Parvilucifera Rostrata Sp. Nov

Protist, Vol. 165, 31–49, xx 2014

http://www.elsevier.de/protis

Published online date 17 October 2013

ORIGINAL PAPER

Parvilucifera rostrata sp. nov.

(Perkinsozoa), a Novel Parasitoid that

Infects Planktonic Dinoﬂagellates

a,b c,d e,b a,b

Frédéric Lepelletier , Sergey A. Karpov , Sophie Le Panse , Estelle Bigeard ,

f a,b a,b,1

Alf Skovgaard , Christian Jeanthon , and Laure Guillou

CNRS, UMR 7144, Station Biologique, Place Georges Teissier, CS90074, 29688 Roscoff

Cedex, France

Université Pierre et Marie Curie (Paris VI), Station Biologique, Place Georges Teissier,

CS90074, 29688 Roscoff Cedex, France

Zoological Institute RAS, St. Petersburg, Russia

St. Petersburg State University, St. Petersburg, Russia

CNRS, FR 2424, Plate-forme Merimage, Station Biologique de Roscoff, Place Georges

Teissier, CS90074, 29688 Roscoff Cedex, France

Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University

of Copenhagen, Frederiksberg, Denmark

Submitted April 8, 2013; Accepted September 15, 2013

Monitoring Editor: Mona Hoppenrath

The diversity and ecological roles of protists in marine plankton are still poorly known. In 2011, we made

a substantial effort to isolate parasites into cultures during the course of blooms of the toxic microalga

Alexandrium minutum (Dinophyceae) in two estuaries (the Penzé and the Rance, Brittany coast, north-

west of France). In total, 99 parasitic strains were obtained. Screening of ribosomal internal transcribed

spacer regions (including ITS1, 5.8S and ITS2) revealed the existence of two ribotypes. Small subunit

and partial large subunit rRNA genes revealed that these two ribotypes belong to different species of the

genus Parvilucifera. The ﬁrst ribotype was tentatively afﬁliated to the species Parvilucifera infectans,

whilst the second represents a new species, Parvilucifera rostrata sp. nov. The new species has several

distinct morphological features in the general organization of its zoospore and in the shape and size of

processes covering the sporangium. Both Parvilucifera species are generalist parasitoids with similar

generation times, and this study thus raises the question of how two parasitoids exploiting similar

ecological resources and infection strategies can coexist in the same ecosystem. Taxonomic relation-

ships between Parvilucifera spp. and other closely related marine parasitoids, such as syndinians, are

discussed.

Key words: Parvilucifera; dinoﬂagellate; parasitoid; Alexandrium minutum; toxic blooms.

Introduction

Photosynthetic dinoﬂagellates are important pri-

1 mary producers in marine ecosystems. However,

Corresponding author; fax +33 2 98 29 23 24

e-mail [email protected] (L. Guillou). some opportunistic and toxic species can cause

32 F. Lepelletier et al.

water discoloration, massive faunal mortality and Marine perkinsozoans (including Perkinsus and

even fatal toxicity in humans. Such phenomena Parvilucifera spp.) and syndinians belonging to

have become an increasing worldwide problem in the division Alveolata, which are characterized

recent decades. There is more and more evidence by the presence of peripheral membranous sacs

that eutrophication of marine coastal waters, global (alveoli) lining the plasmalemma, are monophyletic,

warming, transport of resting stages by cargo bal- and include three very different main lineages,

last, and habitat modiﬁcations are responsible for dinoﬂagellates, apicomplexans and ciliates. Perkin-

the worldwide increase of such mass prolifera- sozoans and syndinians are closely related to

tions (Garcés and Camp 2012; Hallegraeff 2010). dinoﬂagellates according to ribosomal phyloge-

These bloom-forming microalgae have natural ene- nies (Hoppenrath and Leander 2009; Massana

mies, among which various parasitic eukaryotic et al. 2008; Moore et al. 2008; Saldarriaga et al.

microorganisms have been known for many years 2003). Syndiniales are still considered as a highly

(Cachon 1964; Chatton 1920). Hard to detect and specialized order within the dinoﬂagellate lineage,

often neglected, the ecological relevance of such due to a morphological resemblance of their free-

parasitic eukaryotic microorganisms in aquatic living stages with dinoﬂagellates (Cachon 1964).

ecosystems has recently been highlighted by their However, perkinsozoans and syndinians are also

high representativeness in eukaryotic environmen- parasitic lineages that share morphological charac-

tal gene libraries (Guillou et al. 2008; Lefèvre et al. teristics with apicomplexans. Perkinsozoans infect

2008). their host using an apically clustered complex of

Two eukaryotic microparasites infecting bloom- secretory organelles similar to those of apicomplex-

forming marine dinoﬂagellates, Parvilucifera ans. Three different types of secretory organelles

(Perkinsozoa) and Amoebophrya (Syndiniales), have been described in Apicomplexa (micronemes,

have been reported repeatedly in the literature rhoptries and dense granules). These organelles

(Park et al. 2004). Such parasites are highly viru- are structured in the cell throughout a cone-shaped

lent and strategically more related to parasitoids tubulin structure, the conoid. The recent detection

(parasites that ultimately kill or sterilize their host of dense granules and rhoptry-like organelles in

to accomplish their life cycle). The two groups of syndinians (Gestal et al. 2006; Miller et al. 2012)

parasitoids have grossly similar life cycles. Brieﬂy, reopened the discussion on the phylogenetic posi-

a small (3-7 ␮m in size) and short-lived (hours to tion of perkinsozoans and syndinians within the

days) biﬂagellate parasitoid cell penetrates the host Alveolata.

cell. The trophocyte settles either in the cytoplasm Species boundaries of such eukaryotic micropar-

or the nucleus and develops while killing the host asitoids are hard to delimit. Environmental diversity

cell. Then, the trophocyte matures into a sporocyte studies have reported extensive genetic diversity

(either a resting sporangium in Parvilucifera spp. in syndinians (Guillou et al. 2008). Marine Alveo-

or a short-living vermiform stage in Amoebophrya lates (MALV) group II, believed to be synonymous to

spp.) that produces hundreds of new infective the order Amoebophryidae, included more than 45

ﬂagellates within days (Cachon 1964; Norén et al. well-identiﬁed genetic clades based on SSU rRNA

1999). Their impact on host populations is far sequence similarity, a number likely to be far from

from fully understood. Eukaryotic microparasitoids exhaustive. By contrast, the genus Parvilucifera and

may play a fundamental role in maintaining the relatives are rarely observed in environmental SSU

diversity and resilience of marine ecosystems rRNA libraries in marine ecosystems, despite the

by potentially removing proliferating and invasive fact that this genus has regularly been reported dur-

species (Chambouvet et al. 2008). Mathematical ing dinoﬂagellate blooms since its ﬁrst description

models have been used to determine the capacity from the west coast of Sweden (Johansson et al.

of such parasitoids to control their host populations 2006; Norén et al. 1999). This genus is easy to

(Llaveria et al. 2010; Montagnes et al. 2008; maintain in culture, as it produces a resistant stage,

Salomon and Stolte 2010). Several recent studies the sporangium, which can be maintained at cold

highlight co-evolutionary interactions between temperatures for several months. Three species

such parasitoids and their hosts. This could be have been described to date. P. infectans (Norén

evidenced as example by the parasitic adaptation et al. 1999) and P. sinerae (Figueroa et al., 2008)

to its host sexual reproduction. Indeed, parasitoids are closely related according to both SSU rRNA

can survive for several months in host cysts, whilst sequences and their morphologies. These two

sexual reproduction of hosts seems to be promoted species infect a wide range of dinoﬂagellate hosts

in the presence of these parasitoids (Chambouvet (Garcés et al. 2013a; Norén et al. 1999) and they

et al. 2011; Figueroa et al., 2010). potentially act as generalists in the ecosystem. The

Parvilucifera rostrata sp. nov. (Perkinsozoa) 33

third species, P. prorocentri, known to infect Pro- strains of A. minutum were obtained during these

rocentrum fukuyoi (but for which the full extent of two surveys.

host range is unknown), was in the original descrip- These parasitoid strains were examined by

tion suggested to belong to another genus (Leander sequencing of the ribosomal internal transcribed

and Hoppenrath 2008). spacer regions (comprising ITS1, 5.8S and ITS2).

