sign in sign up
<<
Home , Parvilucifera, Theca

Available online at www.sciencedirect.com ScienceDirect

European Journal of 58 (2017) 9–25

Life-cycle, ultrastructure, and phylogeny of Parvilucifera corolla sp. nov. (Alveolata, ), a parasitoid of dinoflagellates Albert René˜ a,∗, Elisabet Alacida, Rosa Isabel Figueroab, Francisco Rodríguezb, Esther Garcésa aInstitut de Ciències del Mar (CSIC), Pg. Marítim de la Barceloneta, 37-49 08003 Barcelona, Catalonia, Spain bCentro Oceanográfico de Vigo, IEO (Instituto Espa˜nol de Oceanografía), Subida a Radio Faro 50, Vigo, Spain

Received 20 July 2016; received in revised form 25 November 2016; accepted 25 November 2016 Available online 13 December 2016

Abstract

Recent studies of marine have revealed parasites to be key components of marine communities. Here we describe a new species of the parasitoid genus Parvilucifera that was observed infecting the dinoflagellate Durinskia baltica in salt marshes of the Catalan coast (NW Mediterranean). In parallel, the same species was detected after the incubation of seawater from the Canary Islands (Lanzarote, NE Atlantic). The successful isolation of strains from both localities allowed description of the life cycle, ultrastructure, and phylogeny of the species. Its infection mechanism consists of a free-living zoospore that penetrates a dinoflagellate cell. The resulting trophont gradually degrades the dinoflagellate cytoplasm while growing in size. Once the host is consumed, schizogony of the parasitoid yields a sporocyte. After cytokinesis is complete, the newly formed zoospores are released into the environment and are ready to infect new host cells. A distinguishing feature of the species is the radial arrangement of its zoospores around the central area of the sporocyte during their formation. The species shows a close morphological similarity with other species of the genus, including P. infectans, P. sinerae, and P. rostrata. © 2016 Elsevier GmbH. All rights reserved.

Keywords: Apical complex; HAB; Parasite; Parvilucifera; Phylogeny; Ultrastructure

Introduction parasitoids on the diversity of the marine community (Hudson et al. 2006) highlight the importance of on the Marine parasitic protists are a key component of plank- structure of food webs (Lafferty et al. 2006). tonic and benthic marine communities (Chambouvet et al. In aquatic ecosystems, living organisms are almost con- 2014; de Vargas et al. 2015) and there is considerable inter- stantly confronted with parasites (Jephcott et al. 2016). est in their diversity, behavior, and ecology. Parasites may act Many marine parasites belong to the eukaryotic lin- as community regulators by causing changes in host popula- eage of , which include , apicomplexans, tion densities (Alacid et al. 2016), thereby indirectly affecting dinoflagellates, marine alveolates (MALV), and the Perkin- non-host species, which in turn alters community composi- sozoa. All groups contain some parasitic representatives, and tion (Hatcher et al. 2012). The strong potential effects of some are exclusively parasitic (, Perkinsozoa,

∗Corresponding author. Fax: +34 93 230 9555. E-mail address: [email protected] (A. René).˜ http://dx.doi.org/10.1016/j.ejop.2016.11.006 0932-4739/© 2016 Elsevier GmbH. All rights reserved. 10 A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25

MALV). The presence of Perkinsozoa has been demon- presented, together with a morphological comparison with strated in freshwater (Bråte et al. 2010), marine waters (de other species of Parvilucifera described to date. Vargas et al. 2015), and sediments (Chambouvet et al. 2014). These parasites infect shellfish (Andrews 1988), tadpoles (Chambouvet et al. 2015), and dinoflagellates (Park et al. Material and Methods 2004). Thus far, the diversity of Perkinsozoa is mainly rep- resented by environmental sequences, with only two genera Detection, isolation, and culture of the parasitoid of Perkinsozoa described to date: (Mackin, Owen, Collier) Levine and Parvilucifera Norén et Moestrup. Rastri- The parasitoid was detected in two locations, in Cata- monas subtilis (=Cryptophagus subtilis) Brugerolle is also lan coastal waters (NW Mediterranean Sea) and along coast included in Perkinsozoa (Brugerolle 2002, 2003), but molec- of the Canary Islands (North Atlantic). Samplings along ular sequences are not available and its taxonomic affiliation the Catalan coast were carried out in May 2015, in the remains to be confirmed. salt marshes of L’Estartit (42◦0144N, 3◦1130E). After The genus Parvilucifera was erected by Norén et al. (1999) several days of incubation, cells of the dinoflagellate Durin- based on the description of P. infectans, detected in waters of skia baltica present in the natural sample and found to be the North Sea. Parvilucifera infections were also reported infected were transferred to well-plates. As cultures of the in the Mediterranean Sea (Delgado 1999), on the French initial host were not available, different dinoflagellates were Atlantic coast (Erard-Le Denn et al. 2000), on the Baltic added to the wells. Those with Alexandrium minutum (strain Sea (Johansson et al. 2006), and in waters as far rang- AMP4) were infected and could be successfully maintained ing as the Indian Ocean, the North American Atlantic, and by the weekly transfer of aliquots (0.5 mL) of the infected Tasmania (Norén et al. 2000). Later, the species P. sin- culture into healthy, exponentially growing A. minutum cul- erae (Figueroa et al. 2008), P. prorocentri (Leander and tures. The parasitoid was also isolated from Canary Island Hoppenrath 2008), and P. rostrata (Lepelletier et al. 2014) samples collected on September 2015 in tidal ponds of Lan- were additionally described. All of these organisms are par- zarote, in a rocky shore area (Charco del Palo, 29◦0450.7N asitic for a wide range of dinoflagellate hosts (Garcés et al., 13◦2707.2W). In the original population, dinoflagellates 2013a; Lepelletier et al. 2014; Norén et al., 1999), with the were present at low density and there was no sign of infection. exception of P.prorocentri, for which the host range could not Thus, to reveal the possible presence of the parasite, 40-mL be checked and its only known host is the benthic dinoflagel- aliquots of the natural samples were inoculated with 1 mL late Prorocentrum fukuyoi (Leander and Hoppenrath 2008). of exponentially growing A. minutum strain AMP4; 3 days However, a partial specialization could exist in Parvilucifera later the cultures were checked for infection and found to be parasitoids, as observed for P. sinerae, with its preference for positive. Parasitoid cultures were subsequently maintained certain dinoflagellate species (Alacid et al. 2016). following this same procedure. Based on previous experi- Parvilucifera species are endoparasitoids. Thus, once a ence with other species (Lepelletier et al. 2014; Norén et al. host is infected by the biflagellated zoospore, the parasitoid 1999; Turon et al. 2015), we attempted to maintain the para- degrades the cellular contents of the host and a sporangium sitoids in the dormant sporangium stage or to slow down their develops inside the host cell. The mature sporangium gives life cycle by storing the cultures at 4 ◦C in the dark. However, rise to numerous zoospores that are eventually released both efforts were unsuccessful. into the environment in response to an external signal, e.g. dimethylsulfide, excreted by hosts (Garcés et al. 2013b). Zoospores contact their potential hosts, with subsequent Optical microscopy penetration and the development of an infection strongly dependent on the degree of host susceptibility (Alacid et al. Infected cells were serially isolated, washed in filtered 2016). seawater, and transferred to well-plates with healthy A. min- Given their capability to kill their hosts, the wide range utum cells to obtain non-contaminated cultures. Parasitoid of dinoflagellates that are able to infect, and the high vir- cultures were followed daily to identify the different life- ulence observed in lab experiments, these parasites have cycle stages. Infected cells were observed directly either on generated interest as potential controllers of harmful algal the well-plates or in settling chambers under a phase-contrast blooms (Erard-Le Denn et al. 2000; Norén et al. 1999; Park Leica DM-IRB inverted microscope connected to a ProgRes et al. 2004). However, their partial specialization (Alacid et al. C10 (JENOPTIK Laser, Optik, Systeme GmbH, Jena, Ger- 2016) and their strain-specific infectivity (Råberg et al. 2014) many) digital camera. For the Canary Islands cultures, the hinder their implementation until the mechanisms underlying procedure was similar but the cells were additionally washed −1 their specificity are better understood. in PBS, stained with DAPI (2 ␮gmL ), and observed at

