Rapaza

Rapaza
LM of R. viridis capturing a Tetraselmis cell (arrowhead) with the anterior part of the cell. Scale bar: 10 μm
Scientific classification
Domain:	Eukaryota
Phylum:	Euglenozoa
Class:	Euglenida
Clade:	Euglenophyceae
Order:	Rapazida Cavalier-Smith, 2016
Family:	Rapazidae Cavalier-Smith, 2016
Genus:	Rapaza Yamaguchi, Yubuki & Leander, 2012
Species:	R. viridis
Binomial name
Rapaza viridis Yamaguchi, Yubuki & Leander, 2012

Rapaza viridis (Latin for 'green grasper') is a species of single-celled algae within the Euglenophyceae. It is the only species within the genus Rapaza, family Rapazidae and order Rapazida. It was discovered in a tide pool in British Columbia. It is the first example of a mixotroph and of a kleptoplastic species wihin Euglenozoa: it feeds on microalgae through phagocytosis, and then steals the prey chloroplasts to use them for photosynthesis, modifying their structure in the process. In particular, Rapaza viridis can only feed on Tetraselmis cells native to its original environment, and will reject any other prey.

Due to its unique mode of nutrition and phylogenetic position, Rapaza viridis is considered an evolutionary step between phagotrophs and phototrophs with permanent chloroplasts. It is hypothesized that the common ancestor of Euglenophyceae was a kleptoplastic phagotroph, much like this species, due to the presence of horizontally acquired genes from many different algae. After the divergence of Rapaza, the remaining Euglenophyceae acquired permanent plastids from Pyramimonas.

Etymology

The genus name Rapaza comes from Latin rapax 'seizing' and 'grasping', in reference to the feeding behavior of the cells. The species epithet viridis, meaning 'green', references the color of the chloroplasts and algal prey cells in the process of being digested. Together, the binomial name means 'green grasper' in Latin.

Taxonomy

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Type locality of Rapaza viridis

The genus Rapaza was described in 2012 by protistologists Aika Yamaguchi, Naoji Yubuki and Brian S. Leander, on a study published in the journal BMC Evolutionary Biology. It was created to accommodate a species of euglenid, Rapaza viridis, isolated in 2010 from marine water samples collected at a tide pool in Pachena Beach, British Columbia, Canada. After cultivation, various growth experiments and DNA analysis, the microorganism was shown to belong to the phototrophic euglenids (Euglenophyceae). The new species had a functioning chloroplast but also exhibited phagotrophy, making it the first and only example of mixotrophic euglenids.

In 2016, protozoologist Thomas Cavalier-Smith assigned this genus to several monotypic higher-level taxa: family Rapazidae, order Rapazida and subclass Rapazia within the then-accepted class Euglenophyceae. His classification scheme was neglected by other authors in favour of treating the entirety of Euglenida (Euglenophyceae plus a variety of heterotrophic flagellates) as a class, and deprecating the use of Rapazia as a subclass. As of 2021, only Rapazidae and Rapazida are accepted taxa.

Biology

Morphology

Transverse TEM image of R. viridis showing mitochondrion (M), Golgi body (G) and 16 pellicle strips (arrows). Scale bar: 1 μm

Rapaza viridis is a unicellular flagellate, a type of protist that is capable of swimming through two flagella that differ in length and in movement. The longer flagellum is around twice the length of the shorter flagellum, but has the same thickness. The cells are slender with a tapered posterior end, measuring approximately 10–38 μm long and 3–15 μm wide. Like other euglenids, cells are surrounded by a pellicle composed of 16 protein strips arranged helically below the cell membrane, and contain mitochondria with discoidal cristae. As in other spirocutes (i.e. flexible euglenids), cells of R. viridis are capable of 'metaboly' or 'euglenoid movement', which allows for active peristaltic deformation of the cell shape. Its feeding apparatus consists of one rod built of four rows of microtubules and a feeding pocket. There is a stigma composed of 1 to over 10 pigmented particles. The cytoplasm contains ellipsoid paramylon grains, as well as polysaccharide grains as a result of photosynthesis.

Predation

LM of R. viridis cells that were starved for 7 days (left) and cells that were fed 24 hours earlier (right). Arrow: stigma, double arrowhead: chloroplast. Scale bar: 10 μm.