Here, we report the presence of two distinct Two different ribotypes were obtained with no

species of Parvilucifera during blooms of the toxic sequence polymorphism, i.e. sequences belonging

dinoﬂagellate species Alexandrium minutum. Com- to one ribotype were strictly identical, indepen-

parative analyses of their ultrastructure, phylogeny, dently of geographical origin and isolation date.

and host speciﬁcity are also presented. Ribotypes 1 and 2 represented 39 and 60 strains,

respectively. Internal transcribed spacer regions

of these two ribotypes could not be aligned. Ribo-

Results types 1 and 2 had only 88 and 50.7% sequence

identities based on their SSU and partial LSU

rRNA sequences, respectively. Small subunit rRNA

Strain Isolation and Phylogeny

gene (SSU rRNA or 18S) sequences of ribotype

In 2011, A. minutum blooms were monitored in the 1 share 98% sequence identity with Parvilucifera

st th

Penzé estuary from the 1 of June to the 11 of infectans (Norén et al. 1999) and 99% sequence

July and in the Rance from the 26 of May to identity with P. sinerae (Figueroa et al. 2008).

the 24 of June. Maximum A. minutum densities Ribotype 2 had 84% sequence identity with the

were observed on the 22 of June in the Penzé SSU rRNA sequence of P. prorocentri (Leander

-1 th

(401,790 cells mL ) and the 27 of May in the and Hoppenrath 2008). The D1 and D2 regions

-1

Rance (431,097 cells mL ). In total, 99 parasitic of the large subunit rRNA gene (LSU rRNA or

SSU gene Pa ral LSU gene

1/99 AY831406_Alexandrium tamarense 1/90 AY831406 _

Alexandrium tamarense

1/100 DQ44429 0

_Alexandrium fundyense

1/98

1/100 AY83140 9 DQ444290_Alexandrium fundyense

_Alexandrium aﬃne 1/10 0

1/98

AY831408_Alexandrium minutum Other AY831409_Alexandrium aﬃne

Other

AF377944_Gonyaulax polyedra dinoflagellates 0.99/71 AY831408_Alexandrium minutum

AY831412

_Akashiwo sanguinea dinoflagellates

1/52 AJ 841810 AY347309_Cochlodinium polykrikoides

_Prorocentrum donghaiense

0.99/90

1/99 AY80373 9_Prorocentrum micans AY822610_Prorocentrum donghaiense

AY347309_Cochlodinium polykrikoides X16108 _Prorocentrum micans

1/10 0 EF065717 ex

_Hematodinium perezi_ Liocarcinus depurator

AF377944_

1/10 0 AF286023_Hematodinium sp. MALV4 Gonyaulax polyedra

DQ14640 4_Syndinium turbo_ex Paracalanus parvus AY831412_Akashiwo sanguinea

1/100 DQ146406_Syndinium sp._ex Corycaeus sp. Ichthyodinium chabelardi_ex Sardina pilchardus

AF472553 sp. ex

0.99/48 _Amoebophrya _ Karlodinium micrum 0.99/77 AB 473667_unpublished parasite_ex

AF472554_Amoebophrya sp._ex Gymnodinium instriatum Phalacroma parvula

AF 472555_Amoebophrya sp._ex Scrippsiella sp. MALV2 AB473666_unpublished parasite_ex Phalacroma parvula MALV1

1/100 AF239260 sp. ex

_Amoebophrya _ Dinophysis norvegica 1/99 AB473665_unpublished parasite_ex Phalacroma lavelata

1/10 0 JN934987

_Euduboscquella cachoni

EU304548_uncultured marine alveolate - environmental samples

1/100 JN934988_Euduboscquella cachoni_ex Eunnnus tenuis

JN60606 5_Euduboscquella crenulata_ex Favella panamensis MALV1 Parvilucifera infectans_ex Protoperidinium sp.

0.99/- 1/99 AB264776_Ichthyodinium chabelardi_ex Thunnus albacares 1/100 Parvil ucifera infectans (RCC2816)_ex Alexandrium minutum

1/100 FJ440623 ex

_Ichthyodinium chabelardi_ Sardina pilchardus Parvil ucifera rostrata Alexandrium minutum Perkinsea

EU502912_Parvilucifera sinerae_ex Alexandrium minutum (RCC2800)_ex

1/100 Parvil ucifera infectans (RCC2816)_ex Alexandrium minutum AY305326_Perkinsus andrewsi

1/100

AF133909_Parvilucifera infectans_ex Protoperidinium sp.

1/99 AF509333_Perkinsus atlancus

1/95 Parvil ucifera rostrata _ Alexandrium minutum Perkinsea

(RCC2800) ex

sp. ex sp.

FJ424512_Parvilucifera prorocentri_ex Prorocentrum fukuyoi 0.99/84 Amoebophrya _ Scrippsiella

0.99/63 AY30532 6_Perkinsus andrewsi HM483395_Amoebophrya sp._ex Akashiwo sanguineaum

MALV2

1/100

1/100 AF509333_Perkinsus atlancus_ex Tapes decussatus HM483394_Amoebophrya sp._ex Gymnodinium instriatum

DQ46245 5

1/91

0.99/86 _Ascogregarina taiwanensis

_ex

AF040725_Cryptosporidium parvum Syndinium turbo Paracalanus parvus MALV4

AF 013418_Theileria parva EF681910_Colpodella ponca

AF026388_Eimeria tenella AF 013419_Theileria parva

AF 434059

1/95 _Sarcocyss singaporensis

1/- L76472 AF101077_Hammondia hammondi

_Sarcocyss capracanis

1/96

1/991/82 AF009244_Frenkelia micro_ex Microtus arvalis Apicomplexa X75453_Toxoplasma gondii

M64244_Sarcocyss muris 1/99 AF 001946

_Neospora caninum

0.99/- L76471

_Isospora felis 0.99/55

AF109678 AF076900_Besnoia besnoi

_Besnoia besnoi

0.99/96

X75453_Toxoplasma gondii 1/56 U85705_Isospora felis Apicomplexa

AJ271354 ex

_Neospora caninum_ Bos taurus AF012883_Sarcocyss muris

0.99/71 AF 096498

_Hammondia hammondi

AF044252

1/10 0 AY142075 sp. _Frenkelia micro _Colpodella

0.99/94

AF330214_Colpodella tetrahymenae AF237617_Sarcocyss singaporensis

AB721057_uncultured freshwater eukaryote AF 012885_Sarcocyss capracanis

AF280076

1/100 _Voromonas ponca

AF026388

1/95 AY078092_Colpodella ponca _Eimeria tenella

DQ17473 2_Chromerida sp. EU106870_Chromera velia

DQ174731_Chromera velia AF 040725_Cryptosporidium parvum

AY23484 3_Colpodella edax

HM245049 sp.