In this study, we used optical and electron microscopy to 1000 × magnification (Leica DMR, Germany) using a micro- characterize the life-cycle stages and the ultrastructural fea- scope camera (Axiocam HRC, Zeiss, Germany). The number tures of a new species of Parvilucifera that infects several of zoospores contained in each sporangium was counted from species of dinoflagellates. Its phylogenetic position is also videos captured during zoospore release. A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 11

Scanning electron microscopy PCR amplification, sequencing, and phylogenetic analysis Two of the Canary Islands samples were processed, one containing free zoospores and the other mature spo- Mature sporangia from the Mediterranean specimens were rangia. A 2-mL sample of the culture was collected isolated, transferred to successive drops of seawater, and when mature sporangia were observed, fixed with 10% placed in 200-␮L PCR tubes. The tubes were subjected to ◦ ◦ glutaraldehyde (v/v), and stored at 4 C until processed. several rounds of freezing/thawing and stored at −80 C until It was then post-fixed with osmium tetroxide at a processed. PCR was conducted with a PCR mixture contain-

◦

final concentration of 2% and stored again at 4 C for ing 5 ␮Lof10× buffer (Qiagen) containing 15 mM MgCl2, 1h. 1.25 U of Taq DNA polymerase (Qiagen), 0.2 mM of each When free-living zoospores had emerged from the sporan- dNTP, and 0.5 mM of each primer. The primer pairs EUK A gia, 5 mL of sample was filtered through a 5-␮m Swinnex (Medlin et al. 1988)—1209R (Giovannoni et al. 1988) and filter. The filtrate was collected in a 2-mL Eppendorf tube, 528F (Elwood et al. 1985)—EUK B (Medlin et al. 1988) were ◦ fixed with 10% glutaraldehyde (v/v), and stored at 4 C until used to obtain the complete SSU rDNA region. The PCR con- processed. Both samples were gravity-filtered through 3.0- ditions were as follows: initial denaturation for 5 min at 95 ◦C, ␮m pore size filters (Nucleopore, Pleasanton, CA, USA) 30 cycles of 45 s at 95 ◦C, 1 min at 55 ◦C, and 3 min at 72 ◦C, ◦ and then washed first in seawater for 15 min followed by followed by a final extension step of 10 min at 72 C. 10 ␮Lof distilled water for 15 min. They were then dehydrated in a PCR products were electrophoresed for 20–30 min at 120 V 25, 50, 75, 90, 95, and 100% ethanol series for ca. 10 min. in a 1.2% agarose gel and then visualized under UV illumi- ◦

The final step of 100% ethanol was repeated twice. The nation. The remainder of the sample was frozen at −20 C filters were critical-point dried, mounted on stubs, sputter- and later used for sequencing. For the Canary Islands strain,

coated with gold-palladium, and examined under a Hitachi the PCR procedure included one primer pair (EUK A–EUK

S-3500N scanning electron microscope (Hitachi High Tech- B) and the amplification reaction mixtures (25 ␮L): 2.5 ␮L

nologies America Inc., Schaumburg, IL, USA). Measurement of 10 × buffer, 1.25 mM MgCl2, 0.75 U of Taq DNA poly- data were collected either directly during observations or merase (Qiagen), 0.6 mM of each dNTP, and 0.2 mM of each thereafter from the images using ImageJ (National Insti- primer. The PCR conditions were the same as detailed above. tutes of Health, USA). Background was removed in some One amplicon from the Canary Islands samples was cloned ® images using Photoshop (Adobe Systems, Mountain View, using the pSpark universal DNA cloning kit (Canvax, Spain) CA). following the manufacturer’s instructions. The PCR prod- ucts were purified and sequenced by Genoscreen (France), in which they were migrated in a 3730XL sequencer, and Transmission electron microscopy by CACTI, University of Vigo (Spain), in which they were migrated in a AB 3130 DNA sequencer. Sequencing was The ultrastructure of the different parasitoid life-cycle done using forward and reverse primers in all cases and par- stages was followed by collecting 2-mL samples of parasitoid tial sequences were merged. The sequences were aligned culture on 3 successive days during the infection process and with those from GenBank using the MAFFT v.6 program then preparing them for transmission electron microscopy (Katoh et al. 2002), under L-INS-i, and manually checked (TEM) as follows. The samples were fixed with 25% glu- with BioEdit v. 7.0.5 (Hall 1999), yielding a final align- taraldehyde at a final concentration of 10% (v:v) and then

ment of ∼1790 positions. Phylogenetic relationships were stored at 4 ◦C until processed. They were then post-fixed determined using maximum-likelihood (ML) and Bayesian at room temperature in an osmium tetroxide solution con- inference methods. For the former, the GTRGAMMA evo- sisting of one volume of 4% osmium and three volumes of lution model was used on RAxML (Randomized Axelerated cacodylate buffer. The pellet was washed twice (10 min each), Maximum Likelihood) v. 7.0.4 (Stamatakis 2006). All model embedded in agar (2.5%), and dehydrated through a graded

parameters were estimated using RAxML. Repeated runs on series of ethanol (30, 50, 70, 85, 2 × 95, 2 × 100%; 10 min distinct starting trees were carried out to select the tree with each) followed by two 10-min rounds of dehydration with the best topology (the one with the greatest likelihood of 1000 100% acetone. Finally, the samples were infiltrated with 2:1, alternative trees). Bootstrap ML analysis was done with 1000 1:1, and 1:2 mixtures of acetone: Spurr’s resin (approx. 2 h pseudo-replicates and the consensus tree was computed with for each dilution) and embedded overnight in 100% Spurr’s the RAxML software. The Bayesian inference was performed resin. The material was sectioned on an Ultracut E (Reichert- with MrBayes v.3.2 (Ronquist et al. 2012), run with a GTR Jung) microtome using a diamond knife (Diatome). Sections model in which the rates were set to gamma. Each analy- were collected on a 200-mesh grid coated with Formvar film, sis was performed using four Markov chains (MCMC), with stained in 2% uranyl acetate and lead citrate following the one million cycles for each chain. The consensus tree was method of Reynolds (1963), and examined in a JEOL JEM- created from post-burn-in trees and the Bayesian posterior 1010 electron microscope operated at 80 kV. Micrographs probabilities of each clade were examined. Sequences gener- were taken using a CCD Megaview 1k × 1k digital camera. 12 A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 ated in this study were deposited in GenBank under accession formed and ready to be released, the sporocyte is referred to numbers KX519758–KX519761. Sequences KX519758 and as the sporangium. During the release process, the zoospores KX519759 correspond to isolates obtained from L’Estartit swim within the sporangium, emerging through several (>6)

