UNIVERSITAT DE BARCELONA FACULTAT DE BIOLOGIA

MÀSTER EN MICROBIOLOGIA AVANÇADA

Host-range determination and specificity of a marine zoosporic parasite species

Work done by

David Verchili Camañ

To obtain the title of

Advanced Microbiology Master 2018-2019

Master’s final project carried out under the supervision of Dr. Rachele Gallisai and Dr. Albert Reñé at the Instituto de Ciencias del Mar (CSIC)

Barcelona, July 9th, 2019

Abstract In the aquatic environment, dinoflagellates are classified as the main cause of the formation of harmful algal blooms (HAB), allowing a great abundance of parasitic infections which infect the host, grow (feeding) and reproduce. Our work is based on the study of the host-range and infection specificity of a new parasite, called Perkinsid sp. 1 (Perkinsozoa, Alveolata). The study starts with a description of the infectious process of two Perkinsid sp.1 strains (Estartit and Sant Pol), followed by the susceptibility infection of 44 dinoflagellate strains. Moreover, the production of resistance structures against parasite infection was studied. From the 44 dinoflagellate strains tested, only 15-16 of them presented infection, showing an intraspecific variability between both parasite strains. Most part of the hosts that were susceptible to the infection belong to Gonyaulacales and Peridiniales orders. We can define Perkinsid sp. 1 as a generalist parasite due to the infective specificity shown.

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Index

Abstract ...... 2 Introduction ...... 3 Parasitism in aquatic environments ...... 3 Perkinsozoa ...... 4 Dinoflagellates ...... 5 Parasite specificity ...... 7 Objectives ...... 8 Materials and Methods ...... 8 Maintenance and growth of Perkinsid sp. 1 parasitoid culture strains ...... 8 Maintenance and growth of hosts culture strains ...... 8 Phytoplankton screening for parasitoid infections...... 9 Determination of resting cysts production by dinoflagellates ...... 11 Host specificity index ...... 12 Results ...... 14 Observations of Perkinsid sp.1 infection in dinoflagellates hosts...... 14 Screening dinoflagellate hosts for Perkinsid sp.1 strains infections ...... 16 Resting cyst observations in dinoflagellates...... 17 Determination of specificity index of Perkinsid sp.1 parasite ...... 18 Discussion ...... 19 Perkinsid sp. 1 host-range in dinoflagellate community ...... 19 Cysts formation ...... 22 Conclusion ...... 23 Acknowledgements ...... 24 References ...... 25

Introduction

Parasitism in aquatic environments Recent studies (Sime-Ngando et al., 2015) confirm that there are more parasitic species than free-living ones in the environment. Zoosporic parasites refer to a category of parasites that can produce numerous motile flagellated asexual zoospores, that are released into the water (Jephcott et al., 2016), which objective is to restart the parasite life cycle into a new host. Zoosporic parasitism plays an important role in the ecosystem, producing adverse effects on their hosts, which can lead to dead or motile variations, leading dinoflagellates into the sediment (Montresor et al., 1998; Reñé et al., 2011). Furthermore, dinoflagellate parasitism exert important top-down controls and, diverse effects on the ecosystem ecology, influencing both structure and function of the microbial food web, specially phytoplankton population dynamics (D. Wayne Coats, 1999; Jephcott et al., 2016).

Diverse dinoflagellates can be target of parasitic infection processes by other parasitic microorganisms, acting as hosts for the infection process (D. Wayne Coats, 1999; Jephcott et al., 2016). In case to provoke the host death, the zoosporic parasite is referred as parasitoid (once the parasite completes its entire life cycle). Many parasites are detected in blooms near coastal and estuarine environments (D. Wayne Coats, 1999), which have generation times of 2-4 days (D. W. Coats & Heisler, 1989; D. Wayne Coats, 1999), being lethal to dinoflagellate hosts. In addition, most part of parasitic dinoflagellates are obligate heterotrophs.