Rapaza viridis is an obligate mixotroph that feeds on algae through phagocytosis. In the same sample where the species was discovered, the microorganism consumed native Tetraselmis algae and grew to distinctly larger and brighter cells in their presence, digesting them completely in the course of around 12 hours. When starved from the algae, cells of R. viridis became smaller and colorless, retaining at least one healthy chloroplast within its cytoplasm. During growth experiments, cells of R. viridis were exposed to a variety of different algae (e.g., Navicula, Pycnococcus, Dunaliella, Scrippsiella and non-native strains of Tetraselmis) while starved from the Tetraselmis strain that the species was found with. However, the mixotroph rejected all other preys, and could not survive for longer than 35 days without being exposed to that specific algal strain. On the other hand, even under constant supply of that strain, the species could not survive for more than a week in the absence of a light source for photosynthesis.

Upon exposition to the native Tetraselmis strain, R. viridis cells enter a feeding frenzy: they capture algae with the anterior part of the cell and drag it swimming backward in a spiral pattern or rotate rapidly. The euglenid can gradually peel away the cell wall of Tetraselmis through repeated peristaltic euglenoid movement (or metaboly), and then engulf the naked prey cell, or engulf the cell with an intact theca and afterwards discharge the theca. The entire process takes between 5 and 40 minutes, but a single R. viridis individual can contain several ingested Tetraselmis cell.

Chloroplasts and kleptoplasty

When describing Rapaza viridis, two types of distinct chloroplasts were reported: one belonging to the ingested green alga Tetraselmis, and one homologous to the chloroplasts seen in phototrophic euglenids. The former were surrounded by two membranes and contained an eyespot and pyrenoids surrounded by starch, without any penetrating thylakoids. The latter were surrounded by three membranes and contained 1–3 pyrenoids, as well as thylakoids in stacks of three that penetrate the pyrenoids. From these observations, it was inferred that R. viridis possesses 'canonical' plastids, i.e. completely functional plastids equivalent to those seen in other Euglenophyceae, which depend on the host cell for survival and multiply and evolve with it. However, studies later revealed that there are no canonical plastids in R. viridis. Instead, the chloroplasts of prey cells are extracted and temporarily retained by R. viridis for its own use in a process known as kleptoplasty ('stealing of plastids'). After phagocytosis of the algal prey, its plastids are separated from the other cellular components, which are later excreted from the host cell. Then, the stolen plastids ('kleptoplasts') are transformed until they resemble canonical plastids: they are divided into smaller fragments by fission, the green algal pyrenoid surrounded by starch disappears, smaller pyrenoids penetrated by thylakoids are formed, the starch grains gradually disappear, and a three-membrane envelope is displayed.

R. viridis requires a regular supply of kleptoplasts through phagocytosis of its prey. The cell is unable to survive for longer than 35 days without acquiring new kleptoplasts. During starvation, the remaining kleptoplasts are gradually degraded, and vacuoles are formed to recycle intracellular substances.

Evolution

According to phylogenetic analyses, Rapaza viridis is the sister group to all other Euglenophyceae. This phylogenetic position is consistent with its place as an evolutionary step between the completely phagotrophic peranemids and the phototrophic Euglenophyceae, because mixotrophy is considered the transitional state during the establishment of the endosymbiotic prey cell and the phagotrophic host cell. It is also consistent with other intermediate characters, e.g. it is the only eukaryovorous euglenid that, instead of gliding on the substrate, is capable of swimming in the water column, a pattern only seen in phototrophs.

Spirocuta Euglenophyceae Euglenales Eutreptiales Pyramimonas chloroplasts Rapaza kleptoplasty, HGT from algae Peranemida Anisonemia "Ploeotiida" Petalomonadida

Phylogenetic position of Rapaza within the Euglenida, with sublabels indicating evolutionary steps towards permanent chloroplasts.

Rapaza viridis is the first case of kleptoplasty within Euglenozoa. Particularly, its chloroplasts are obtained from the green alga Tetraselmis. Transcriptomic and genomic analyses revealed that there are genes encoded in the nucleus of R. viridis and other Euglenophyceae for plastid-targeted proteins acquired from chloroplasts of many different algae (including algae from the "red lineage", i.e. red and chromalveolate algae) through multiple ancient events of horizontal gene transfer. Due to these discoveries, the leading hypothesis is that the last common ancestor of all Euglenophyceae was not a phototroph, but an algivorous phagotroph without permanent plastids which could have exhibited kleptoplasty, much like Rapaza viridis. This common ancestor horizontally acquired the protein targeting system from many algae after prolongued coexistence (from both kleptoplasty and predation). This targeting system could have been involved in the establishment of permanent plastids in the remaining Euglenophyceae, which originated from the green alga Pyramimonas. Additionally, Tetraselmis-derived genes are abundant in other Euglenophyceae, while Pyramimonas-derived genes are minor in Rapaza, meaning that the close association with Pyramimonas began after the divergence of Rapaza.