Journal of

Plankton Research

plankt.oxfordjournals.org

J. Plankton Res. (2013) 35(5): 1167 – 1171. First published online July 16, 2013 doi:10.1093/plankt/fbt066

The formation of the twin resting cysts in the dinoflagellate Dissodinium pseudolunula, a parasite of copepod eggs ´ MEZ1,2* AND LUIS FELIPE ARTIGAS1 FERNANDO GO 1

ˆ TE D’OPALE, MREN ULCO, 32 AV. FOCH, WIMEREUX LABORATOIRE D’OCEANOLOGIE ET GEOSCIENCES (CNRS UMR 8187 LOG), UNIVERSITE´ DU LITTORAL CO 62930, FRANCE AND 2LABORATORY OF PLANKTON SYSTEMS, OCEANOGRAPHIC INSTITUTE, UNIVERSITY OF SA˜O PAULO, PRAC¸A DO OCEANOGRA´FICO 191, SALA 100, SA˜O PAULO SP 05508-120, BRAZIL

*CORRESPONDING AUTHOR: [email protected] Received April 8, 2013; accepted June 15, 2013 Corresponding editor: Roger Harris

The dinoflagellate Dissodinium pseudolunula is the most common and widespread ectoparasite of copepod eggs in neritic waters. When the host is absent, the species survives with a distinctive pair of twin resting cysts described as Pyrocystis margalefii. Based on live samples, the formation of the twin resting cysts is illustrated here for the first time. The gymnodinioid infective cells did not form overwintering cysts under unfavourable conditions. These are formed inside the secondary lunate sporangium. KEYWORDS: Dinophyceae; life cycle; overwintering cyst; parasitism; resting spores Copepods are the most abundant metazoans in the sea and represent a key trophic link in pelagic food webs (Mauchline, 1998). Numerous parasites have been shown to influence the mortality and fecundity of copepod populations (The´odoride`s, 1989). The lipid-rich copepod eggs are the target of several specialized parasites (Cachon and

Cachon, 1987; Coats, 1999). The parasitic dinoflagellate Dissodinium pseudolunula has a world-wide distribution in marine neritic habitats. This species has been known since the beginning of plankton studies (Pouchet, 1885), but the parasitic stage of Dissodinium upon copepod eggs was not discovered until 1978 (Drebes, 1978). The gymnodinoid

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dinospores infest planktonic crustacean eggs, absorb the host contents and form two successive sporangia (a globular primary sporangium followed by eight crescent lunate secondary sporangia which develop from the former) that produce new infective gymnodinioid dinospores (Drebes, 1978). The species survives the host-free season as resting cysts. Drebes (Drebes, 1981) was able to germinate a dinoflagellate resting cyst, known as Pyrocystis margalefii, in culture and he observed that the gymnodinioid dinospores were identical to those of Dissodinium pseudolunula. However, he was unable to verify whether the dinospores were able

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to infect copepod eggs. These distinctive pairs of twin resting cysts are widely distributed in the plankton of the eastern North Atlantic and North Sea with a peak in numbers in August and September (John and Reid, 1983). Molecular phylogeny has revealed that Dissodinium and Chytriodinium, both ectoparasites of crustacean eggs, are close relatives within the group of Gymnodinium sensu stricto (Go´mez et al., 2009). Drebes (Drebes, 1981) demonstrated that the infective dinospores that emerged from the twin resting cyst resemble those of Dissodinium pseudolunula. However, the stage of Downloaded from http://plankt.oxfordjournals.org/ at Universidade de São Paulo on August 28, 2013

Fig. 1. Light micrographs of life stages of live specimens of Dissodinium pseudolunula collected in the coastal NE English Channel in 2010 and 2011. (A– E) Infected copepod eggs. (F–Q) Developmental stages of the globular primary sporangia. (R– AE) Developmental stages of the lunate secondary sporangia. (AE– AH) Infective dinospore enclosed in an outer hyaline membrane. fl, flagellum. Scale bar: 10 mm. See online supplementary data for a colour version of this figure.