Fig. 1. Description of a general life cycle from an aquatic zoosporic parasite. (Jephcott et al., 2016)

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The life cycle of zoosporic parasites is mainly divided into three stages (Fig. 1): host infection by the zoospore, consumption of the host cellular material by the trophocyte or feeding stage, and the production of the new generation of zoospores by the sporocyst (D. Wayne Coats, 1999; Jephcott et al., 2016). Infections begin when a free- living zoospore penetrates the host cell, developing to a trophocyte. This stage starts feeding on the host digesting the entire cell contents while start growing, occupying the most part of the host intermembrane space. Then, the trophocyte divides its nucleus, forming the sporocyte stage, where the sporogenesis takes part and, the new generation of zoospores can be visualized. Finally, the new generation of zoospores determines the third phase of the parasite infection cycle, they produce a break in the cell membrane and they are released into the environment to find a new host to infect, restarting the infection cycle (Jeon & Park, 2019). Therefore, the main goal of parasitoids is to infect and kill a host to be able to feed on its cellular material, while generating a new generation of zoospores than can infect a new host in the environment. The infection process has several consequences for diverse capacities, such as stop swimming and sinking immediately (Alacid et al., 2015).

The main zoosporic parasites of dinoflagellate correspond to Amoebophrya, which corresponds to a syndinian parasite capable to infect free-living dinoflagellates, considered both, generalist (Kim, 2006) and specialist parasites (Chambouvet et al., 2008). Amoebophrya consisted of seven described species (Jephcott et al., 2016). Next, Chytrids (Chytridiomycota), which includes a diverse saprotrophs, well studied obligate parasites of phytoplankton community in freshwater (Jephcott et al., 2016), and scarce knowledge for their presence in the marine environment (Gleason et al., 2011; Jephcott et al., 2016). And finally, Perkinsozoa, which belongs to Alveolata superphylum, including many unclassified microorganisms (Aurélie Chambouvet et al., 2014).

Perkinsozoa The first description of Parvilucifera genus was in 1999 (Norén et al., 1999). It includes five known species until the date: Parvilucifera infectans (Norén et al., 1999), Parvilucifera sinerae (Figueroa et al., 2008), Parvilucifera rostrata (Lepelletier et al., 2014), Parvilucifera corolla (Reñé, et al., 2017) and Parvilucifera prorocentri (Leander & Hoppenrath, 2008). Further studies erected the new genera Dinovorax and Snorkelia, the family of Parviluciferaceae and reclassified P. prorocentri as S.

4 prorocentri (Reñé et al., 2017). Nowadays, known species of parasitoids of dinoflagellates belonging to Perkinsozoa are grouped into the three genera Parvilucifera, Dinovorax and Snorkelia (Reñé et al., 2017a) .

All three genera share their main features, except for the zoospore discharge (Mangot et al., 2011), which is the characteristic that differentiates them. Perkinsids present different stages during its life cycle, among which can highlight a free-living stage that belongs to the zoosporic flagellates. Furthermore, the infectious stage is characterized by the presence of two flagella located on the apical complex of the cells (Mangot et al., 2011). Lastly, a high number of zoospores grow up inside the host cell. Moreover, Perkinsozoa life cycle presents an intracellular stage named trophont, which represent the initial stage of infection once the parasite zoospores have penetrated the host cell. Perkinsus spp. and Parvilucifera spp. trophont generate a “sporangium” inside the host cell. Once the adhesion of the parasite into the host takes part, the zoospore penetrates the host cytoplasm, breaking the cell membrane. Then, the trophont grows inside within the host cytoplasm, consuming all cellular material and increasing in size. The trophont undergoes multiple divisions either in sporangia, generating zoospores that will be released outside the host structure (Alacid et al., 2015; Mangot et al., 2011).

Dinoflagellates Dinoflagellates represent an important group of microorganisms that are able to produce a significant amount of organic matter, catalogued as important primary producers, as well as host for a large variety of parasites (Perkinsids incl.), in tropical, subtropical and Mediterranean aquatic environments (Jephcott et al., 2016). They are a large group of flagellate eukaryotes, which belongs to Dinoflagellata phylum, within the Alveolata superphylum. Half community of dinoflagellates are photosynthetic, while the other half is considered as heterotrophic, feeding via osmotrophy and phagotrophy, which classify them as members of both phytoplankton and zooplankton of marine and freshwater ecosystems (Smayda & Reynolds, 2003). The morphological diversity and, adaptative characteristics permit them to colonize diverse aquatic environments (Smayda & Reynolds, 2003). Half part of known dinoflagellate species are considered as “free-living” heterotrophic organisms (Sherr, 2011). A small proportion of them are defined as “potent toxins producers” (~60 spp. from both, estuarine and marine species), and harmful producers (~185 spp.). Besides that, dinoflagellates are the major group of eukaryotic algae in aquatic ecosystems, producers of harmful algal blooms (HAB’s), with a total of 300 hundred species of micro algae (reported to