salt marshes strain, while KX519760 and KX519761 corre- apertures (Figs 1H, 2H, 3 A). These are round, 2.0–2.6 ␮min spond to isolates obtained from Lanzarote strain. KX519761 diameter, and covered by an operculum, which is lost at the was obtained from clonal products. moment of zoospore release (Fig. 3B). The surface of the spo- rangium is ornamented with numerous processes (Fig. 3C) but the apertures have a peripheral smooth area that lacks pro- cesses (Fig. 3A, C). A net of star-shaped filamentous material Results connects the processes and covers the sporangium (Fig. 3D). Rarely, a round body, the remnant of the amorphous hya- Time course of infection: life-cycle of the line material found in the trophont, is observed in the empty parasitoid and the morphology of its developing sporangium, after all zoospores have been released (Fig. 1I). stages When infecting A. minutum, each sporangium can contain up to 150 zoospores and the release of most zoospores lasts less The complete life-cycle of the parasitoid was studied under than 10 min. It is common that few zoospores are not able to light microscopy and TEM using A. minutum (strain AMP4) exit through the apertures and rest in the sporangium. as the host. The entire life-cycle in A. minutum was completed in 3–4 Nuclear divisions days. Infection occurs when a free-living zoospore pene- trates the host cell, which loses its flagella, and sinks. In the A new infection begins with host penetration by the early stages of infection, the immature trophont, which is the zoospore and formation of the trophont. The host nucleus feeding stage of the parasitoid, develops inside the host cell is still visible, together with the recently introduced para- (Fig. 1A, B), within the (Fig. 2 A). site nucleus, in the center of the spherical structure of the Multiple simultaneous infections (2–3 trophonts) of the same immature trophont (Fig. 4A). Occasionally, two or even three host cell were rarely observed (Fig. 2B). After degrading parasitoid nuclei are observed inside the host cell, suggesting the cytoplasmic contents of the host, the parasitoid gradually multiple infections (Fig. 4B, arrows). As the trophont grows grows in size (Fig. 1C, D). At this stage, the trophont is recog- and matures, the central area is occupied by a large vacuole nizable by the pale color and the large number of lipid droplets and both the host nucleus and the chloroplasts are displaced in its cytoplasm (Fig. 2A, B). Once the host cytoplasm is to the periphery of the cell (Fig. 4C, D). The host nucleolus completely consumed, the parasitoid, which by then has is still commonly seen during early stages of zoospore devel- reached the size of the host, stops growing, having become opment (Fig. 4D). During schizogony, parasite nuclei begin the mature trophont (Fig. 2C). If multiple trophonts develop to replicate, as evidenced by the presence of mitotic nuclei at within the same host at the same time, they are smaller (data the sporocyte periphery (Fig. 4E, F). During zoospores mat- not shown). At this stage, all of the trophont’s cytoplasmic uration, the newly formed parasite nuclei become radially material (including, mitochondria and Golgi body) and its organized, and they are round, gradually shrink in size, and unique double-membraned, ovoid nucleus is found at the become elongated (Fig. 4G). When the zoospores are fully cell periphery (Fig. 2C). The central area of the trophont is mature and ready to be released, the nuclei have a thin and occupied by a large vacuole containing amorphous hyaline elongated shape (Fig. 4H). material (Figs 1D, 2C). This stage is followed by the early sporocyte. Schizogony results in the production of new nuclei arranged radially in Zoospores ultrastructure

the sporocyte periphery (Fig. 2D) and cellular organelles in

the inner sporocyte (Fig. 2E). With the initiation of cytokine- Zoospores are teardrop shaped, 2.9 ± 0.4 ␮m long, and sis, the zoospores begin to differentiate from the periphery to 1.7 ± 0.4 ␮m wide (n = 7) (Fig. 3E, F). They possess two

the center of the sporocyte (Fig. 2E). This is accompanied by dissimilar flagella, a long (up to 13.5 ␮m in length) hairy

a reduction in the amount of the central, amorphous hyaline anterior one and a short (3.5–4 ␮m in length) naked poste- material, and the surrounding cytoplasm (Figs 1E, 2F). As rior one (Fig. 3E, F). The insertion of both flagella occurs the zoospores mature, they are distributed radially around the at a mid-cell position (Fig. 3F). Zoospores contain a mito- central body (Figs 1F, 2F). At this stage, the sporocyte has a chondrion with tubular cristae and the mitochondrial matrix grayish color (Fig. 1F). The organelles migrate to the periph- is seldom observed (Fig. 5A, C). There is also a single Golgi ery where the zoospores are being formed and the nuclei body with six cisternae (Fig. 5B) that is connected to an gradually elongate, becoming smaller in size (Fig. 1G). Once ovoid vacuole that also makes contact with the mitochon- completely formed, zoospores detach from the central undif- drion (Fig. 5A–C). The elongated nucleus of the zoospore ferentiated cytoplasm and remain in the sporocyte, randomly has condensed material along its periphery (Fig. 5A). The distributed (Figs 1G, 2G). When the zoospores are fully nucleus is laterally located on one side of the zoospore, and A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 13

Fig. 1. Light microscopy micrographs showing the life-cycle stages of Parvilucifera corolla during host infection. (A) Early trophont (arrow) degrading a Durinskia baltica cell. (B) Early trophont (arrow) degrading an Alexandrium minutum cell. (C) Late trophont after having completely consumed its host. Numerous lipid droplets are visible. (D) Late trophont detached from the host theca; all of the cytoplasmic material has migrated to the periphery. The central area is completely occupied by hyaline material (hy). (E) Early sporocyte, corresponding to the early stage of zoospores. (F) Late sporocyte, during which immature zoospores in the periphery of the cell are arranged radially around the central vacuole. (G) Newly formed zoospores in the sporangium (late sporocyte) are ready to be released. (H) Empty sporangium with its processes and an operculum (arrowhead). (I) Empty sporangium and the remnants of the central vacuole (c).