1/100 X54512_Tetrahymena thermophila _Chromerida

0.99/100 X5617 1_Tetrahymena pyriformis Ciliates X54512_Tetrahymena thermophila

AF149979

_Paramecium tetraurelia X54004_Tetrahymena pyriformis

Ciliates

X80344

1/100 _Hyphochytrium catenoides 1/100

AF149979

AY485471_Cylindrotheca closterium _Paramecium tetraurelia

1/92 AF054832_Bigelowiella natans Outgrou p HQ395663_Saprolegnia parasica

X57162_Guillardia theta 1/99 FR 696318_Eurychasma dicksonii Outgroup

0.05 0.1

Figure 1. Bayesian inferences of small (left, 1444 bp) and D1 and D2 domains of the large (right, 314 bp) subunit

rRNA gene sequences belonging two parasites isolated during A. minutum (dinoﬂagellate) blooms and related

organisms. On the nodes are indicated the bayesian posterior probabilities followed by maximum-likelihood

bootstrap values.

34 F. Lepelletier et al.

28S) of ribotype 1 were 100% identical to a strain less than 10 host cells survived to the infection

described as P. infectans (Norén, pers. comm). after 10 days), 3) the host is moderately resistant

Phylogenies of both SSU and LSU rRNA gene (sporangia are observed but more than 10 host cells

sequences provided congruent tree topologies remained after 10 days), 4) the host is resistant (no

(Fig. 1). Within the Alveolata, these two ribotypes evidence of infection after 10 days).

grouped with all other Parvilucifera sequences in all Host strains were susceptible to moderately

phylogenies, with strong statistical supports for ML resistant in most cross-infections (Table 1). Both

and Bayesian inferences. Perkinsozoans (Parvilu- parasitoids efﬁciently infected a wide range of

cifera and Perkinsus) formed an independent and Alexandrium species (including A. minutum, A.

monophyletic group in all cases, but with low sta- catenella, A. tamarense, A. ostenfeldii, and A.

tistical supports (Fig. 1). The sister lineages of tamutum) as well as several other dinoﬂagel-

perkinsozoans are certain marine alveolate clus- late species (namely Scrippsiella spp., Hete-

ters (called MALV-1, -2 and -4) including syndinians rocapsa triquetra, Gonyaulax spinifera, Gymno-

(represented by several genera such as Syndinium, dinium aureolum, Kryptoperidinium foliaceum, and

Amoebophrya, and Ichthyodinium). Perkinsozoans Akashiwo sanguinea). However, infections were

and syndinians always branched at the basal part never observed on Prorocentrum micans (Table 1).

of dinoﬂagellate clade with moderate support. In the presence of zoospores, Heterocapsa rotun-

data cells rapidly enlarged and exploded within

Long-term Storage of Sporangia a few hours, without evidence of infection (likely

caused by an allelopathic signal). Strong intraspe-

For long-term storage of parasitoids, the efﬁ-

ciﬁc variability was observed. For example, no

ciency of the two cryoprotectants dimethyl sulfoxide

Parvilucifera strains were able to infect the 11

(DMSO) and methanol was ﬁrst tested using differ-

A. minutum strains tested. Conversely, only two

ent ﬁnal concentrations of 5%, 10% and 15% on

A. minutum strains (RCC3019 and RCC3021)

2-3 representatives of both ribotypes. Parasitoids

were susceptible to all Parvilucifera strains tested.

did not survive using methanol and 15% DMSO. By

Hence, virulence of the parasitoid depends to a

visual inspection, more sporangia were observed

greater extent on the strain than on the parasitoid

using 10% DMSO after 7 days of incubation (com-

species. Of all strains tested, RCC2902 (ribotype

pared to 5%). All parasitic strains were therefore

1) and RCC2842 (ribotype 2) were the most vir-

cryopreserved using 10% DMSO. For each strain,

ulent. According to previous observations by Toth

a control sample was cryopreserved and the via-

◦ et al. (2004), the production of temporary cysts in

bility of the parasitoid tested (stored at -150 C for

the presence of the parasitoid was observed from

at least 10 days before thawing, see Methods). In

time to time (no quantitative data available).

total, 92% of the control samples (91 of 99) revived

15 days after their inoculation into an exponentially

growing A. minutum culture. All parasitoid strains Characteristics of the Parasitoid

were able to infect A. minutum after 6 months in Trophocytes and Sporangia

◦

the dark at 4 C.

Both ribotypes have similar generation times and

general appearance in light microscopy (Fig. 2).

Cross-infection Speciﬁcity

Generally, only one trophocyte is formed per host

Three strains of each parasitoid ribotype were (Fig. 2A, G). However, two (less often 3 to 5)

tested for cross-infection using a wide range of trophocytes can develop within a same host in

dinoﬂagellate hosts (Table 1 both ribotypes (Fig. 2B). Trophocytes are actively

), including 11 strains of A. minutum originating growing during the ﬁrst 36 hours by digesting their

from different locations (see also Supplementary host. A sporangium is then released out of the host

Table S1). Incubation time for cross-infection tests cell (Fig. 2D, I). Sporangia ﬁrst appear whitish, but

was ﬁxed at 10 days (corresponding to about 2 acquired a typical blackish coloration, similar in both

generations). Virulence could be deﬁned both by ribotypes, after 120-132 hours (Fig. 2F, I).

the capacity of the parasite to kill their host and Using electron microscopy, young, almost spher-

to exceed their growing rate. Indeed, responses ical trophocytes were clearly visible in the host

to infection were classiﬁed into the following cat- cytoplasm 12 hours after inoculation of the para-

egories: 1) the host is susceptible (the host is well sitoid (Fig. 3A, D). In both ribotypes, the cytoplasm

infected, numerous sporangia are observed and no of the parasitoid contains a peripheral nucleus

host cells persist after 10 days), 2) the host is mod- with an eccentric nucleolus, mitochondria, a Golgi

erately susceptible (sporangia are observed and apparatus (not shown), lipid inclusions, and starch

Parvilucifera rostrata sp. nov. (Perkinsozoa) 35

Table 1. Infection capability of the two Parvilucifera species on various dinoﬂagellate strains. White: the host

is susceptible (no host survives the infection); light-gray: moderately susceptible (the host is well infected and

less than 10 host cells survive the infection); dark-grey: moderately resistant (the host is well infected but more

than 10 host cells remained after 10 days); black: the host is resistant (no infection is observed, see text for

explanations). ND: Not Determined. Shading: host cells enlarged and exploded in few hours without evidence

of infection.