5 generate algal blooms). A quarter part of the total (~ 80 species from different known genera) is considered as producers of a wide variety of toxins (Smayda & Reynolds, 2003) that are responsible of diverse human syndromes known as: paralytic shellfish poisoning (PSP), produced by dinoflagellates of the genera Alexandrium, Pyrodinium and Gymnodinium, and some species of Prorocentrum; neurotoxic shellfish poisoning (NSP), whose some of their toxin producers are Heterosigma akashiwo, Chatonella marina or Karenia brevis; amnesic shellfish poisoning (ASP), produced by species of diatom named Pseudo-nitzschia; diarrheic shellfish poisoning (DSP), produced by species of Dinophysis and Prorocentrum lima; ciguatera fish poisoning (CFP), with Gambierdiscus, Ostreopsis and Prorocentrum as toxin producers (Lachmann Johannes & Claparède, 2015; Wang, 2008; Burkholder et al., 2006), caused by the consumption of seafood contaminated by the algal toxins.

Within the HAB’s, two mainly groups of organisms are distinguished: i) toxin producers, capable of contaminate the fauna in the environment; ii) Producers of a high biomass content, which can kill most part of the flora and fauna. Some HAB’s have both groups of microorganisms, making them quite harmful.

Most part of harmful events take place in estuaries and coastal marine waters. The particularity of these zones is the high contributions of nutrients from land and, the appearance of diverse upwellings, (Burkholder et al., 2006; Graham et al., 2009).

The HAB’s along the Catalan coast has been gaining prominence during the recent decades. Dinoflagellates account for 75% of all HAB (Smayda, 1997; Vila, 2001), being able to grow up in environments with low turbulence due to the increase in anthropic activity. Despite HAB’s formation in open ocean produced by natural causes and seasonal cycles (Garcés et al., 2012), there has been an increment of HAB’s across the Mediterranean coast, in near shore waters (like estuaries, ports, bays, etc.) where the level and the availability of all nutrients allow them to proliferate. Phytoplankton abundance and harmful algal species (i.e. Alexandrium catenella) are much higher in coastal than in open waters, where the turbulence and others fluctuations are more notable (Garcés et al., 2012). More than 40 harbours have been built along 400 km of Catalan coast (Vila, 2001), which can be the cause of an increment of A. catenella (PSP and high biomass producer) HAB’s in the Catalan coast (Tarragona and Barcelona harbours) (Vila et al., 2001), similar to Alexandrium minutum. These species are responsible of recurrent algal blooms in confined areas such as the port of Arenys and the port of Tarragona, respectively (Vila, 2001).

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Parasite specificity Parasitism has an important role for the ecology of the community. This is particularly evident on their host, which can lead to irreversible effects, including the death. The specificity in parasitic interactions (host-parasite) is determined by host genotypes which are resistant to a unique subset of parasite genotypes and, on the other hand, by parasite genotypes that are infective on a subset of host genotypes (Little et al., 2006). However, the parasite specialization could be observed within the community, as shown by different parasite strains that are specifically specialized to infect some particular host genotypes (Carius et al., 2001; Little et al., 2006).

In addition, the host-specificity of a huge number of dinoflagellates is difficult to study due to geographical and taxa variations, which is why, as we have mentioned previously, parasites can be divided into generalists or specialists (D. Wayne Coats, 1999; Little et al., 2006). Parvilucifera parasitoids described until the date are considered host-generalist parasitoids due to their capability to infect a high range of dinoflagellates under laboratory conditions. Different hosts have been studied by the scientific community, allowing to reach this conclusion (M.M.,Neus, 2017; Reñé et al., 2017b).

A new Perkinsid species (named as ‘Perkinsid sp.1) has been discovered recently during two Alexandrium taylori blooms occurred in the Catalan coast (L’Estartit and Sant Pol beaches). This Perkinsid species is currently in process of being described, what means that there are no ecological data about their life cycle and no studies have been carried out yet about their ecology, specificity and the dinoflagellate (host) susceptibility to be infected.

In order to categorize the impact of the parasites in the community, host specificity is arguably one of the most important properties for a parasite because it can determine, i.e. the potential to invade a new habitat or, if a parasite can survive the extinction of a host species (Poulin et al., 2006). Parasites can be divided into two groups: (i) Specialists, which can infect a determined number of host range and, (ii) Generalists, able to infect a wide variety of hosts.

Previous studies of host specificity were taken. Dinovorax pyriformis was studied in order to categorize the specificity of the parasitoid (M.M.,Neus, 2017); and Parvilucifera specificity, like P. infectans (Norén et al., 1999), P. rostrata (Lepelletier et al., 2014), which are examples of generalist aquatic parasites in laboratory conditions.