In this specimen, the dinoflagellate theca is still present (th). Scale bars = 10 ␮m. the on the opposite side. The Golgi body is A), followed by a transverse partition. In the basal body, a positioned between the two organelles (Fig. 5B). A lenticular dense globule is adjacent to a less conspicuous dense area lipid droplet is visible in the posterior part of the zoospore, (Fig. 6A). The anterior flagellum has long, thin hairs grouped just below the nucleus (Fig. 5B, C). Zoospores contain a small on one side of the flagellum (Fig. 6A, C). The two central refractile body located beneath the nucleus and close to the microtubules of the axoneme are distinct in size; the smaller lenticular lipid droplet (Figs 2G, 5B). one is commonly filled with dense material (Fig. 6B). The The posterior flagellum shows a dense structure at the kinetosome of the anterior flagellum is shorter and lacks an proximal end of the central axoneme microtubules (Fig. 6 electron-dense area (Fig. 6A). 14 A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25

Fig. 2. Transmission electron microscopy (TEM) micrographs of P. corolla showing its life-cycle stages during host infection. (A) Early trophont, inside a parasitophorous vacuole (arrow), degrading the cytoplasm of an A. minutum cell. (B) Cell infected by two trophonts. (C) Late trophont, after having consumed all of the host cytoplasm. (D) Early sporocyte, with peripherally arranged nuclei. A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 15

Fig. 3. Scanning electron microscopy (SEM) micrographs of the sporangia and zoospores of P. corolla. (A, B) Empty sporangium with opened apertures. A remnant of the A. minutum theca and a still-closed aperture can be seen (arrow). (C, D)

Details of the processes and the filamentous star-shaped filaments connecting them. (E, F) Dorsal and ventral views of a zoospore. The anterior and posterior flagella are visible. Scale bars: (A, B) = 5 ␮m, (C–F) = 1 ␮m. Abbreviations: af = anterior flagellum; ap = aperture; pf = posterior flagellum; pr = process; th = dinoflagellate theca.

The anterior side of the zoospore contains an apical com- surrounded by a double membrane. An open pseudoconoid plex composed of several different structures, including is formed by a curved row of 4–5 microtubules (Fig. 6E, numerous (ca. 20) with bulbous ends (Fig. 6D). F). Thin, conoid-associated micronemes (Fig. 6E, F) and run longitudinally to the posterior side of the subtending alveolar vesicles in the apical area (Fig. 6E) are cell (Fig. 6D) but they are difficult to distinguish from additional features of the zoospore. micronemes (Fig. 6E). Both rhoptries and micronemes are

(E) Detail of the area marked in (D). Arrowheads indicate early zoospore cytokinesis. (F) Late sporocyte, with zoospores radially arranged around the central area. (G) Sporangium containing many mature zoospores (arrows). Arrowheads indicate the refractile bodies of the zoospores. (H) Empty sporangium.

Arrows indicate the apertures, arrowheads the opercula. Abbreviations: hy = amorphous hyaline material; l = lipids; m = mitochondrion; n = nucleus. All scale

bars = 5 ␮m, except (E) = 1 ␮m and (F) = 2 ␮m. 16 A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25

Fig. 4. Epifluorescence microscopy micrographs of the sporangia and zoospores of DAPI-stained P. corolla. (A, B) Recently infected cells (trophonts), in which one (A) or three (B) zoospores (arrows) are located close to the host nucleus (hn). (C, D) Two stages during trophont maturation, during which the host nucleus (hn) and putative host nucleolus (pn) are pushed to the cell periphery. (E, F) Parasite nuclei in are peripherally distributed. When replication is completed, the parasite nuclei organize radially (H) and

become elongated (G). Scale bar = 10 ␮m. A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 17

Fig. 5. TEM micrographs showing the general features of P. corolla zoospores. (A) Longitudinal section of a zoospore. The elongated nucleus (n) is located on one side of the cell. Condensed material is seen in its periphery. The mitochondrion (m) is located on the opposite side. The matrix (*) is visible. Close to the mitochondrion, an ovoid vacuole (v) is present. A cluster of rhoptries (rp) cross the cell longitudinally. (B) Transverse section of a zoospore. The nucleus (n) and mitochondrion (m) are located laterally on opposite sides of the cell. The Golgi body is located between them, has six cisternae (arrowheads), and is connected to the ovoid vacuole (v). A cluster of vesicles are visible in the anterior part of the cell representing some apical complex components, probably micronemes (arrow) and rhoptries (rp). A lenticular lipid droplet (l) is located laterally in the posterior part of the zoospore, close to the nucleus. A refractile body (b) is visible posteriorly. (C) Section of a zoospore. The nucleus (n) and mitochondrion (m), with the matrix (*) and ovoid vacuole (v) attached, are located laterally. The lenticular lipid droplet (l) is located below the nucleus. A cluster of rhoptries (rp) is observed. Scale bars = 500 nm. Development of the sporangium wall zoospore (Fig. 7E). Following zoospore release, the opercula detach from the sporangium wall (Fig. 2H) and the typi- During the initial stage of infection, the trophont gradually cal three-layered wall of the empty sporangium is observed increases in size within the parasitophorous vacuole, sepa- (Fig. 7F). rated from the host cytoplasm (Fig. 2A, B). At this stage three layers can be recognized: the parasitophorous vacuole mem- Phylogeny brane, a heterogeneous opaque intermediate layer, and the plasma membrane of the parasitoid (Fig. 7A, B). The tropho- The SSU rDNA sequences showed some variability. cyte has a folded surface with numerous alveoli underneath Excluding ambiguous positions, sequences from the Mediter- the plasma membrane (Fig. 7B) and endo/exocytosis vesicles ranean Sea were identical (KX519758 and KX519759), while overlying the folds (Fig. 7A, B). At the mature trophont stage, KX519760 from the Atlantic Ocean showed 99.8% simi- the host is completely consumed and the parasitoid wall is larity with Mediterranean ones, and KX519761 also from marked by processes. The folded surface disappears and the the Atlantic Ocean had a 99.5% similarity with Mediter- rest of this layer is electron-translucent. At this stage, the spo- ranean ones and 99.3% with KX519760. In the phylogenetic rangium wall is composed of three layers: an inner thin dense tree constructed from the SSU rDNA sequences (Fig. 8) all layer, a medium thick and loose layer, where the base of the sequences obtained in this study clustered together (100%/1). processes are attached, and an outer layer that extends over They were included in a clade (99%/1) that also contained P. the top of the processes and connects them (Fig. 7C). Alve- rostrata and P. infectans/P. sinerae sequences. Parvilucifera oli and the plasma membrane are located beneath the dense prorocentri clustered independently at a distance, forming internal layer. Also, during this stage, invaginations of the a cluster containing all Parvilucifera species (84%/1). All inner layer and the plasma membrane are observed in some members of Parvilucifera clustered within the Perkinsozoa areas (Fig. 7D). These invaginations will later give rise to group (96%/1), standing as a sister group with the rest of the the sporocyte opercula (Fig. 2H). The processes (n = 10) are , i.e., dinoflagellates, MALV groups, apicomplex-

0.3–0.7 ␮m long, measured at different life-cycle stages. Pro- ans, and colpodellids. cesses of differing lengths are seen on the same sporangium and during the same life-cycle stages. The continuous plasma Host range membrane of the sporocyte becomes invaginated and sur- rounds the immature zoospores. After cytokinesis it will be Preliminary screening of susceptible dinoflagellate hosts the source of the plasma membrane of each newly developed for infection by the Canary Islands strain suggested that the 18 A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25