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performed in a large pool (50 m diameter, 1 m depth) that formed during low tide along the shore at Wimereux. In addition, intertidal surface sediments at Wimereux were examined following the sampling procedure described by Go´mez et al. (Go´mez et al., 2011). Plankton and benthos live samples were settled in Utermo¨hl chambers and examined with an inverted microscope (Nikon Eclipse TE2000-S) equipped with a Nikon Digital Sight DS-2M camera. Infected eggs and globular and lunate sporangia of D. pseudolunula were common in live samples from April to September, with more specimens in June (Fig. 1). The fast-swimming gymnodinioid dinospores were difficult to record, and the micrographs were taken when they were enclosed inside a thin hyaline outer membrane (Fig. 1AE – AG). Only 10 pairs of twin resting cysts were observed between July and September in 2010 and 2011

Fig. 2. Light micrographs of the lunate sporangium and the resting cyst of live specimens of Dissodinium pseudolunula collected in the coastal NE English Channel in 2010 and 2011. (A–E) Lunate secondary sporangia. (F –J) Formation of a pair of twin resting cysts inside a lunate sporangium. (K –N) Pairs of twin resting cysts. (O– R) Single resting cyst. (S– T) Pairs of cells with the appearance of the resting cyst. Scale bar: 10 mm

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the life cycle of Dissodinium in which the resting cysts are formed remains unknown. This fact also represents a way to confirm the relationship between the twin resting cyst and Dissodinium. The most intuitive explanation is that the infective dinospore forms a resting cyst under unfavourable conditions (when it is unable to find the host). In fact, the infective dinospore forms an outer membrane (Drebes, 1981). However, this corresponds to a temporary cyst which differs from the twin resting cysts of Dissodinium in size, shape and cell content. The present study identifies the missing link between the known life stages of Dissodinium and its overwintering resting cyst. Live phytoplankton samples were collected weekly with a 20 mm mesh net in 2010 and 2011 in coastal waters of the NE English Channel (off Wimereux, France), which are characterized by recurrent blooms of diatoms and the haptophyte Phaeocystis globosa. Daily sampling was also

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Based on cultures, Drebes (Drebes, 1978) observed that the infective dinospores formed an outer hyaline membrane, and they eventually divided inside. This has been also observed in natural samples (Fig. 1AH). In parasites, as a general trend, it is considered that the infective dinospores form the resting cysts when they are unable to find a host (Drebes, 1984). However, the twin resting cysts of D. pseudolunula differed from the infective dinospore in size, shape and cell content. It is difficult to accept that the latter should be able to form twin resting cysts with a biomass five to eight times greater than the infective dinospore itself. In addition, the exhausted infective dinospore has to accumulate the energetic reserves to survive the autumn–winter period, and to form the cellular functions of a new infective dinospore for the next spring. Drebes (Drebes, 1981) hypothesized that the primary sporangium develops a certain number of resting cysts instead of secondary lunate sporangia, as a possible reaction to changing environmental conditions. This study has revealed that the twin resting cysts are formed inside the secondary lunate sporangium (Fig. 2F–J). The pair of twin resting cysts receives 1/8 of the lipid reserves of the infected copepod

Fig. 3. Life cycle of Dissodinium pseudolunula.

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(Fig. 2K – R). In sand samples, a single resting cyst was also found on 17 September 2010 (Fig. 2R). Several lunate sporangia and twin resting cysts were observed on 8 July 2010. As a general trend, each recently released lunate sporangium contained a hyaline mass and a prominent nucleus. This content transformed by binary fission into eight gymnodinioid cells that acquired a green-brownish pigmentation (Fig. 1, see Supplementary data, Video, http://www.youtube.com/watch?v=YGA6U3EhdU). However, some lunate sporangia showed an intense pigmentation before the formation of the first pair of daughter cells (Fig. 2E). One of these lunate sporangia contained a pair of the twin resting cysts. They lacked the distinctive orange granules that characterized the typical twin resting cysts (Fig. 2F–J). This is the first direct evidence that the twin resting cysts are formed inside a lunate secondary sporangium of Dissodinium. In addition, a pair of cells inside an outer hyaline membrane was observed in a sand sample collected on 17 May 2010 (Fig. 2S–T). The grainy appearance of the cell contents and the presence of an orange granule strongly resembled a cell that germinated from a twin resting cyst.

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S U P P L E M E N TA RY DATA Supplementary data can be found online at http://plankt.oxfordjournals.org.

of the weekly monitoring supported by the INTERREG IVA “2 Seas” DYMAPHY project, co-funded by E.R.D.F. funds.