36 F. Lepelletier et al.

Table 1 (Continued )

granules. The central part of trophocytes is ends of processes (Fig. 4H). At this stage, the mem-

occupied by vacuoles with amorphous electron branes of alveoli coaslesce and underlie nearly the

translucent material, and lipid globules. These vac- entire plasma membrane forming a true pellicle

uoles become bigger as the parasitoid grows and (Fig. 4H). Sporangium wall formation is accompa-

seem to fuse with each other to form a large cen- nied by endocytotic activity (Fig. 4H, arrowheads),

tral vacuole (compare Fig. 3A with 3B and 3D with which seems to remove the extra membrane of the

3E). Kinetosomes and alveoli were not observed in folds made by the plasma membrane (which disap-

trophocytes. pears) and perhaps the electron opaque material

The membrane of the parasitophorous vacuole as well.

seems to be formed by the host, and had a typical Sporangia are released from the totally disin-

three-layered structure (Fig. 4 G-H). The trophocyte tegrated host and its parasitophorous membrane

has a folded surface, which is separated from the (Fig. 4I). At this stage, the sporangium is covered

parasitophorous membrane by an opaque interme- with its own extracellular wall, which consists of an

diate layer (Fig. 4G-H). Alveoli appear at the top inner thick bilayered material at the bases of the

of each fold at the ﬁrst stage of sporangium mat- processes, and a thin outer layer connecting the

uration, while the rest of the cell is covered by the distal ends of processes (inserts of Fig. 3C, F). The

plasma membrane (Fig. 4G). The opaque interme- inner thick bilayered material consists of an inner-

diate layer is rather homogenous at this stage. As most thin dense layer and an outer thick and loose

maturation of the sporocyte proceeds, this layer layer of ﬁbrous material. Membrane and alveoli

becomes heterogeneous (Fig. 4H) with concen- are just beneath this dense layer which is clearly

trations (future processes) in between the former visible at the operculum maturation stage (marked

folds, which gradually disappear (Fig. 4G-I). The in Fig. 3B). This means that this bilayered material

rest of this layer becomes electron translucent with has a non-membraneous nature and lies above

the exception of a thin layer connecting the outer the pellicle. Further sporangium wall maturation

Parvilucifera rostrata sp. nov. (Perkinsozoa) 37

Figure 2. Maturation processes of Parvilucifera rostrata (ribotype 2, A-F) and P. infectans (ribotype 1, G-I).

One to several trophocytes per host cell (A-B, G-H) grow within parasitophorous membrane (arrow in I). Young

whitish sporangia released from the host cell (G, I). Mature sporangia appear blackish (F, I). Scale bars: A-D

and G-I, same as on A = 10 ␮m. E-F, same as on F = 10 ␮m.

involves the gradual degradation of the external whole sporangium envelope and their number

layer (Fig. 4A, D) and more or less complete and arrangement are similar in both ribotypes

denudation of the processes (Fig. 4B, C, E, F). The (Figs 3, 4). However, processes are smaller in

␮

sporangium becomes covered with a continuous ribotype 2 (0.4-0.45 m in length) whereas they are

␮

layer of thick bilayered material, cementing the much more elongated (0.55-0.65 m in length) and

bases of processes. This external wall cannot resemble amphora in ribotype 1 (Fig. 3C, F; Fig. 4C,

be stained by calcoﬂuor. Processes cover the F). Opercula, like processes, are well developed

38 F. Lepelletier et al.

Parvilucifera rostrata sp. nov. (Perkinsozoa) 39

in mature sporangia (Fig. 3C,F). They are formed forming the peripheral microtubular corset of the

by discontinuities throughout the innermost layer zoospore (Figs 5, 6). The kinetosome of the long

of the sporangium wall, and attached to the spo- ﬂagellum has a composite root formed by a band

rangium wall by ﬁbrous material that is continuous of 5 microtubules underlined with ﬁbrillar material

to the thick ﬁbrous opaque layer (Fig. 3C, F). (Fig. 6C).

Mature sporangia acquire their blackish col- In general, zoospores of both ribotypes have sim-

oration when zoospores are fully developed and ilar organelle disposition. The elongated nucleus is

differentiated (Figs 2, 3). Zoospores, released into normally shifted to the ventral side in the middle

the water through opercula, are similar in size in of the cell. This nucleus has peripheral condensed

both ribotypes (Fig. 3C, F; Fig. 5A, E). Inside the heterochromatin, and the nucleolus is not observed

sporangium, a residual body is observed in both at this stage (Fig. 5B, F). A large mitochondrion

ribotypes. At the end of sporogenesis, this resid- with tubular cristae is located at the opposite dorsal

ual body seems either to be empty or to contain side more anteriorly to the nucleus. A rather large

amorphous opaque material (Fig. 3C, F). vacuole with electron translucent contents occupies

most of time the posterior end of zoospores in ribo-

type 2, whereas it is generally located in the central

Zoospores

part of the cell in ribotype 1 (Fig. 5B, F). Several

The zooids of ribotype 2 (6 ␮m ± 0.5 ␮m in length, lipid globules were observed in zoospores of both

1.8 ␮m ± 0.2 ␮m in width; n = 46) are slightly larger ribotypes.

and more elongated than those of ribotype 1 Several structures of the apical complex extend

␮

(5.5 m 0.6 ␮m in length, 1.6 ␮m ± 0.1 ␮m in throughout the entire zoospore of both ribotypes.

width; n = 37, Fig. 4). The zoospore of ribotype 2 has Numerous micronemes (up to 20 and 26 proﬁles

a rostrum, which is absent in the round zoospore in cross sections of ribotypes 1 and 2, respec-

of ribotype 1. In both ribotypes, zooids have two tively) and several rhoptry-like structures ﬁll the

ﬂagella of unequal size. The long anterior ﬂagel- central part of the cell passing from the posterior

lum terminates in a short conical tip and carries to the anterior pole (Figs 5, 6). Micronemes have a

unilateral hairs in both ribotypes (Fig. 5A, E). This broader posterior part narrowing towards the ante-

ﬂagellum is two to three times longer than the cell rior end. Most of them terminate at the anterior

body. A short acronematic posterior ﬂagellum is pole of the cell. A long band of ﬁve microtubules

as long as the body and has a proximal paraxial accompanies the micronemes passing along the

swelling which contains ﬁbrillar material (Fig. 5G). longitudinal axis of zoospores (Fig. 6B). In cross

The short ﬂagellum adheres to the cell body, and sections, these are arranged in a slightly curved

is hardly visible using light microscopy. The kineto- line, and each of the microtubules has two ﬁlaments

some of the short ﬂagellum contains an electron directed towards its convex side (Figs 5, 6A, B).

dense core below the partition in both ribotypes Additional ﬁlamentous structures appear at the

(Fig. 5C, D, G). The kinetosome of the long ﬂa- anterior end of this band (Fig. 6A, C). The struc-

gellum is much shorter and does not contain an ture and composition of this cytoskeletal element

electron-dense core (Fig. 5D, H). Orthogonal kine- differ from the roots passing under the plas-

tosomes have well developed microtubular roots malemma. It is associated with micronemes, and

comprising 4-5 microtubules (Figs 5, 6). They were we consider this structure, which was found in

not investigated in detail, but all roots certainly pass both ribotypes, as a pseudoconoid, or as an open

from kinetosomes under the plasma membrane conoid.

➛

Figure 3. Transmission electron micrographs of a late trophocyte and an early sporangium of Parvilucifera

rostrata (ribotype 2, A-C), and P. infectans (ribotype 1, D-F). Abbreviations: Golgi apparatus (ag), host cell (h),

lipid globule (l), large vacuole (lv), mitochondrion (m), nucleus (n), nucleolus (nu), parasite 1 (P1), parasite

2 (P2), residual body (rb), starch granules (st), vacuole (v), zoospores (zo). A. P. rostrata trophocyte inside

cytoplasm of Alexandrium minutum at 24 hours. B. Late stage of P. rostrata trophocyte still partially retained

inside the host’s envelope at 36 hours. Note the presence of large vacuole and operculum formation (bracket

at the right corner). C. Sporangium of P. rostrata with zoospores and residual body at 132 hours. Note that the

sporangium envelope has mature processes, as well as opercules (arrowheads). D. Two trophocytes (P1, P2)

of P. infectans developing inside the same host cell at 24 hours. E. Trophocyte of P. infectans in A. minutum at

36 hours. F. Mature sporangium of P. infectans with zoospores, and two opercules (arrowheads) at 120 hours.