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Objectives The objectives of the present study were: (i) Establish the infection specificity of two Perkinsid sp.1 strains (Estartit and Sant Pol) (ii) Study the high/low susceptibility of dinoflagellates to be infected and, the prevalence of infection of both parasite strains. iii) The formation of resting cyst as a defence mechanism against infection was also explored.

Materials and Methods

Maintenance and growth of Perkinsid sp. 1 parasitoid culture strains Perkinsid sp. 1 parasitoid was isolated during Alexandrium taylori bloom, on July 2017 in the Estartit beach (42° 3' 5" N; 3° 12' 9" E) and on July 2018 on St. Pol beach, in Sant Feliu de Guíxols (41° 47' 26" N; 3° 3' 5.4" E). Both locations are located in the Catalan Coast, NW Mediterranean Sea (Reñé et al. in prep.).

Both parasitoid strains were isolated and maintained in 6-well plates by Reñé et al. in prep. Twice a week, some infected cells were transferred into new wells and healthy host were added. A mix of Alexandrium minutum and Alexandrium mediterraneum was used to sustain the parasitoid infection.

Maintenance and growth of hosts culture strains Different species of phytoplankton cultures were acquired from the culture collection of the Centro Oceanográfico de Vigo (CCVIEO), Spain, and the culture collection of the Institut de Ciències del Mar, Barcelona, Spain. A total of 44 strains (Table 1) were used, studying the host specificity in different phytoplankton species; including different strains within the same species (i.e. A. minutum)

40 mL of each phytoplankton cultures were maintained in polystyrene tissue culture flasks with L1 medium (Guillard, R.R.L. & Hargraves, P.E. 1993) without silica. In case of Diatoms phylum, silica was added into the medium for their maintenance. All stock cultures were grown at 21ºC, (except Dinophysis acuminata and Alexandrium catenella which were maintained at 16ºC) in culturing chambers with L:D cycle of 10:14h at 100 µmol photons m2 s-1.

The experiment was carried out by duplicate, in order to study the host-range of both Perkinsid sp. 1 strains Estartit and Sant Pol.

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Phytoplankton screening for parasitoid infections. Different taxa were used in order to study the host-range of Perkinsid sp. 1 strains (Table 1). The infection susceptibility to the Perkinsid sp. 1 parasitic infection of 44 phytoplankton strains belonging to Dinophyta (31 strains) and other classes (13 strains) of phytoplankton was tested. In general, 5 orders from Dinophyta division were studied (Dinophysiales, Gonyaulacales, Gymnodiniales, Peridiniales and, Prorocentrales) with a total of 14 genera, and 7 different classes including Diatoms (3 orders, and 3 genera), Clorophyta (2 orders, and 3 genera), Haptophyta (3 orders, and 3 genera), Ochrophyta (1 order, and 2 genera) and Cryptophyta (1 order, and 1 genera) were studied in order to determine the specificity of the Perkinsid sp.1 parasitoid (Table 1).

Table 1. Dinoflagellate strains infected by both Perkinsid sp.1 strains (Estartit and Sant Pol).. Y/N indicate host infection. Percentage of infection: Low = 1-3 infected cells; Medium = Less than 50% of infected cells; High = more than 50% of infected cells. “-“ represents a non-infection level. Abbreviations: Y: Yes; N: No.

Class Ordre Family Genus Species Perkinsid % sp.1 strain infection EST St Pol Dinophyceae Dinophysiales Dinophysiaceae Dinophysis acumiata N N - Gonyaulacales Gonyaulacaceae Alexandrium affine Y Y Medium andersonii Y Y Medium catenella Y Y Low margalefi N N - mediterran Y Y Medium eum minutum Y Y Medium (AMP4) minutum Y Y Medium (Arenys) minutum Y Y Medium (VGO) tamarense Y Y Medium taylori Y Y Medium Goniodomataceae Gambierdiscus excentricus N Y Low Ostreopsidaceae Coolia monotis Y Y Low Coolia tropicalis Y Y Low Ostreopsis ovata N N - Gymnodiniales Amphidiniaceae Amphidinium carterae N N - Gymnodiniaceae Barrufeta bravensis N N - Gymnodinium catenatum N N - Gymnodinium impudicum N N - Gymnodinium litoralis N N - Kareniaceae Karlodinium veneficum N N -