Fig. 6. TEM micrographs of the flagellar and apical complex structures of P. corolla zoospores. (A) Longitudinal section of the insertion area of the anterior (af) and posterior (pf) flagella. Central axoneme microtubules can be observed in the posterior flagellum, with its dense structure at the proximal end (arrowhead). A transverse sections shows a dense globule (*) next to A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 19 parasitoid is a generalist (F. Rodríguez, pers. comm.), as Etymology: corolla, referring to the radial arrangement are most Parvilucifera species. Both field samples of Durin- of zoospores around a central round body, resembling the skia baltica and strains of Alexandrium minutum maintained corolla of a flower. in the laboratory were successfully infected. An ongoing Type host: Durinskia baltica (Levander) Carty et Cox study is devoted specifically to determine the host range (Alveolata, Myzozoa, Dinoflagellata, Dinotrichales) of the parasitoid exposed to a wide selection of microalgal strains belonging to different groups, with a special focus on Ecology: Detected in coastal salt marshes of the NW Mediter- − dinoflagellates. Thus far, the results show that our strains ranean Sea in May 2015 during a bloom (>106 cells L 1) are able to infect several strains of dinoflagellate genera, of the dinoflagellate Durinskia baltica. At that time, the including Prorocentrum, which is not the case for other salinity of the water was 31.8‰ and the water temperature ◦ Parvilucifera species (except P. prorocentri). was 21.1 C. Table 1 summarizes the prominent characters of the studied organism and compares this species with related ones. Our results support the description of the studied Discussion organism as a new species belonging to the genus Parvilu- cifera. Morphological and ultrastructural comparisons

Parvilucifera corolla showed the same life-cycle and Species description stages as other species of the genus, sharing more charac- teristics with P. sinerae, P. infectans and P. rostrata than with Alveolata Cavalier-Smith, 1991 P. prorocentri. The main differences refer to the features of Myzozoa Cavalier-Smith et Chao, 2004 the mature sporangium and the free-living zoospores (Garcés Perkinsozoa Norén et Moestrup, 1999 and Hoppenrath 2010; Lepelletier et al. 2014; Norén et al. 1999). Parvilucifera corolla René,˜ Alacid, Figueroa, Rodríguez, The zoospores of P. corolla are teardrop-shaped, while Garcés sp. nov. those of P.sinerae and P.infectans are described as elongated.

Diagnosis: Parasitoid of dinoflagellates. Free-living They differ from those of P.rostrata, which show a rostrum in zoospores (2.9 ␮m long; 1.7 ␮m wide) are teardrop shaped, the apical region (Lepelletier et al. 2014), and those of P.pro- with two unequal flagella. Zoospores have an open conoid, rocentri, which are reniform (Leander and Hoppenrath 2008). rhoptries, and micronemes. The cells contain one mitochon- Both the length and the width of the zoospores (Table 1) are drion, a Golgi body, a nucleus, a refractile body, and lipid similar in P. corolla, P. sinerae (Figueroa et al. 2008), and P. droplets. After infection, the trophocyte digests the host infectans (Norén et al., 1999). The zoospores of P.rostrata are cytoplasm and is composed of a peripheral area, containing larger. Lepelletier et al. (2014) reported longer dimensions for the organelles, and a central undifferentiated mass. Its P.infectans zoospores; however, according to direct measure-

size is variable and limited to the host size. It undergoes ments using published images of P. infectans zoospores the schizogony, matures to form a sporocyte, and produces new length is ∼3 ␮m. The dimensions of P.prorocentri zoospores zoospores that are released via apertures. The sporocyte is were within the same range (Leander and Hoppenrath 2008). ornamented with numerous processes. Under optical microscopy, cells from the early stages of infection show an opaque hyaline coloration in all species. Hapantotype: SEM stubs containing free-living zoospores However, the mature sporangium of all Parvilucifera species and mature sporangia and resin-embedded samples of (except P.prorocentri) has been described as blackish (Alacid the type locality used for TEM containing all life-cycle et al. 2015; Lepelletier et al. 2014; Norén et al., 1999), while stages of the parasitoid and have been deposited in the in P.corolla it is grayish. This could be due to the lower num- Electronic Microscopy Laboratory of the Institut de Cièn- ber of zoospores contained in the sporangium of the newly cies del Mar (ICM-CSIC) from Barcelona, under the code described species: while infection of A. minutum by P. sin- ICM–SEM–2016 AR 01 to 02 and ICM–TEM–2016 AR 03 erae and P. infectans sporangia results in approximately 300 to 14 respectively. and 500 zoospores, respectively, in P. corolla infections of Type locality: Fra Ramon coastal lagoon, L’Estartit, Catalo- the same host species around 150 zoospores are produced. A nia, NW Mediterranean Sea (42◦0144N, 3◦1130E) remarkable difference between P. corolla and related species an electron-dense area (arrow). The anterior flagellum lacks both the dense globule and the dense area but is characterized by thin hairs (h). (B) Transverse section of the anterior flagellum axoneme, showing a heteromorphic pair of central microtubules. (C) Transverse section of the anterior flagellum, with thin hairs grouped on one side. (D) Longitudinal section of the anterior end of a zoospore, showing micronemes (mn) with bulbous posterior ends and the nucleus (n). (E) Transverse section of the apical area of a zoospore, showing a curved row of four microtubules (arrowheads), conoid-associated micronemes (arrow), rhoptries (rp), and possible micronemes. Subtending alveolar vesicles are observed in the apical region (*). (F) A curved row of five microtubules (arrowheads), representing the pseudo- (open) conoid, a group of pseudo-conoid associated micronemes (arrow), and a cluster of rhoptries and probably micronemes. Scale bars (A, D–F) = 200 nm, (B, C) = 100 nm. 20 A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25

Fig. 7. TEM micrographs showing different stages in the development of P. corolla membranes. Early stage of trophont development showing (A) endo/exocytotic vesicles (arrowhead). (B) The surface of the parasitoid membrane is folded, with endo/exocytotic vesicles (arrowheads) along the top of the folds. (C) Late stage of the trophont, with already-formed processes. (D) A late- stage trophont, showing invaginations (arrowheads) that will form opercula in successive stages. (E) Sporocyte membranes during zoospore formation. (F) Wall of an empty sporangium after zoospore release. Scale bars = 500 nm. Abbreviations: a = alveoli; dm = dinoflagellate membrane; il = intermediate layer; ml = medium layer; nl = internal layer; n = nucleus; ol = outer layer; pm = parasitoid plasma membrane; pr = process; th = dinoflagellate theca; vm = parasitophorous vaculole membrane. A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 21