Inserts in C and F show typical shape and dimension of the processes with three layers of sporangium wall (i

– innermost, m – middle, o – outer). Scale bars: A-F = 2 ␮m. Inserts: in C = 300 nm, in F = 250 nm.

40 F. Lepelletier et al.

Figure 4. Scanning electron micrographs of Parvilucifera spp. sporangium (A-C. P. rostrata, ribotype 2, D-F. P.

infectans, ribotype 1) and different stages of the sporangium wall maturation in P. rostrata (G-I). Abbreviations:

alveoli (al), amorphous material (am), aperture (ap), thin outer non-membrane layer of sporangial wall (o), host

cell (h), mitochondrion (m), pellicle (pe), plasma membrane (pl), membrane of parasitophorous vacuole (mp),

process (pr), starch granules (st). A, D. Progressive disintegration of thin outer layer (o) of sporangial wall

(arrows on D show the folded pellicle of parasite under an outer layer). B, E. Mature sporangium with open

Parvilucifera rostrata sp. nov. (Perkinsozoa) 41

Discussion more closely resemble those of P. infectans than

those of P. sinerae. Based on the last two features

Species Delineation within the Genus (cytoplasmic infection in thecate dinoﬂagellates and

Parvilucifera shape of processes), we tentatively assigned our

ﬁrst ribotype as P. infectans. Nevertheless, the rela-

Previous studies have highlighted the strong

tionship between P. infectans and P. sinerae, hardly

resemblance between P. sinerae and P. infectans

separated using their respective SSU rRNA gene

(Figueroa et al. 2008; Garcés and Hoppen-

sequence, requires clariﬁcation.

rath 2010). P. sinerae was separated from P.

The differentiation of our second ribotype from

infectans based on their respective SSU rRNA

all other Parvilucifera species described to date

gene sequences, which differ by more than 20

is clearly marked by its genetic distance from all

nucleotides. The SSU rRNA gene sequence of ribo-

other described species. The shape and size of

type 1 differs in only 8 nucleotides from that of

sporangium ornamentation appear also to be key

P. sinerae and in 27 nucleotides from that of P.

features allowing the distinction of this ribotype.

infectans. The LSU rRNA gene sequence of ribo-

Processes are very short in ribotype 2 (0.4-0.45 ␮m

type 1 had 100% identity with that of a P. infectans

in length), while they are more elongated in P.

specimen collected at the same location as the type

infectans (0.6-0.8 ␮m in length, in the size range

species (Kristineberg Marine Research Station,

of ribotype 1) as well as in P. sinerae (0.7-0.8 ␮m

Sweden). However, since the SSU rRNA gene of

in length), and absent in P. prorocentri. In addition,

the latter specimen has not been sequenced, its

this ribotype can be clearly differentiated from P.

identity cannot be ascertained. Ribotype 1 could

infectans and P. sinerae by the general morphology

not be identiﬁed by its host range, as both P.

of its sporozoite (having a rostrum in our ribotype

infectans and P. sinerae are generalists, infecting

2, whilst zoospores are rather rounded to elongated

a wide range of dinoﬂagellate hosts. Interestingly,

(this study, Figueroa et al. 2008, Norén et al. 1999)

Prorocentrum micans could not be infected by

in the two other species. This ribotype is described

any of the six Parvilucifera strains tested during

here as a new species, P. rostrata.

this study and by the strain of P. sinerae (Garcés

et al. 2013a). However, resistance is highly vari- Diagnosis

able among host strains, as observed in Akashiwo

sanguinea (resistant in Garcès et al. 2013a, but

resistant to susceptible in this study using differ-

Parvilucifera rostrata sp. nov. Karpov and

ent parasitoid strains), and should be tested on

Guillou (Alveolata, Perkinsozoa) (Figs. 2A-F,

several parasitic strains. Two routes of infection

4A-C, 5A-C).

(either cytoplasmic in athecate host species or

Type illustration: (Figs. 4A-C, 5A-C), from

nuclear in thecate host species) were described in

material originally collected in the Penzé estu-

P. sinerae (Figueroa et al. 2008). Only cytoplasmic

ary on the Brittany coast (English Channel,

infections were observed in our isolates, even in

France) in June 2011.

the thecate A. minutum host. P. infectans and P.

Diagnosis: Endoparasitoid infecting the

sinerae can also be differentiated by the shape and

cytoplasm of a wide range of dinoﬂagel-

ornamentation of processes. P. sinerae has ﬁbrous

lates. The trophocyte develops inside the host.

material, similar to that of the two ribotypes from

The sporangium is released into water. Spo-

French ecosystems. However, this ﬁbrous material

rangium wall covered by numerous processes

has an outer thickening, as long as the process

0.4-0.45 ␮m in length. Zoospores (6 ␮m in

itself, which are not always connected with pro-

length, 1.8 ␮m in width) with two unequal ﬂa-

cesses (see ﬁg. 9 in Figueroa et al. 2008). This

gella, anterior rostrum, are released from spo-

extended material is absent in our species. Ribo-

rangium via opercules. Zoospore has open

type 1 in this study shows processes that clearly

conoid, rhoptry, microneme-like structures

➛

apertures (ap), almost totally lost the outer layer of sporangium. C, F. Surface of mature sporangia at higher

magniﬁcation. G. Folded covering of parasite under the parasitophorous membrane (mp), alveoli at the top of

the folders and amorphous material in between. H. Next stage of maturation with processes appearance, more

developed alveoli, and endo/exocytotic activity of rufﬂed pellicle (arrowheads). The developing outer thin layer

of sporangium wall pointed with arrows. I. Nearly formed sporangial wall with developed pellicle and processes,

but thin outer non-membrane layer (o) still present (this stage is just before to stage at A). Scale bars: A, B, D,

E = 10 ␮m, C, F = 800 nm, G, H = 500 nm, I = 1 ␮m.

42 F. Lepelletier et al.

Figure 5. Zoospore structure of Parvilucifera rostrata (ribotype 2, A-D) and P. infectans (ribotype 1, E-H).

Abbreviations: acroneme (ac), kinetosome of short ﬂagellum (ks), kinetosome of long ﬂagellum (kl), lipid globule

(l), long ﬂagellum (lf), mitochondrion (m), microneme (mi), nucleus (n), paraxial swelling (pa), rostrum (r),

short ﬂagellum (sf), vacuole (v). A. In toto zoospore of P. rostrata with rostrum, long hairy and short smooth

ﬂagella. B. Longitudinal section of P. rostrata zoospore. Note the presence of micronemes (mi) and large

vacuole (v). C. Longitudinal section of P. rostrata zoospore. Insert corresponding to arrow shows a putative

pseudoconoid in transversal section. Arrowheads show the v-shaped ﬁbers of microtubules. D. Longitudinal

section of both ﬂagella of P. rostrata, with ﬂagellar roots (arrowheads). Note the presence of electron dense core

below the transversal plate. E. In toto zoospore of P. infectans. F. Longitudinal section of P. infectans zoospore. G.