FUNDING F.G. was supported by a UL1 post-doctoral grant and a CNRS convention of research on phytoplankton. F.G. is currently supported by the Brazilian contract BJT 370646/ 2013-14 of Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico.

REFERENCES Cachon, J. and Cachon, M. (1987) Parasitic dinoflagellates. In Taylor, F. J. R. (ed.), The Biology of Dinoflagellates. Blackwell, Oxford, pp. 571 – 610. Coats, D. W. (1999) Parasitic life styles of marine dinoflagellates. J. Eukaryot. Microbiol., 46, 402– 409. Coats, D. W., Kim, S., Bachvaroff, T. R. et al. (2010) Tintinnophagus acutus n. g., n. sp. (Phylum Dinoflagellata), an ectoparasite of the ciliate Tintinnopsis cylindrica Daday 1887, and its relationship to Duboscquodinium collini Grasse´ 1952. J. Eukaryot. Microbiol., 57, 468–482. Drebes, G. (1978) Dissodinium pseudolunula (Dinophyta), a parasite on copepod eggs. Br. Phycol. J., 13, 319–327. Drebes, G. (1981) Possible resting spores of Dissodinium pseudolunula (Dinophyta) and their relation to other taxa. Br. Phycol. J., 16, 207–215. Drebes, G. (1984) Life cycle and host specificity of marine parasitic dinophytes. Helgola¨nder Meeresunters., 37, 603–622. Go´mez, F., Lo´pez-Garcı´a, P. and Moreira, D. (2011) Molecular phylogeny of the sand-dwelling dinoflagellates Amphidiniopsis hirsuta and A. swedmarkii (Peridiniales, Dinophyceae). Acta Protozool., 50, 255–262. Go´mez, F., Moreira, D. and Lo´pez-Garcı´a, P. (2009) Life cycle and molecular phylogeny of the dinoflagellates Chytriodinium and Dissodinium, ectoparasites of copepod eggs. Eur. J. Protistol., 45, 260– 270. John, A. W. G. and Reid, P. C. (1983) Possible resting cysts of Dissodinium pseudolunula Swift ex Elbra¨chter et Drebes in the Northeast Atlantic and the North Sea. Br. Phycol. J., 18, 61–66. Mauchline, J. (1998) The biology of calanoid copepods. Adv. Mar. Biol., 33, 1 –710.

AC K N OW L E D G E M E N T S We thank the crew of the R/V Sepia II (INSU) and UMR LOG technicians and students for their help in the field sampling of the Wimereux-Slack transect, which was part

Pouchet, G. (1885) Nouvelle contribution a` l’histoire des Pe´ridiniens marins. J. Anat. Physiol., 21, 28– 88. The´odoride`s, J. (1989) Parasitology of marine zooplankton. Adv. Mar. Biol., 25, 117– 177.

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egg. A host infection could provide 8 lunate sporangia and the subsequent 8 pairs (16 cells) of resting cysts. Drebes (Drebes, 1981) observed that the cells that germinated to form the twin resting cysts were eventually able to divide. This may be the case in the specimens illustrated in Fig. 2S– T. Consequently, each infected egg may provide 32 new infective dinospores for the following spring (Figs 1 and 2). Intuitively, one might suggest that the factor that triggers the formation of the twin resting cysts is the absence of copepod eggs. However, the secondary lunate sporangium may be unable to directly sense the presence of hosts in the surrounding waters. It is uncertain whether the decrease in the size or quality of the eggs induces the formation of the resting cysts as a mechanism to avoid the failure of the next generation of infective dinospores. Another possibility would be that the twin resting cyst is produced at a constant rate throughout the year, in order to provide a permanent seeding bank. However, the active Dissodinium form is common in April and May, while the resting cysts were mainly observed in July–September (John and Reid, 1983). The twin resting cysts are preferentially formed in the host-free season. This study provides new insights into the life strategies of this important parasite group (Fig. 3). The production of cysts has been reported in parasites of tintinnid ciliates (Coats et al., 2010). However, we know little about the overwintering cysts in parasitic dinoflagellates. Dissodinium and other parasites on copepod eggs may be a contributory factor to the large year-to-year variability in the standing stock of copepods. It is important to understand their life cycles as a first step and to include parasitism in the models of secondary production in the ocean.