A multifunctional organelle coordinates phagocytosis and chlorophagy in a marine eukaryote phytoplankton Scyphosphaera apsteinii

Introduction

Haptophyte microalgae, including biomineralizing coccolithophores and naked flagellates, comprise 30–50% of the standing stock of microbial primary producers in the world's ocean (Liu et al., 2009). As mixotrophs, haptophyte flagellates are responsible for, on average, 40% of bacterivory in oligotrophic ecosystems (Unrein et al., 2014). Mixotrophy is a collection of functional physiological traits defined by a combination of autotrophic and phagotrophic carbon acquisition (Raven et al., 2009; Flynn et al., 2019) distinct from osmotrophy, which is the uptake of dissolved organic molecules (Flynn et al., 2013). Mixoplankton are important drivers of carbon flow in pelagic microbial communities (Mitra et al., 2014, 2016; Ward & Follows, 2016), with significant contributions to bacterivory and carbon transfer by pico- and nanoplankton (Zubkov & Tarran, 2008). Mixotrophy also allows for life-history, phenotypic, and habitat flexibility, including the ability to thrive in oligotrophic regions or survive subphotic conditions and periods of darkness (Brutemark & Granéli, 2011; Anderson et al., 2018; Wilken et al., 2020). Experimental and modeling studies suggest mixotrophy traits, including increased phagotrophy, may be favored under ocean warming scenarios, for example if metabolic rate responses of phagotrophy are greater than those of photosynthesis (Gonzalez et al., 2022; Lepori-Bui et al., 2022).

Studies directly testing phagocytosis in haptophytes have been conducted primarily on motile representatives (Anderson et al., 2018), including 3 species from the calcifying subclass Calcihaptophycidae (de Vargas et al., 2007), the coccolithophores (Parke & Adams, 1960; Houdan et al., 2006; Avrahami & Frada, 2020). In addition to two flagella, motile haptophytes have a characteristic haptonema, a unique microtubule-based appendage located between the flagella. The haptonema intercepts small prey particles in the flagella-driven feeding currents and deposits them on the posterior portion of the cell, opposite the flagellar and haptonemal roots, where phagocytosis occurs (Parke & Adams, 1960; Kawachi et al., 1991; Kawachi & Inouye, 1995; Dölger et al., 2017). By contrast, the toxic species Prymnesium patellifera appears to immobilize or kill large prey before engulfing them by pseudopodia that also form at the posterior pole (Tillmann, 1998). Additionally, putatively flagellated and mixotrophic heterococcolith-bearing species have been described from the fossil record (Gibbs et al., 2020) based on the arrangement of interlocking coccoliths around a regularly shaped opening that would surround the flagella and haptonema. Extant members of the Syracophaerales and some Zygodiscales (e.g. Helicosphaera spp.) have similar hetercoccolith arrangments, are flagellated in both the haploid and diploid phases, and are ecologically active with distributions along seasonal and environmental gradients (Šupraha et al., 2016; D'Amario et al., 2017).

The haplodiplontic life history of coccolithophores links motile, holococcolith-bearing haploids and nonmotile heterococcolith-bearing diploids (Houdan et al., 2004; Young et al., 2005), suggesting that mixotrophy is likely widespread across the haptophytes. However, in the absence of flagella, a haptonema, and nonoverlapping holococcoliths to enable phagotrophy, nonmotile diploids encased in interlocking heteroccoliths were thought to confine their heterotrophic nutrition to osmotrophy through the uptake of species-specific suites of dissolved organic sources of nitrogen and phosphorus for growth (Benner & Passow, 2010; Godrijan et al., 2020). Transcriptomic studies of both haploid and diploid Gephyrocapsa huxleyi (as Emiliania huxleyi) indicated that they possess the molecular machinery necessary to phagocytose and enzymatically degrade potential prey particles (Rokitta et al., 2011); this was corroborated by low levels of phagotrophy (Ye et al., 2024), despite recent genetic modeling suggesting that G. huxleyi is purely autotrophic (Koppelle et al., 2022). Limited phagotrophy has been reported in 3 additional nonmotile species clad in interlocking heterococcoliths (Avrahami & Frada, 2020).

Scyphosphaera apsteinii Lohmann lies within the order Zygodiscales (Young & Bown, 1997). The coccosphere of nonmotile, heterococcolith-bearing S. apsteinii is formed by two types of noninterlocking coccoliths, plate-like muroliths and urn-shaped lopadoliths, underlain by a layer of overlapping body scales. The chloroplasts are large with lobes and finger-like extensions. The phylogenetic placement, structure, and amenability to culture has made S. apsteinii a model organism in studies of calcification and membrane physiology (Drescher et al., 2012; Durak et al., 2016; Langer et al., 2023). During those studies, decalcified S. apsteinii were observed using lamellipodia to move across a Petri dish and to interact with particles in the media, suggesting that it had the capacity for phagocytosis (see Supporting Information Video S1). Here, we comprehensively tested calcified S. apsteinii for phagocytosis and the physiological status of the hypothesized digestive vacuole using functional assays, relevant prey and visualization of ingestion, particle processing, and ultrastructure of the organelle. During these experiments, we unexpectedly observed chlorophagy. Both prey and chloroplast fragments were processed by the same prominent acidic vacuole that was constitutively expressed and was conserved throughout the cell cycle. We conclude that the vacuole is a novel multifunctional organelle likely playing an essential role in nutrient acquisition and recycling.