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Levanderinaceae Levanderina fissa N N - Peridiniales Peridiniaceae clade I Heterocapsa triquetra Y Y Medium Peridiniaceae clade Kryptoperidinium foliaceum Y Y Low II Peridiniaceae clade Scrippsiella trochoidea Y Y Low IV Prorocentrales Prorocentraceae Prorocentrum hoffmanian N N - clade I um Prorocentraceae Prorocentrum micans N N - clade II minimum N N - rathymum Y Y Low triestinum N N - Prorocentraceae Prorocentrum lima N N - clade III Diatoms Coscinodiscales Coscinodiscaceae Coscinodiscus radiatus N N - Thalassiosirales Thalassiosiraceae Thalassiosira weissflogii N N - clade III Chaetocerotales Chaetocerotaceae Chaetoceros affinis N N - Pyramimonadal Pyramimonadaceae Pyramimonas sp. N N - es Chlorodendrale Chlorodendraceae cl Tetraselmis convulatae N N - s ade IV suecica N N - Coccolithales 1. Calyptrosphaeracea Holococcolithoph sphaeroide N N - e ora a

➔ Pleurochrysidaceae Pleurochrysis elongata N N -

Isochrysidales ➔ Noelaerhabdaceae Emiliania huxleyi N N -

Prymnesiales ➔ Prymnesiaceae Prymnesium faveolatum N N -

Raphidophyce Chattonellales ➔ Chattonellaceae Chattonella subsalsa N N - ae Heterosigma akashiwo N N - Cryptophyceae Pyrenomonadal➔ Pyrenomonadaceae Rhodomonas baltica N N - es

The susceptibility of phytoplankton strains to parasitoid infections was tested in 24 well- plates, in a final volume of 2 mL and a ratio of 1:10 (host/zoospores). Newly produced Perkinsid sp. 1 zoospores were added to each phytoplankton culture at a final concentration of 105 cells/ml-1. The Perkinsid sp. 1 parasitoid completes its life-cycle in approximately 4 days (Reñé et al. in prep). So, the parasitoid culture was observed by optical microscopy during one week before the establishment of the experiment. When the zoospores number was high, the culture was filtered using a 5µm filter, which permitted to separate zoospores from the rest of cells present in the culture. The flow- through containing the zoospores was collected and 1 mL subsample was fixed with

10 formaldehyde solution at 4% final concentration and counted with a Sedgwick-Rafter counting chamber. Phytoplankton cultures were used for this experiment at a final concentration of 104 cells ml-1. All phytoplankton strains were fixed with iodine Lugol’s solution and quantified using the same method than the zoospores.

The presence of infections on phytoplankton strains was examined every 3 days, by inverted light microscopy. When infections were present, healthy host cells were added after 4 days, allowing a second cycle of infections, in order to check the viability of infections. On the other hand, if infections were not detected, more zoospores were added to the host culture after 7 days of the first inoculation in order to confirm its resistance to the parasitoid.

The observation of host-parasitoid infections was conducted during a period of 15 days. During this time, observations and controls of the different infection levels were carried out in order to allow the development of the new zoospore generation. Furthermore, if a low prevalence of dinoflagellate was observed by inverted microscopy, an additional volume of host that would allow infection by the new generation of zoospores of the parasite was added.

The phytoplankton cultures were categorized as resistant (if not infections were observed) or as susceptible. Once the cultures were infected, some checks were carried out to control the abundance of both dinoflagellates and parasites by the use of the two criteria to be taken into account: the swimming capacity, presence of sporangia or new zoospores. Furthermore, an infection level was stablished. The levels of infection "Low", "Medium" and "High" were assigned according to the number of infected cells visualized during the infection period. "Low" was for those strains in which only solitary infected cells were observed; "Medium" was categorized for those strains were the infection had a higher percentage of infection than “Low” level; and, "High", for those strains were most of the cells were infected.

Determination of resting cysts production by dinoflagellates In some cases, phytoplankton strains produced cysts when their susceptibility to infection was tested. A specific experiment was conducted in order to explore whether it was a response to the presence of parasitoid zoospores (Table 2). The procedure was like the experiment previously explained. A control treatment (only host) and an infection treatment (host + zoospore) were prepared by duplicate for the 6 phytoplankton strains, where cyst production was detected, following the same

11 procedure than before. The test was performed in 24 well-plates. The samples were fixed after 3 days with formaldehyde solution at 4% final concentration. The number of vegetative cells and cysts was counted using a 1mL Sedgewick-Rafter chamber and compared between treatments.