100/1 HQ219336 Uncultured AY2009C17 100/1 HQ219457 Uncultured alveolate AY2009E17 AY642738 Uncultured eukaryotic A48 AB721034 Uncultured freshwater RW10 2010 89/1 AB721061 Uncultured freshwater eukaryote SB20 2010 95/1 AB901649 Uncultured eukaryote AOox H 2012Dec 126 93/1 100/1 AB902201 Uncultured eukaryote AOox H 2012Mar 87 100/1 DQ244034 Uncultured alveolate PAG2AU2004 100/1 AB771865 Uncultured freshwater eukaryote K2JAN2011 AB771904 Uncultured freshwater eukaryote K11JAN2012 100/1 100/1 DQ244021 Uncultured alveolate PAD10AU2004 DQ244035 Uncultured alveolate PAD7AU2004 AF530536 Uncultured alveolate AT4-98 100/1 EF196722 Uncultured eukaryotic picoplankton B26 88/1 EF527175 Uncultured marine eukaryote SA2 99/1 AY642744 Uncultured eukaryotic picoplankton A31 100/1 EF196703 Uncultured eukaryotic picoplankton BA330 EF196680 Uncultured eukaryotic picoplankton BA1 - /1 DQ455739 Uncultured eukaryote BB01-172.24w - /0.98 DQ103802 Uncultured marine eukaryote M2 18C03 Perkinsozoa 100/1 AY919735 Uncultured freshwater eukaryote LG15-08 EF196788 Uncultured eukaryotic picoplankton B95 DQ244038 Uncultured alveolate PAB5AU2004 100/1 EU162629 Uncultured alveolate PAA8SP2005 100/1 EF196689 Uncultured eukaryotic picoplankton BA70 AF324218 KX519759 Parvilucifera corolla Estartit F10-11 KX519760 Parvilucifera corolla Lanzarote 2

100/1 KX519758 Parvilucifera corolla Estartit F5-12 Parvilucifera 96/1 99/1 KX519761 Parvilucifera corolla Lanzarote 3 KF359485 Parvilucifera infectans 100/1 100/1 EU502912 Parvilucifera sinerae 84/1 AF133909 Parvilucifera infectans KF359483 Parvilucifera rostrata FJ424512 Parvilucifera prorocentri 100/1 EU162625 Uncultured alveolate PAG5SP2005 - /0.99 EU162627 Uncultured alveolate PAD12SP2005 93/1 100/1 AY919809 Uncultured freshwater eukaryote clone LG36-11 AY919821 Uncultured freshwater eukaryote LG53-06 KJ757341 Uncultured eukaryote clone SGYY1526 100/1 EU162622 Uncultured alveolate PAB8SP2005 EF196688 Uncultured eukaryotic picoplankton BA43 90/0.99 96/1 U27499 AMU27499 Alexandrium minutum JX262491

95/1 Azadinium spinosum KP790156 Gyrodinium cf. spirale - /0.99 AF286023 sp. AF069516 ex - /0.97 Amoebophrya Akashiwo sanguinea Marine Alveolates AB264776 Ichthyodinium chabelardi (Group I, II & IV) 96/1 L24381 100/1 AF009244 Myzozoa 100/1 microti Apicomplexa AF013418 parva DQ174731 AF330214 tetrahymenae Colpodellids AY078092 Colpodella pontica U57765 Entodinium caudatum 100/1 AY102613 tetraurelia Ciliates X53486 Oxytricha granulifera 100/1 AY485471 Cylindrotheca closterium X77704 Grammonema striatula Outgroup

0.2

Fig. 8. Maximum likelihood tree inferred from the SSU rDNA sequences of representatives of the Alveolata phylum. Sequences of the Grammonema striatula and Cylindrotheca closterium served as the outgroup. Sequences of Parvilucifera corolla obtained in this study are indicated in bold. The bootstrap (BS) and Bayesian posterior probabilities (BPP) are provided at each node (BS/BPP). Only BS and BPP values >80% and >0.95, respectively, are shown. 22 A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25

Table 1. Comparison of the morphological characters of Parvilucifera corolla, other Parvilucifera species, and Perkinsus.

P. corolla P. infectansa,b P. sineraec P. rostratad P. prorocentrie Perkinsusf

Alveoli + + + + + +

a b

Length × width of 2.9 × 1.7 4 × 1.5 /5.5 × 1.6 2.7 × 2.2 6 × 1.8 4 × 1.5 4.5 × 2.9 zoospores (␮m) No. of zoospores 150 500 250–300 ? ? several hundred per sporangium in A. minutum Mitochondrion ++ + + + + with tubular cristae

Golgi with 6 ++ ? ? + − cisternae Micronemes + + + + + + Rhoptries + + + + + + Reduced + (4–5) + (5) + (5) + (5) + (4–6) (20–39) pseudoconoid (no. of microtubules) Conoid-associated + + + + +? + micronemes Refractile body in ++ + + + + zoospore

Two dissimilar ++ + + + − flagella Heteromorphic pair of central microtubules in ++ + + + + anterior axoneme

Dense globule in ++ + ? − +

the basal body

Dense area in the ++ + + − −/+ axoneme

Residual body ++ + + − ? inside the

sporangium

Length of 0.3–0.7 0.6–0.8 0.7–0.8 0.4–0.45 − − sporangial

processes (␮m)