Longitudinal section of short ﬂagellum with paraxial swelling and electron dense core in kinetosome. Arrowheads

show microtubules of ﬂagellar roots. H. Longitudinal section of long ﬂagellum. Scale bars: A, E = 1 ␮m, B,

F = 200 nm, C, D, G, H = 400 nm, insert = 100 nm.

Parvilucifera rostrata sp. nov. (Perkinsozoa) 43

and large vacuole with electron translucent

contents.

Etymology: rostrata referring to the rostrum

of zoospores.

Type species: RCC2800, cryopreserved

strain deposited in the Roscoff Culture Col-

lection (http://www.sb-roscoff.fr/Phyto/RCC/)

Structural Similarities and Evolutionary

Relationships

In typical dinoﬂagellates, the nuclear envelope

remains intact during mitosis and chromosomes

are permanently condensed and attached to the

inner membrane (dinokaryon). Nuclear division

occurs by closed extranuclear pleuromitosis with-

out centrioles, with no obvious organizing center for

mitotic spindle. They have at least one nucleolus in

to which some chromosomes penetrate. This con-

ﬁguration may be deeply modiﬁed in trophozoites

of parasitic dinoﬂagellates, such as Blastodinium

spp. (Skovgaard et al. 2012). In these organisms,

the nucleus of trophocytes includes a very large

nucleolus (generally absent in the free-living stage),

many ribosomes, and chromosomes that are more

or less decondensed. This is a typical synergid

nucleus according to the Chatton’s description

(Chatton 1920). In such parasites, the typical

dinokaryon is only observed in sporocytes, after

several rounds of cellular division. Considering this

strong morphological adaptation to the parasitic life

style, only free-living and dispersive stages (sporo-

zoites in parasites, see Table 2) can be compared

among taxa. Indeed, zoospores of Amoebophrya

(Syndiniales MALV2), Parvilucifera, and Perkin-

sus are characterized by the absence of packed

condensed chromosomes, and the presence of

strongly condensed DNA material observed at the

periphery of the nucleus. A nucleolus is absent,

or rarely observed, at this stage. The “nucleolus-

like” structure observed in mature sporangium of

P. prorocentri (ﬁg. 32 in Leander and Hoppenrath

2008) could probably be re-interpreted as con-

densed chromatin emerging from the periphery of

➛

(fr), kinetosome (k), mitochondrion (m), microneme

(mi), nucleus (n), pseudoconoid (pc), rhoptries (rh). A.

Pseudoconoid and ﬂagellar root structure in transver-

sal section: each microtubule has V-shaped ﬁber;

arrows show ﬁbrillar elements of pseudoconoid. B.

Figure 6. Details of pseudoconoid and ﬂagellar root Longitudinal section of pseudoconoid (arrowheads).

structure in Parvilucifera rostrata (ribotype 2, A, B) C. Longitudinal section of pseudoconoid (arrow-

and P. infectans (ribotype 1, C). Abbreviations: ﬂa- heads) and transversal section of composite ﬂagellar

gellum (f), ﬂagellar composite root (fcr), ﬂagellar root root (fcr). Scale bars: A = 100 nm, B, C = 400 nm.

44 F. Lepelletier et al. in

and

nucleus

conoid spp. ) )

periphery

the

Azevedo Brugerolle condensed 1989 2002 Perkinsus ( - open + + ball - ? - Rastrimonas ( the of

Parvilucifera and

nucleus

conoid ) periphery

the

Leander ? of Hoppenrath 2008 condensed open - + + - ? + Parvilucifera prorocentri ( the (Syndiniales),

) and

MALV Norén nucleus

1999

conoid

periphery

al. the

Garcés of Hoppenrath 2010; Parvilucifera spp. (present paper, ( condensed - open + + cylinder + posterior ﬂagellum - et (5 microtubules) the including

) the

and

1974 Ris

lineages,

macro).

Hollande Merodinium ( Kubai 1974; + ? - - ? ? + ? nuclear membrane packed condensed chromosome attached and

alveolate

) the

and

(micro

1974

ﬂagellum marine

) (ant?

Appleton Soyer Vickerman 1998 + - - - ? - + MALV4 Hematodinium ( Syndinium ( packed condensed chromosome attached post?) nuclear membrane long produced

several

of are

al.

the

et the

Nass sizes,

Miller and

zoospores )

of (hyposome)

different

1992; MALV2 Amoebophrya (Fritz 2012 + - + condensed - - + - periphery nucleus transversal ﬂagellum with

al. )

al.

1988;

et characteristics Harada

2007

) zoospores, condensed

al.

in in

of Gestal Coats

not - 2006 Euduboscquella Ichthyodinium + 2012; MALV1 Ichthyodinium ( - + + Coats et Euduboscquella ( - - - posterior ﬂagellum * types

spp.

one Morphological

two

core 2.

the least the

least

genetic material ﬂagellar kinetosome dimorphisms of at structure like of swellings ﬂagellar hairs at Zoospore Rhoptry-like conoid-like Micronemes- Organization Flagellar Nontubular Trichocysts Dense Perkinsus * Table

Parvilucifera rostrata sp. nov. (Perkinsozoa) 45

the nucleus towards a more central region forming However, trichocysts were observed in P. prorocen-

indentations (repeatedly observed in enlarged ﬁg. tri. Similarly, a dense core is observed in at least

28 in Leander and Hoppenrath 2008). DNA conden- one of the ﬂagellar kinetosomes of Perkinsus spp.

sation seems to be absent in MALV1 syndinians, and Parvilucifera spp. with the notable exception of

such as Ichthyodinium (Gestal et al. 2006) and P. prorocentri (Table 2). These latter two charac-

Euduboscquella (Coats et al. 2012; Harada et al. teristics, conﬁrmed in this study, are probably the

2007). This clearly contrasts with the description strongest arguments supporting the reclassiﬁcation

of “packed chromosomes” attached to the nuclear of P. prorocentri into a different genus.

membrane in syndinians belonging to MALV4 Within the Alveolata, both SSU and LSU rRNA

such as Hematodinium (Appleton and Vickerman phylogenies converged to place both perkinso-

1998), Syndinium (Soyer 1974) and Merodinium zoans and syndinians as independent lineages

(Hollande 1974; Ris and Kubai 1974). DNA organi- located at the basal part of the dinoﬂagellate clade.

zation in syndinians/perkinsozoan zoospores could Syndinians do not form a monophyletic lineage, and

be a diagnostic feature to separate orders or typical features of the different marine alveloate lin-

families among these parasitic lineages. Addi- eages should be carefully compared in the light of

tional observations of more specimens/species are their genetic afﬁliation. More genes should also be

required. compared.