Table 2. Strains of dinoflagellate species infected by Perkinsid sp.1 to study the production of resting cysts. C = Control treatment (host); Q = Infections treatment (host + zoospores). Cysts increment column represent the differences between both treatments in cyst abundance.

Class Ordre Genus Species Cyst Cysts increment C Q Dinophyceae Gonyaulacales Coolia tropicalis N Y 14,4 % Ostreopsis ovata Y Y 0 % Gymnodinium litoralis Y Y 15,5 % Gymnodiniales Kryptoperidiniuim foliaceum Y Y 7,1 %

Host specificity index

Host Specificity Index (STD index) was chosen to measure the phylogenetic specificity; it measures the taxonomic average distinction of all host species infected by a given parasite. The index STD was determined following (Poulin et al., 2003) for both Perkinsid sp. 1 strains (Estartit and Sant Pol). Hosts species are located within a taxonomic hierarchy, according to the Linnean classification in Phyla, Class, Order, Family, and Genera (Fig. 2). The taxonomic distinction is simply the average number of steps in the hierarchy that should be given for a common taxon of two host species, calculated in all possible pairs of host species (Fig. 2). Dinophysiales were established into one family (Claparède E. & Lachmann J. 1859); Gonyaulacales (Gottschling et al., 2012; Kim and Park, 2017) and Peridiniales into 3 families (Anglès et al., 2017), while Gymnodiniales into 4 families (Larsen, 2000; Reñé et al., 2015), Prorocentrales into 3 (Hoppenrath et al., 2013), Coscinodiscales (Sancetta, C. 2006), Thalassiosirales (Hoppenrath et al., 2007), Chaetocerotales (Stachura-Suchoples K. & Williams, D.M., 2009), Pyramimonadales (Not et al., 2012), Chlorodendrales, Isochysidales, Prymnesiales, Chattonellales and Pyrenomonadales into one family, and, finally Coccolithales into two.

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Goniodomataceae Gambierdiscus

Gonyaulacales Gonyaulacaceae Alexandrium Ostreopsidaceae Coolia

Peridiniaceae clade I Heterocapsa Peridiniales

Peridiniaceae clade III Kryptoperidinium Dinophyceae Peridiniaceae clade V Scrippsiella Prorocentrales Prorocentraceae clade II Prorocentrum

Fig. 2. Organigram, which represent the phylogenetic jumps of each species needed to calculate the STD index, for both Perkinsid sp. 1 strains (Estartit and Sant Pol).

The dinoflagellate host species infected by both Perkinsid sp. 1 strains (Table 1) explained above were used to determine the specificity index.

The STD index was calculated using this equation:

∑ ∑푖<푗 휔푖푗 STD = 2 푠(푠−1)

Where s represents the number of hosts species used by a parasite, the double summation is over the set {i=1, … s; j=1, … s, such that i

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Results

Observations of Perkinsid sp.1 infection in dinoflagellates hosts. Live and detailed observation development by inverted microscopy of dinoflagellates hosts allowed the identification of cell infection (Fig. 3), making a comparison between infected cells and healthy ones.

The presence of the diverse Perkinsid sp.1 life-cycle stages was a criteria recorded for the experiment. The first life-cycle stages of the parasite were visible during the first or second day of the infection. Once the zoospore penetrates the dinoflagellate cell membrane, trophont is the first life-cycle stage of the parasite (Fig. 3 E, M). The trophont begins to consume and degrade the host cytoplasm and cellular material. Then, the trophont grows inside the dinoflagellate until all the space is occupied, yielding the sporocyte stage (Fig 3 A-D, G, I, J, N). Sometimes, during the infection process, the outer membrane may become degrade (Fig. 2 I), while in the internal part, the trophont takes part. Subsequently, the differentiation of the new mature zoospores takes place (Fig. 3 H, K) predisposed to occupy most part of the internal cell space (Fig. 3 F, G, L, O). Once the mature zoospores are formed, they cause a break in the cell membrane being released into the environment in search of a new dinoflagellate host to repeat the infection cycle.

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Figure 3. Dinoflagellates strains infected by Perkinsid sp.1 parasitoid under laboratory conditions. (A) Alexandrium affine; (B) Alexandrium andersonii; (C) Alexandrium catenella; (D) Alexandrium mediterraneum; (E) Alexandrium minutum AMP4; (F) Alexandrium minutum Arenys; (G) Alexandrium minutum VGO; (H) Alexandrium tamarense; (I) Coolia monotis; (J) Coolia tropicalis; (K) Gambierdiscus excentricus; (L) Heterocapsa triquetra; (M) Kryptoperidinium foliaceum; (N) Scrippsella trochoidea; (O) Prorocentrum rathymum. Scale bar = 10 µm.