Germ tube − − − − ++

−

Bipartite − − − + −

−

Nucleus − − − + − syndinean-like

a Norén et al. (1999). b Lepelletier et al. (2014). c Garcés and Hoppenrath (2010). d Lepelletier et al. (2014). e Leander and Hoppenrath (2008). f Azevedo (1989). is the radial disposition of the zoospores during maturation large and conspicuous lenticular lipid droplet is seen in the of the former. This regular pattern around the central body is zoospores of P. corolla, in contrast to the scattered, round not observed in the other Parvilucifera species. lipid bodies of other species of the genus Parvilucifera. The refractile body of all previously described Parvilu- The lengths of the processes vary (Table 1), and overlap cifera species is large, round, and occupies the posterior end with those of P. rostrata (Lepelletier et al. 2014), P. sinerae of the zoospores; in P.corolla it is much smaller and not easily (Figueroa et al. 2008), and P. infectans (Lepelletier et al. observed on ultrastructure sections. Although the genus name 2014). Star-shaped filaments covering the sporangium have Parvilucifera refers to the refractile zooids, the zoospores been observed in all three of these Parvilucifera species. P. of P. corolla are not refractile under light microscopy. A A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 23 prorocentri lacks processes and filamentous material on the syndinian-like nucleus, such as that of Amoebophrya (Miller sporangium and germ tube (Leander and Hoppenrath 2008). et al. 2012) and Psammosa (Okamoto et al. 2012) para- The apical complex structures and their arrangement did sites. In addition, they are released through a germ tube, a not differ from those described in the other species. character shared with Perkinsus taxa. The other four Parvilu- cifera species cluster together with high support, whereas P. prorocentri clusters independently at the base of this group Ecology of the parasitoid (Hoppenrath and Leander 2009). These differences are sig- nificant enough to consider that it belongs to a different genus. Parvilucifera corolla was detected in coastal salt However, we believe advisable to wait until new organisms marshes of the NW Mediterranean Sea during a bloom sharing such characters with P. prorocentri, i.e. germ-tube (>106 cells L−1) of the dinoflagellate Durinskia baltica. formation, are hopefully discovered to check their phyloge- These coastal lagoons are isolated but receive occasional netic position before conducting any taxonomic change. seawater inputs during sea storms, such that their salinity varies from 10 to 40‰ throughout the year (López-Flores et al. 2006). The temperature range is from 6 to 30 ◦C (X. Quintana, pers. comm.). The parasitoid’s primary host pro- Host range duces resting cysts, which were abundant in the analyzed samples (data not shown). Cysts allow the dinoflagellate Most Parvilucifera species are able to infect A. minutum or to survive under adverse conditions and to bloom when other Alexandrium species (Garcés et al. 2013a; Lepelletier optimal conditions are restored. Similarly, the parasitoid et al. 2014; Norén et al., 1999). The host range of P.prorocen- might withstand a large range of temperatures and salin- tri has not been evaluated (Leander and Hoppenrath 2008). ities even in the absence of a host, probably by entering In fact, although the species of the genus are regarded as a dormancy stage in the sediment, as suggested for the generalist parasitoids, able to infect a wide range of dinoflag- other Parvilucifera species (Garcés et al. 2013b). Alterna- ellate hosts, they show a preference for phylogenetically close tively, new parasitoid populations may be introduced when species. P. infectans, P. sinerae, and P. rostrata seem to pre- the coastal marshes are flooded by seawater. In the case of fer Alexandrium and related species. Alacid et al. (2016) the Canary Islands (Atlantic Ocean) detection, the environ- demonstrated the partial specialization of P. sinerae, which ment harboring P. corolla was a tidal pond with no entries asymmetrically infects its hosts, and this may have eco- of inland waters. Annual seawater temperatures in Lanzarote logical implications for both parasite and host populations. were in the range of 18–22 ◦C, but those of isolated tidal Ongoing work on the selectivity of P. corolla will provide ponds are typically higher (23.5 ◦C at the time of sampling a more in-depth characterization of its host range and shed for this study). The sample from Charco Palo was domi- light on the differences between this and other Parvilucifera nated by a mixed assemblage of ciliates (e.g., spp.) species. As noted above, the susceptibility of some Pro- together with a minor community of benthic dinoflagellates, rocentrum strains to P. corolla infection distinguishes this namely, members of the genera and Proro- parasitoid from others in the same genus. In particular, given centrum. the shallow environments in Mediterranean and Lanzarote Island, where the two strains originated, it would be interest- ing to determine whether P. corolla is prone to develop on Phylogenetic position of Parvilucifera corolla benthic microalgal hosts that could coexist in these environ- ments. The SSU rDNA sequences obtained in this study from the Mediterranean differed slightly from those of the Atlantic strains. By contrast, the sequences of several tested genes of P. sinerae strains from different years and sampling Acknowledgements sites, including Mediterranean and Atlantic locations, were identical. However, the prevalence and virulence of this This study was supported by the Consejo Supe- species differed significantly (Turon et al. 2015). No sig- rior de Investigaciones Científicas (CSIC) through the nificant differences in the morphology of P. corolla were project ECOPAR (CSIC Intramural Project) granted to observed among strains, but the phenotypic traits of this E. Garcés. Dinoflagellate cultures were provided by species should be closely examined to determine whether the culture collection of CCVIEO (Vigo, Spain). We they account for the physiological variability within P. thank the Unitat de Microscopia Electronica, Facultat de corolla. Medicina-SCT and Universitat de Barcelona for techni- Parvilucifera prorocentri closely resembles other Parvilu- cal assistance with the TEM analyses, and J. M. Fortuno˜ cifera species both morphologically and phylogenetically (ICM-CSIC) for technical support with the SEM anal- but it differs in some of its morphological characteristics. yses. We also thank Á. Sebastián and V. Outeirino˜ The sporangium wall is smooth and shows no processes. from CACTI, Universidade de Vigo, for DNA sequenc- Zoospores contain numerous bipartite trichocysts and a ing. 24 A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25