Reduced apical complex structures (compared The presence of Parvilucifera-like parasitoids

to apicomplexans) are observed in perkinsozoans in the Penzé estuary was ﬁrst reported in 1997

(grouping Perkinsus spp. and Parvilucifera spp.). (Erard-Le Denn et al. 2000). Although the para-

This structure includes a pseudo-conoid, rhoptry- sitoid species cannot be clearly identiﬁed using

and microneme-like vesicles. Similar vesicles have information provided in the original report, this study

been observed in syndinians such as Ichthyodinium demonstrated that both the microalga and this kind

and Amoebophrya (Fritz and Nass, 1992; Gestal of parasitoid coexisted at this location 14 years

et al. 2006; Miller et al. 2012). Again, such struc- before our sampling. Observations that Parvilu-

◦

tures are clearly observed only in zoospores. They cifera sporangia can be stored at 4 C for months

likely play a fundamental role during the ﬁrst steps suggest that this stage is likely used for survival in

of infection, as is the case for apicomplexans. sediments in the absence of hosts and/or during

Infection also results in the construction of a para- winter. These sporangia may then be reactivated

sitophorous vacuole membrane, derived from the in the presence of their potential host, possibly

host cell membrane, which protects the parasite via chemical signals such as dimethylsulphide, that

from the host cytoplasm (Hausmann et al. 2003). occur at relatively high concentration during blooms

This feature has been observed in Parvilucifera (Garcés et al. 2013b). A recent study reported high

spp., Perkinsus spp. and in the syndinians Amoe- prevalence (up to 40%) of infection of A. minu-

bophrya sp. infecting dinoﬂagellates (Miller et al. tum populations by an Amoebophrya-like parasitoid

2012) and Euduboscquella sp. infecting ciliates in the Penzé estuary (during three consecutive

(Coats et al. 2012). Interestingly, this membrane years, 2004 to 2006, Chambouvet et al. 2008).

disappears in the host nucleus infected with Amoe- This species seems to have a different strategy

bophrya sp. (Miller et al. 2012), and is absent to survive in the ecosystem, as the intracellular

in the perkinsid Rastrimonas subtilis infecting a trophocyte can survive through the host encyst-

Cryptophyceae (Brugerolle 2002). Observations of ment and propagate, following germination of the

process maturation made in P. rostrata and P. cyst (Chambouvet et al. 2011). These studies high-

infectans during this study suggest that the ﬁbrous light the diversity of parasitoids and of the strategies

material observed above processes could have they adopt to survive and adapt to their hosts. They

been closely connected with the parasitophorous also reﬂect long-lasting coevolution between such

membrane, which disappears in the sporangia. microalgae and their protistan parasitoids.

SEM observations seem to conﬁrm that this ﬁbrous The possibility for two parasitic species of

material remains attached to processes in the Parvilucifera to coexist in the same ecosystem

mature sporangium of P. sinerae (see ﬁg. 4 from at the same time is demonstrated in this study.

Garcés and Hoppenrath 2010), although this spe- Coexistence of several genotypes of Amoebophrya

ciﬁc point requires more detailed observations. infecting the same host species has also been

Trichocysts, which are supposed to be involved in reported (Salomon et al. 2003). The two species

host/prey capture in Alveolata, are widely observed of Parvilucifera have similar life cycles, genera-

in dinoﬂagellates and syndinians species (Table 2), tion times, host spectra, and general ultrastructural

but are absent from Parvilucifera and Perkinsus. organization, and they apparently differ only in

46 F. Lepelletier et al.

some relatively minor morphological characteristics All strains used in this study have been deposited in

the Roscoff Culture Collection (RCC, http://www.sb-roscoff.fr/

of their zoospores and sporangia. Although more

Phyto/RCC/).

cross-infection studies are required to compare

Sequencing: The identity of newly isolated host and par-

their virulence, highly virulent strains have been

asitoid strains was revealed by sequencing of the internal

detected in both species. How these closely related transcribed spacer regions ITS1, 5.8S, and ITS2. Zoospores of

species of parasitoid that compete for the same parasitoids were separated from host cells by ﬁltration through

␮

a 5 m cellulose acetate ﬁlter (Minisart, Sartorius, Germany).

resources using the same ecological strategies can

Cells were then harvested by centrifugation and stored at -

coexist in the same ecosystem is an interesting ◦

20 C. DNA was extracted using a modiﬁed GITC (guanidinium

open question.

isothiocyanate) protocol (Chomczynski and Sacchi 2006). Cells

were submerged in 50 ␮L of the GITC extraction buffer and incu-

◦

bated at 72 C for 20 min. One volume of cold isopropanol was

◦

Methods then added and samples stored at -20 C overnight for DNA

precipitation. Tubes were then centrifuged (20,000 g, 15 min at

◦

4 C) and supernatants removed. The DNA pellet was cleaned

Strain isolation and cultivation: Infected and uninfected

with 70% ethanol (100 ␮L), followed by a last centrifugation

host cultures were maintained in F/2 medium (Marine Water

(20,000 g, 10 min). The supernatant was removed and the DNA

Enrichment Solution, Sigma) prepared with autoclaved natu-

pellet was hydrated in 20 ␮L of sterile distilled water and stored

ral seawater from the Penzé estuary (salinity 27) collected at ◦

at -20 C until used.

least 3 months prior to use and stored in the dark. This medium

The PCR mix (15 ␮L ﬁnal volume) contained 1 ␮L of the

was supplemented with 5% (v/v) soil extract (Starr and Zeikus

DNA extract, 330 ␮M of each deoxynucleoside triphosphate

1993). A ﬁnal ﬁlter sterilization step using a 0.22 ␮m pore size

(dNTP), 2.5 mM of MgCl2, 1.25 U of GoTaq® DNA poly-

ﬁlter was performed under sterile conditions. All stock cultures

◦ merase (Promega Corporation), 0.17 ␮M of both primers (see

and experiments were conducted at 19 C and on a L:D cycle of

2 −1 list Supplementary Table 2), 1X of the PCR buffer (Promega

12:12 h at 80 ␮mol photons m s . Dinoﬂagellate strains orig-

Corporation). PCR cycles, run in an automated thermocycler

inated from different regions of the French Brittany coasts or

(GeneAmp®PCR System 9700, Applied Biosystem), were pro-

from other geographical locations (either isolated in the context ◦

grammed to give an initial denaturating step at 95 C for 5 min,

of this study or obtained from culture collections, Supplemen- ◦ ◦

35 cycles of denaturating at 95 C for 1 min, annealing at 55 C

tary Table S1). Dinoﬂagellate cultures isolated during this study ◦

for 45 s and extension at 72 C for 1 min 15 s, and a ﬁnal exten-

were established by transferring a single cell into fresh medium ◦

sion step at 72 C for 7 min.

(1 mL, in 24 well plates) using a glass micropipette.

PCR products were puriﬁed (ExoSAP-IT® for PCR Product

Parvilucifera strains were isolated either from the Penzé

◦ ◦ Clean-Up, USB®) and sequenced using the Big Dye Termina-

estuary (north-west France, English Channel, 48 37 N; 3 56 W)

tor Cycle Sequencing Kit version 3.0 (PE Biosystems®) and

or the Rance estuary (north-west France, English Channel,

◦ ◦ an ABI PRISM model 377 (version 3.3) automated sequencer.