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Screening dinoflagellate hosts for Perkinsid sp.1 strains infections 44 host strains were tested in order to study their susceptibility to the Perkinsid sp.1 infection. Both Perkinsid sp.1 strains (Estartit and Sant Pol) were able to infect 15 species, with no strain differences, except from G. excentricus which was only infected by Sant Pol parasitoid strain, belonging to the genera Alexandrium, Heterocapsa, Kryptoperidinium, Scripsella, Prorocentrum (Table 1, Fig. 3).

In reference to the dinoflagellates used as hosts, different orders were tested. A total of 14 different strains of Gonyaulacales were studied; 11 - 12 presented infections by parasite strains (G. excentricus varies according to the parasite strain; infected by Sant Pol; Fig. 3 L). From all Gonyaulacales strains, 10 of them belonged to Alexandrium genera, where 9 of them were infected, except A. margalefi, which did not present any stage of infection. It was possible to verify the intraspecific variability in A. minutum studying 3 strains, being all of them infected by both parasite strains. Different infection stages were showed during the infection cycle: ; A. affine (Fig. 3 A), A. andersonii (Fig. 3 B), A. catenella (Fig. 3 C), A minutum VGO (Fig. 3 G) showed a sporocyst stage; A. minutum AMP4 (Fig. 3 E) and Coolia tropicalis (Fig. 3 J) showed an early trophont stage; In A. mediterraneum (Fig. 3 D) and A. minutum arenys (Fig. 3F) it is possible to observe the zoospores accumulation inside the host cell membrane; In A. tamarense (Fig. 3H) and G. excentricus (Fig. 3L) the differentiation of the new mature zoospore is showed; 2 Coolia strains, both infected (Fig. 3 I and 3 J); 1 strain of Gambierdiscus; and 1 Ostreopsis strain, without visible infection. From the 3 Peridiniales strains tested, all of them were infected by both parasite strains. K. foliaceum (Fig. 3 N), H. triquetra (Fig. 3 M) and S. trochoidea (Fig. 3 O). 7 Gymnodiniales were tested and none of them presented infection stages for any parasite strain. In reference to Prorocentrales, we had a total of 6 strains and only P. rathymum (Fig. 3 P) was infected. Finally, one strain of Dinophysiales was studied (D. acuminata), which no infection was appreciate.

A group of strains that did not belong to Dinoflagellates were tested, 10 of these strains belonged to diatoms. None of them (Coscinodiscales, Thalassiosirales, Chaetocerotales, Pyramimonadales, Chlorodendrales, Coccolithales, Isochrysidales and Prymnesiales) were infected after their exposure to both parasite strains (Table 1).

In general terms, 11 out of 14 Gonyaulacales, 3 out of 3 Peridiniales and, 1 out of 6 Prorocentrales strains were susceptible to be infected by both parasite strains, except G. excentricus that was only infected by Sant Pol parasite strain.

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Resting cyst observations in dinoflagellates. From the 44 dinoflagellates strains tested in the experiment, 4 strains (Table 2) were used to test the production of cysts in response to the parasite infection. Both, two controls (C) and two infections treatments (Q) were observed by inverted microscope. It was observed that C. tropicalis produced cysts (Fig. 4 B) when zoospores were added. However, it did not generate cysts in their absence (Fig. 4 A). C. tropicalis represents the most notable difference between both treatments. The percentage of cysts in this host varies by 14,4%. In the control treatment, no cysts were detected, while when zoospores were added, the cyst number increased considerably (Fig. 4 B – Fig. 5). Ostreopsis ovata did not present significative differences between both treatments, whose percentage of cysts where 12,9% (Fig. 4 E-F), opposite to G. litoralis which produces high abundances of cysts in both treatments (Fig. 4 C, D), showing significant differences between both treatments. Almost twice as many cysts could be counted in presence of zoospores (Fig. 5). In control treatment, G. litoralis showed a 15,5% of cysts, which means a quite remarkable percentage to be a control (Fig. 4 C). In addition, when zoospores were added, the number of cysts increased considerably (Fig. 4 D), up to 31%. Infections were observed in control treatment, which made us think that the first screening (Table 1) of Perkinsid sp.1 infections presented cysts, which came from the mother culture. It represents the increment of cysts between both treatments, where the controls had less abundance of cysts. Finally, K. foliaceum showed an important presence of cysts in both treatments. This rate included a high number of cysts, reaching even 57,6% of cysts when the zoospores were added (Fig. 4 G-H), with an increment of 7,1% between both treatments. Likewise, there were no significant differences between both treatments (Fig. 5)

