Background

Sitting at the evolutionary crossroads between photosynthesis and parasitism, Chromera velia (Apicomplexa, Apicomonada [1]) is a photoparasitic alga and the closest known photosynthetic relative of apicomplexan parasites, the group encompassing the causative agents of malaria (Plasmodium) and toxoplasmosis (Toxoplasma). Chromera velia has been isolated from the scleractinian coral Plesiastrea purpurea using a method developed for isolating intracellular coral symbionts. These symbionts, usually dinoflagellates, reside within coral tissue and support the host by supplying photosynthetically derived carbon, receiving carbon dioxide and protection in return. Chromera velia was initially presumed to have a similar life strategy [2]. However, Cumbo et al. [3] demonstrated that, unlike dinoflagellate symbionts that invade the endoderm of coral larvae via the oral pore, C. velia was also found in the ectoderm, indicating active penetration from the outside. Additionally, transcriptomic analyses of C. velia–infected coral larvae revealed expression patterns indicative of a host–pathogen interaction rather than a mutualistic association [4].

Chromera velia has a complex life cycle that is fully maintained under laboratory conditions (Figure 1). It includes immotile stages such as coccoid cells (Figure 2A), autosporangia, zoosporangia (Figure 2B), and cysts, as well as highly motile, biflagellate zoospores (Figure 2C). The process that produces zoospores is called zoosporogenesis [5]. The zoospore is the only life stage believed to penetrate host cells, as it contains a primitive apical complex, in the form of a conoid (pseudoconoid), a structure known from parasitic apicomplexans [6,7]. Thus, C. velia plays a significant role in our understanding of the evolutionary origin of parasitism [8].

Beyond C. velia, zoosporogenesis and the release of motile cells depend heavily on the biology and life cycle of each organism and its adaptation to its environment. The release of zoospores can be triggered by various biotic and abiotic factors, including osmotic stress, salinity, temperature, pH, nutrient availability, wave motion, and light intensity [9]. In C. velia, zoosporogenesis is induced particularly in response to specific light wavelengths and medium composition [5,10]. Appearance of zoospores has been well documented under 12/12 h light/dark (LD) conditions [2,5]. Zoospore numbers peak between 7 and 10 days after culture inoculation and 6 h after the onset of illumination [6,10]. Except under red light, C. velia zoospores display negative phototaxis in response to all tested light spectra, actively swimming away from the light source. Their average swimming speed is estimated at approximately 2 cm/min [10]. A persistent challenge in the study of C. velia has been the reliable isolation of clean zoospore populations, free from coccoid and other non-motile stages, which is crucial for downstream applications such as gene expression analysis, behavioral assays, and physiological studies. The phototactic behavior of zoospores has been characterized in several taxa, including Ulkenia sp. zoospores [11], Allomyces sp. [12], and Ulva sp. [13]; however, these studies focused on identifying the wavelengths eliciting positive or negative phototaxis and did not exploit this response as a separation principle. To our knowledge, the present protocol is the first to use phototaxis directly as a means of purifying motile zoospores from a mixed culture. Previously reported methods for recovering zoospores or other flagellated stages in related systems rely instead on physical or chemical principles. Passive filtration through nylon or polycarbonate meshes has been used to collect zoospores in oomycetes and chytrids [14,15], differential centrifugation has been applied to actinomycete zoospores [16], and column-based purification, such as anion-exchange chromatography of Eimeria sporozoites [17], has been conducted, all of which can compromise zoospore motility and viability by mechanical shear or osmotic change. By contrast, our method is non-invasive, and its selectivity is driven entirely by the behavior of the zoospores—negative phototaxis. Using a directional light source and a standard U-shaped glass tube, only actively swimming zoospores migrate into the collection arm of the U-tube, yielding a population of intact, motile cells suitable for live-cell imaging, motility assays, and downstream molecular analyses. Furthermore, this low-cost approach may be adapted for other flagellated protists exhibiting light-responsive motility.

Figure 1. Representative stages of Chromera velia cultures and isolated zoospores. (A) A 31-day-old stationary culture (OD₆₀₀ = 0.78) was used as inoculum, compared with a 7-day-old early exponential culture (OD₆₀₀ = 0.05) enriched in zoospores used for isolation. (B) Concentrated zoospore fraction visible as a brownish layer at the tip of the tube after isolation (arrow).

Figure 2. Brightfield light microscopy and chlorophyll autofluorescence of Chromera velia developmental stages. Upper row: Brightfield images. Lower row: Corresponding chlorophyll autofluorescence (red signal). (A) Coccoid cell containing a single, well-developed plastid. (B) C. velia zoosporangium containing six zoospores. (C) Isolated zoospores fixed with 4% paraformaldehyde.