48 38 N; 2 02 W, Supplementary Table S1). The two estu-

Sequences were edited using the BioEdit 7.0.5.3 program and

aries are located about 200 km apart. Each sampling day,

complete sequences deduced from runs using both external

1 mL aliquots of sampled water were incubated in 24 well

and internal primers (Supplementary Table S2).

plates together with 1 mL of exponentially growing Alexandrium

The LSU rDNA gene sequence of P. infectans was kindly

minutum cultures (13 different strains in total, see Supplemen-

provided by Fredrik Norén. The sample originated from the

tary Table S1). The presence of parasites was screened by

Kristineberg Marine Research Station on the Swedish west

microscopy until 15 days of incubation. Monoclonal Parvilu- ◦ ◦

coast (58 15 3.67“N 11 26 46.76 W), the sampling location of

cifera were then established using a glass micropipette to

the type species of P. infectans (Norén et al. 1999).

transfer a single sporangium to 1 mL of exponentially growing

New sequences are available from the GenBank database

host culture (same strain as that in which the parasitoid was

under the following accession numbers: KF359483-KF359489.

established). Strains were re-isolated three times using this pro-

LSU rDNA sequences of Syndinium turbo ex Paracalanus

cedure. During this period, strains were maintained by weekly

parvus and Ichthyodinium chabelardi ex Sardina pilchardus

transfer of 100 ␮L of infected host culture into 1 mL exponen-

isolate PT4 were obtained from same individuals than SSU

tially growing host culture. All monoclonal Parvilucifera strains

sequence available in GenBank, DQ146404 and FJ440625,

were transferred into the same host strain (A. minutum, strain

respectively. Sequences AB473665 to AB473667 are available

RCC3018). All these strains were cryopreserved a few months

in GenBank, but unpublished. They have been isolated from

later. For this, mature sporangia were obtained after 3-5 days of

individual identiﬁed as Phalachroma spp. Because of their close

incubation in 6 mL well plates by inoculating 500 ␮L of infected

relationship with a MALV1 sequence, they have been tentatively

culture into 5 mL of exponentially growing A. minutum culture.

assigned to syndinians in this study.

Before freezing, a cryoprotectant was added, either methanol

Phylogenetic analyses: Alignments were obtained using

or dimethyl sulfoxide (DMSO), at different ﬁnal concentrations.

◦ the online version of MAFFT (http://mafft.cbrc.jp/alignment/

Temperatures were then decreased at a rate of 1 C per minute

◦ server/, Katoh and Toh 2010) using the secondary structure

to -40 C using a cryo-freezer (Kryo 360, Planer, Sunbury-on-

of RNA (Q-INS-I option). Non-informative sites were removed

Thames, UK). Strains were stored both in liquid nitrogen and at

◦ using Gblocks (http://molevol.cmima.csic.es/castresana/

-160 C for long-term preservation. Parasitoids were reactivated

◦ Gblocks server.html, Castresana 2000) using the less strin-

by thawing the cells in a water bath at 25 C followed by transfer

gent conditions. A Bayesian phylogenetic tree was constructed

to an exponentially growing host culture (1 mL of the parasitoid

with MrBayesv3.1.2 (Ronquist and Huelsenbeck 2003) using a

preparation to 5 mL of A. minutum culture). Parasitoid cultures

◦ GTR substitution model with gamma-distributed rate variation

were then incubated at 19 C in the dark for 24 hours and subse-

across sites (GTRCI) as suggested as the best-ﬁt model in

quently in similar culture conditions as those described above.

◦ JModeltest v2.1.1 (Darriba et al. 2012). Four simultaneous

Sporangia can be also stored at 4 C during at least 6 months

Monte Carlo Markov chains were run from random trees for

in dark.

Parvilucifera rostrata sp. nov. (Perkinsozoa) 47

a total of 1,000,000 generations in two parallel runs. A tree Acknowledgements

was sampled every 100 generations, and a total of 2,500 trees

were discarded as ‘burn-in’ upon checking for stationarity

We warmly thank the Ifremer laboratory in Dinard

by examination of log-likelihood curves over generations,

(France) and especially Claude Lebec and Claire

and posterior probabilities were calculated in MrBayes. A

consensus tree (50% majority rule) was constructed from the Rollet for hosting us during the A. minutum bloom

post-burn-in trees and posterior probabilities were calculated in the Rance. Thanks to the “Service Mer et

in MrBayes. Maximum Likelihood analyses are performed with

Observation” of the Roscoff Biological Station

MEGA5.1 (Tamura et al. 2011) using the GTR substitution

and all partners of the Paralex project for their

model with gamma-distributed rate variation across sites.

help with sampling, and to Nicolas Gayet from

Bootstrap values were estimated from 1,000 replicates.

Cross infection: Exponentially growing hosts (40 strains in the “Laboratoire Environnement Profond”, Ifremer

4 -1

total, cell concentrations about 10 cells mL ) were obtained in Brest (France), for his technical assistance during

vented culture ﬂasks by diluting once a week and during three

SEM analyses. Thanks to Ian Probert to carefully

consecutive weeks 5 to 10 mL into 30 to 40 mL of fresh medium.

5 read this manuscript. Sequences were performed

Freshly produced zoospores (concentration ≥ 10 zoospores

-1

mL ) were obtained after 3-5 days of incubation in 6 mL well by Morgan Perennou and Gwenn Tanguy at the

␮

plates, by inoculating 500 L of infected culture into 5 mL of Roscoff Biological Station (Ouest-Génopole). Cryo-

exponentially growing A. minutum culture (strain RCC3018).

preservation was performed by Roseline Edern

In order to remove remains of the initial host, zoospores were

(Roscoff Culture Collection). Strains SZN030 CC1

ﬁltered through a 5-␮m cellulose acetate ﬁlter (Minisart, Sar-

and SZN030 CC2 were provided by Marina Mon-

torius, Germany). Aliquots (100 ␮L) were then inoculated into

1 mL of dinoﬂagellate host culture. For a given parasitoid strain, trésor and strains CBA38 and CBA42 by Antonella

tests of the different hosts were undertaken on the same date Penna. The LSU sequence of P. infectans origi-

and using the same initial parasitoid batch culture. Intra-speciﬁc

nated from Swedish west coast was provided by

variability of answers was deduced from the use of three dif-

Fredrik Norén. Funding for SAK was granted by

ferent strains per parasitic species. Results of cross-infections

the CNRS-INEE (France) and by the RAS Presid-

were recorded by visual inspection under microscopy after 10

days. The presence of sporangia as well as of remaining swim- ium program “Problems of life origin and biosphere

ming host cells were the two criteria recorded. development”. FL was supported by a grant of the

Ultrastructure: For negative staining, samples were sedi-

French Direction Générale de l’Armement (Gilles

mented onto formvar coated copper grids. These were

Vergnaux) and the Région Bretagne. This work

then incubated with osmium tetroxide vapour, rinsed in dis-

was ﬁnancially supported by the CNRS program

tilled water, dried and examined using a JEOL JEM-1400

transmission electron microscope (Jeol, Tokyo, Japan). For EC2CO, the French ANR project PARALEX (“The

◦

scanning electron microscopy (SEM), cultures were ﬁxed in sixth extinction”, N 2009 PEXT 01201) and the

2% glutaraldehyde (ﬁnal concentration) for 2 hours. Cells were

European Project MaCuMBa (FP7-KBBE-2012-6-

␮ ␮

subsequently ﬁltered onto a nuclepore ﬁlter (10 m and 0.8 m 311975).

pore size; Whatman, Maidstone, UK), washed in distilled water

for 2 hours and dehydrated in a graded ethanol series (25%,

50%, 70%, 99%) for 15 min each, and then rinsed three times

in 100% ethanol for 15 min. Samples were critical point dried Appendix A. Supplementary data

in liquid CO2 using a BALZERS UNION CPD 020. Filters were

subsequently glued to SEM stubs with colloidal silver, sputter-

Supplementary material related to this arti-

coated with gold palladium, and examined with a FEI - QUANTA

cle can be found, in the online version, at

200 scanning electron microscope operating at 5 kV. For trans-

http://dx.doi.org/10.1016/j.protis.2013.09.005.

mission electron microscopy (TEM), the intracellular stages of

P.rostrata and P. infectans (strains RCC2800 and RCC2816)

in A. minutum (strain RCC3018) was followed every 12 hours

◦

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