table S8). Further contrast analyses using linear models also showed that the non-PCD/PCD difference was significant between the PCD-induced strain CCMA-UFSCar-3 and the non-A. densus strains but not within theA. densus clade (p < 0.05, electronic supplementary material, table S9). Also, the non-A. densus strains CCMA-UFSCar-277, 605 and CB 2009-30 had a significant reduction in maximum biomass with the PCD supernatant compared to the HC treatment, whereas two (CCMA-UFSCar-003 and 239) of the threeA. densus had significantly increased biomass with PCD compared to the HC.

Figure 2.

Figure 2.

Bayesian phylogenetic tree oftufA showing relationships betweenAnkistrodesmus strains (left) and the estimated differences between maximum biomass of non-PCD and PCD treatments for each strain (right). In the phylogenetic tree, the open circle is the outgroupChlorolobion braunii CCMA-UFSCar-137 and the numbers by nodes are Bayesian posterior probabilities (PP > 0.6). In the barchart, statistically significant comparisons arep < 0.01. Comparisons betweenA. densus CCMA-UFSCar-3 and all other strains were calculated using a linear model. Comparisons betweenA. densus strains and non-A. densus strains were computed using a linear mixed-effects model.

4. Discussion

We found that 23% ofA. densus CCMA-UFSCar-3 cells underwent PCD by nitrogen deprivation, as shown previously [12]. This proportion is consistent with the observation that only 2% to 30% of cells stained positively for FITC-Annexin V due to the short duration of the early apoptotic-like period [33]. Unlike the PCD and HC treatments, most cells in the non-PCD cultures were necrotic (53.3% PI positive). Therefore,A. densus CCMA-UFSCar-3 supernatants were suitable to test for intra and interspecific effects of PCD and non-PCD cell death.

Interestingly, despite their nitrate concentration being lower by 35–40%, PCD supernatants positively affected growth rates of allA. densus strains compared to HC supernatants although statistically significant only for CCMA-UFSCar-003 and 239 (figure 1b). This supports previous assertions that PCD effects can be species-specific [10,11,34]. On the other hand, it also highlights some intraspecific variability (e.g. between 128 and the otherA. densus strains), despite their identicaltufA sequences (figure 2). This suggests that these strains may be different ecotypes. In the highly variable freshwater environments whereA. densus thrives, the species-specific effects of PCD on population fitness could be an advantage even over short timescales [35]. Also, the fast harvesting of these organic materials by conspecifics might prevent their utilization by co-occurring organisms like bacteria [34] and other algae.

The positive effects of the microwaved treatment were expected. Although non-PCD death is reported to release toxic compounds and enzymes (e.g. photoactivated chlorophyll, cellular proteases) [36,37] which have detrimental effects on neighbours, these materials are inactivated by heat [10]. Thus, the microwaved lysate likely resulted only in readily available goods. In spite of that, and despite the lower NO−3 concentrations in PCD cultures, average growth rates were higher only inA. densus.

Nonetheless, considering that the highest growth for all strains occurred in the non-PCD treatment (figure 1c), biomass accumulation using the PCD supernatants was closer to this optimum forA. densus than for the other species. The difference between maximum biomass of non-PCD and PCD cultures was statistically significant betweenA. densus and related species and seems to reflect phylogenetic relationships withinAnkistrodesmus (figure 2). Thus, the effects of PCD supernatants suggest a nutrient recycling directed towards relatives. The nature of the recycled substances remains unknown, but were suggested to be simple molecules (e.g. phosphonucleosides, small peptides, signalling compounds) inC. reinhartii [10]. They also appear to inhibit the growth of other species [11]. In our previous work, we showed that PCD induced greater chloroplast alterations in nitrogen-deprived cells compared to darkness [12]. Therefore, it is possible that PCD materials might have originated from tightly regulated plastid degradation via autophagic, vacuolar PCD. In plants, autophagy is involved in the degradation of proteins and remobilization to other cells (e.g. [38,39]). Indeed, the ability to use organic compounds as nitrogen sources has been shown inAnkistrodesmus [40], as well as for the relatedPediastrum duplex (Sphaeropleales) [41].

Although in multicellular plants PCD is related to the homeostasis and fitness of the individual [42], in unicells it could play a role in kin selection [43]. The observation that PCD materials fromA. densus did not benefit non-A. densus strains suggests a mechanism targeting these substances (possibly nitrogen-containing compounds) to kin instead of losing them as public goods. [14,44]. Although microwave disruption is an artificial stimulus, in nature viral-induced cell lysis also releases dissolved organic matter (DOM) and may lead to similar outcomes to our non-PCD treatment [14,44]. Thus, PCD could promote conspecific growth crowding out unrelated competitors, while non-PCD materials become readily available to the whole community. However, further research with direct DOM measurements and metabolomics should be performed to elucidate which signals/molecules are involved in the process and how they interact with cells to provide possible PCD benefits. Nevertheless, it seems that the mode of cell death may differently affect the fate of organic matter in aquatic ecosystems. InC. reinhardtii, where PCD was also reported as species-specific [11], membrane-bound vesicles were observed in the extracellular space [45]. We speculate that these extracellular vehicles, also known as nanoalgosomes [46] could be the PCD materials delivery vehicles taken up by relatives.

5. Concluding remarks

PCD has species-specific fitness effects inAnkistrodesmus, as suggested by the higher growth rates and maximum biomass yields of conspecifics of the strain induced to PCD. These results support the view that the ecological role of PCD is an inclusive fitness driven mechanism.

Acknowledgements

We thank Prof. Hugo Sarmento from UFSCar for providing access to the flow cytometer facility. We are also thankful to Prof. Pierre Durand from the University of Witwatersrand for his valuable comments and suggestions.

Data accessibility

Data are available from the Dryad digital repository:https://doi.org/10.5061/dryad.9s4mw6mkf [47].

The data are provided in electronic supplementary material [48].

Authors' contributions

M.M.B.F.: conceptualization, data curation, formal analysis, investigation, methodology, visualization, writing—original draft, writing—review and editing; H.H.V.: conceptualization, methodology, writing—review and editing; J.J.M.: formal analysis, investigation, visualization, writing—review and editing; I.L.B.: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, visualization, writing—original draft, writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

This work was supported by the Brazilian funding agencies: The National Council for Scientific and Technological Development, through a grant to I.L.B. (CNPq, project 427777/2018-6); and Coordination for the Improvement of Higher Education Personnel (CAPES), through a scholarship to M.M.B.F.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

  1. Barreto Filho MM, Vieira HH; Morris JJ, Bagatini IL. 2022. Data from: Species-specific effects and the ecological role of programmed cell death in the microalgaeAnkistrodesmus (Sphaeropleales, Selenastraceae).Dryad Digital Repository. ( 10.5061/dryad.9s4mw6mkf) [DOI] [PMC free article] [PubMed]
  2. Barreto Filho MM, Vieira HH; Morris JJ, Bagatini IL. 2022. Data from: Species-specific effects and the ecological role of programmed cell death in the microalgaeAnkistrodesmus (Sphaeropleales, Selenastraceae).Figshare. ( 10.6084/m9.figshare.c.6238476) [DOI] [PMC free article] [PubMed]

Data Availability Statement

Data are available from the Dryad digital repository:https://doi.org/10.5061/dryad.9s4mw6mkf [47].

The data are provided in electronic supplementary material [48].

Articles from Biology Letters are provided here courtesy ofThe Royal Society

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