References sp. nov. (Perkinsozoa) and of its host–specific and strain selective mechanism of infection. Protist 159, 563–578. Alacid, E., Park, M.G., Turon, M., Petrou, K., Garcés, E., 2016. Garcés, E., Alacid, E., Bravo, I., Fraga, S., Figueroa, R.I., 2013a. A game of Russian roulette for a generalist dinoflagellate para- Parvilucifera sinerae (Alveolata, Myzozoa) is a generalist para- sitoid: host susceptibility is the key to success. Front. Microbiol. sitoid of dinoflagellates. Protist 164, 245–260. 7, 1–13. Garcés, E., Alacid, E., René,˜ A., Petrou, K., Simó, R., 2013b. Host- Alacid, E., René,˜ A., Garcés, E., 2015. New insights into the par- released dimethylsulphide activates the dinoflagellate parasitoid asitoid Parvilucifera sinerae life cycle: the development and Parvilucifera sinerae. Isme J. 7, 1065–1068. kinetics of infection of a bloom-forming dinoflagellate host. Garcés, E., Hoppenrath, M., 2010. Ultrastructure of the intracellular Protist 166, 677–699. parasite Parvilucifera sinerae (Alveolata, Myzozoa) infecting Andrews, J.D., 1988. Epizootiology of the Disease Caused by the the marine toxic planktonic dinoflagellate Alexandrium minutum Oyster Pathogen Perkinsus marinus and Its Effects on the Oyster (). Harmful 10, 64–70. Industry, 18. American Fisheries Society Special Publication, pp. Giovannoni, S.J., deLong, E.F., Olsen, G.J., Pace, N.R., 1988. 47–63. Phylogenetic group-specific oligodeoxynucleotide probes for Azevedo, C., 1989. Fine structure of Perkinsus atlanticus n. sp. (Api- identification of single microbial cells. J. Bacteriol. 170, complexa: ) parasite of the clam Ruditapes decussatus 720–726. from Portugal. J. Parasitol. 75, 627–635. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence align- Bråte, J., Logares, R., Berney, C., Ree, D.K., Klaveness, D., Jakob- ment editor and analysis program for Windows 95/98/NT. Nucl. sen, K.S., Shalchian-Tabrizi, K., 2010. Freshwater Perkinsea and Acids. Symp. Ser. 41, 95–98. marine-freshwater colonizations revealed by pyrosequencing Hatcher, M.J., Dick, J., Dunn, A.M., 2012. Diverse effects of par- and phylogeny of environmental rDNA. Isme J. 4, 1144–1153. asites in ecosystems: linking interdependent processes. Front. Brugerolle, G., 2002. Cryptophagus subtilis: a new parasite of cryp- Ecol. Environ. 10, 186–194. tophytes affiliated with the Perkinsozoa lineage. Eur. J. Protistol. Hoppenrath, M., Leander, B.S., 2009. Molecular phylogeny of 37, 379–390. Parvilucifera prorocentri (Alveolata, Myzozoa): insights into Brugerolle, G., 2003. Apicomplexan parasite Cryptophagus perkinsid character evolution. J. Eukaryot. Microbiol. 56, renamed Rastrimonas gen. nov. Eur. J. Protistol. 39, 101. 251–256. Chambouvet, A., Berney, C., Romac, S., Audic, S., Maguire, F., de Hudson, P.J.,Dobson, A.P.,Lafferty, K.D., 2006. Is a healthy ecosys- Vargas, C., Richards, T.A., 2014. Diverse molecular signatures tem one that is rich in parasites? Trends Ecol. Evol. 21, 381–385. for ribosomally ‘active’ Perkinsea in marine sediments. BMC Jephcott, T.G., Alves-de-Souza, C., Gleason, F.H., Van Ogtrop, Microbiol. 14, 110. F.F., Sime-Ngando, T., Karpov, S., Guillou, L., 2016. Ecological Chambouvet, A., Gower, D.J., Jirk, M., Yabsley, M.J., Davis, A.K., impacts of parasitic chytrids, and perkinsids on pop- Leonard, G., Maguire, F., Doherty-Bone, T.M., Bittencourt- ulations of marine photosynthetic dinoflagellates. Fungal Ecol. Silva, G., Wilkinson, M., Richards, T.A., 2015. Cryptic infection 19, 47–58. of a broad taxonomic and geographic diversity of tadpoles by Johansson, M., Eiler, A., Tranvik, L., Bertilsson, S., 2006. Dis- Perkinsea protists. PNAS, E4743–E4751. tribution of the dinoflagellate parasite Parvilucifera infectans de Vargas, C., Audic, S., Henry, N., Decelle, J., Mahé, F., Logares, (Perkinsozoa) along the Swedish coast. Aquat. Microb. Ecol. R., Lara, E., Berney, C., Le Bescot, N., Probert, I., Carmichael, 43, 289–302. M., Poulain, J., Romac, S., Colin, S., Aury, J.-M., Bittner, L., Katoh, K., Misawa, K., Kuma, K., Miyata, T., 2002. MAFFT: a Chaffron, S., Dunthorn, M., Engelen, S., Flegontova, O., Guidi, novel method for rapid multiple sequence alignment based on L., Horák, A., Jaillon, O., Lima-Mendez, G., Lukes,ˇ J., Malviya, fast Fourier transform. Nucleic Acids Res. 30, 3059–3066. S., Morard, R., Mulot, M., Scalco, E., Siano, R., Vincent, F., Zin- Lafferty, K.D., Dobson, A.P., Kuris, A.M., 2006. Parasites dom- gone, A., Dimier, C., Picheral, M., Searson, S., Kandels-Lewis, inate food web links. Proc. Natl. Acad. Sci. U. S. A. 103, S., Acinas, S., Bork, P., Bowler, C., Gorsky, G., Grimsley, N., 11211–11216. Hingamp, P., Iudicone, D., Not, F., Ogata, H., Pesant, S., Raes, J., Leander, B.S., Hoppenrath, M., 2008. Ultrastructure of a novel tube- Sieracki, M.E., Speich, S., Stemmann, L., Sunagawa, S., Weis- forming, intracellular parasite of dinoflagellates: Parvilucifera senbach, J., Wincker, P., Karsenti, E., 2015. Eukaryotic plankton prorocentri sp. nov. (Alveolata, Myzozoa). Eur. J. Protistol. 44, diversity in the sunlit ocean. Science 348, 1–11. 55–70. Delgado, M., 1999. A new diablillo parasite in the toxic dinoflag- Lepelletier, F., Karpov, S.A., Le Panse, S., Bigeard, E., Skovgaard, ellate Alexandrium catenella as a possibility to control harmful A., Jeanthon, C., Guillou, L., 2014. Parvilucifera rostrata sp. algal blooms. Harmful Algae News 19, 1–3. nov. (Perkinsozoa): a novel parasitoid that infects planktonic Elwood, H.J., Olsen, G.J., Sogin, M., 1985. The small-subunit dinoflagellates. Protist 165, 31–49. ribosomal RNA gene sequences from the hypotrichous ciliates López-Flores, R., Garcés, E., Boix, D., Badosa, A., Brucet, S., Masó, Oxytricha nova and pustulata. Mol. Biol. Evol. 2, M., Quintana, X.D., 2006. Comparative composition and dynam- 399–410. ics of harmful dinoflagellates in Mediterranean salt marshes and Erard-Le Denn, E., Chrétiennot-Dinet, M.J., Probert, I., 2000. First nearby external marine waters. Harmful Algae 5, 637–648. report of parasitism on the toxic dinoflagellate Alexandrium min- Medlin, L., Elwood, H.J., Stickel, S., Sogin, M.L., 1988. The utum Halim. Estuar. Coast. Mar. Sci. 50, 109–113. characterization of enzymatically amplified eukaryotic 16S-like Figueroa, R.I., Garcés, E., Massana, R., Camp, J., 2008. First rRNA-coding regions. Gene 71, 491–499. description of the dinoflagellate parasite Parvilucifera sinerae A. René˜ et al. / European Journal of Protistology 58 (2017) 9–25 25

Miller, J.J., Delwiche, C.F., Coats, W., 2012. Ultrastructure of Reynolds, E.S., 1963. The use of lead citrate at high pH as an Amoebophrya sp. and its changes during the course of infection. electron-opaque stain in electron microscopy. J. Cell Biol. 17, Protist 163, 720–745. 208–212. Norén, F., Moestrup, Ø., Rehnstam-Holm, A.-S., Larsen, J., 2000. Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D.L., Darling, Year Worldwide occurrence and host specificity of Parvilu- A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, cifera infectans: a parasitic dinoflagellate capable of killing toxic J.P., 2012. MrBayes 3.2: efficient Bayesian phylogenetic infer- dinoflagellates. In: Harmful Algal Blooms 2000, Tasmania, Aus- ence and model choice across a large model space. Syst. Biol. tralia. 61, 539–542. Norén, F., Moestrup, Ø., Rehnstarn-Holm, A.-S., 1999. Parvilu- Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood- cifera infectans Noren et Moestrup gen. et sp. nov. (Perkinsozoa based phylogenetic analyses with thousands of taxa and mixed phylum nov.): a parasitic flagellate capable of killing toxic models. Bioinformatics 22, 2688–2690. microalgae. Eur. J. Protistol. 35, 233–254. Turon, M., Alacid, E., Figueroa, R.I., René,˜ A., Ferrera, I., Bravo, I., Okamoto, N., Horák, A., Keeling, P.J., 2012. Description of Two Ramilo, I., Garcés, E., 2015. Genetic and phenotypic diversity Species of Early Branching Dinoflagellates, Psammosa pacifica characterization of natural populations of the parasitoid Parvilu- n. g., n. sp. and P. atlantica n. sp. PLoS One 7, e34900. cifera sinerae. Aquat. Microb. Ecol. 76, 117–132. Park, M.G., Yih, W., Coats, D., 2004. Parasites and phytoplankton, with special emphasis on dinoflagellate infections. J. Eukaryot. Microbiol. 51, 145–155. Råberg, L., Alacid, E., Garcés, E., Figueroa, R.I., 2014. The poten- tial for arms race and Red Queen coevolution in a protist host–parasite system. Ecol. Evol. 4, 4775–4785.