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Figure 4. Resting cyst observations of different dinoflagellates hosts. (A-B) Coolia tropicalis; (C-D) Gymnodinium litoralis; (E-F) Ostreopsis ovata; (G-H) Kryptoperidinium foliaceum. Scale bar = 10 µm.

Figure 5. Resting cysts percentages depiction for both treatments; Each bar show the percentage of cyst for each dinoflagellate tested in both, control and infection treatments. Whiskers represent the standard deviation. C = Control; Q = Host + Zoospores.

Determination of specificity index of Perkinsid sp.1 parasite Estartit strain of Perkinsid sp. 1 was capable to infect at least 15 species of the genera Alexandrium, Coolia, Heterocapsa, Kryptoperidinium, Scrippsiella and, Prorocentrum, belonging to three different orders (Gonyaulacales, Peridiniales and Prorocentrales), resulting in a STD specificity index of 2,74.

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The other Perkinsid sp.1 strain (Sant Pol) was able to infect 16 dinoflagellate species from Gambierdiscus, Alexandrium, Coolia, Heterocapsa, Kryptoperidinium, Scrippsiella and, Prorocentrum, which belongs to three different orders (Gonyaulacales,

Peridiniales and Prorocentrales), with a STD index of 2,81.

Both parasite strains showed a similar STD index, only varying in the infection of G. excentricus. The results showed a low difference between them, with similar STD values, showing the same number of infected species and percentage of infection, except for G. excentricus.

Discussion

Perkinsid sp. 1 host-range in dinoflagellate community The study allowed us to classify Perkinsid sp. 1 as a generalist parasitoid for dinoflagellate species. Both strains tested showed and STD of 2,74 and 2,81 (Estartit and Sant Pol respectively) which means that they can infect a wide range of dinoflagellate hosts, under laboratory conditions. Both parasite strains were tested using the same host species, Dinophysiales (0/1 strain infected), Gonyaulacales (11-12 of 14 strains infected), Gymnodiniales (0/7 strains infected), Peridiniales (3/3 strains infected), Prorocentrales (1/6 strains infected) and non-dinoflagellate (0/13 strains infected). On the one hand, Estartit strain infected several orders of dinoflagellates, such as Gonyaulacales (Alexandrium and Coolia genera), Peridiniales (Heterocapsa, Kryptoperidinium and Scrippsiella), and Prorocentrales, where only one strain of Prorocentrum genera showed infection. On the other hand, Sant Pol strain from Perkinsid sp. 1 parasitoid infected the same number of dinoflagellate strains, but also an additional species of Gonyaulacales, Gambierdiscus excentricus. Therefore, these infections allow us to have some knowledge about the infective specificity of this Perkinsid parasite for dinoflagellates. The remaining strains, including all species not belonging to dinoflagellates, did not show infections. Consequently, Perkinsid sp. 1 can be defined as a generalist species for dinoflagellates, despite not causing infections in other phytoplankton groups.

The results of this study show that Perkinsid sp. 1 has low prevalence, due to the low percentage of infected dinoflagellate cells (Table 1). While in other studies it was observed that Parvilucifera species (P. sinerae, P. infectans, P. rostrata) show high prevalence (>80%) when infecting susceptible strains under laboratory conditions (Garcés et al., 2013; Norén et al., 1999 Lepelletier et al., 2014). Perkinsid sp. 1 only

19 infects a small proportion of the host population. In addition, regarding the prevalence of infections, it has been observed that it never reached "high" prevalence levels. In most infected strains, the prevalence of infections was “low” or even punctual (Table 1).

The experimental design stablished in the screening experiment did not allow us to generalise about the behaviour of Perkinsid sp. 1 strains under field conditions. These results permit the study of the infection cycles of several dinoflagellates under laboratory conditions, so it is unknown until the date if these infections would also occur in the environment. In addition, the host-range studied is quite limited, so it could be expanded by adding new species (hosts) enabling to visualize the presence, or not, of infection stages.

The specificity of the infection reflects the mutual evolution of both, parasites and host(s). Moreover, a higher STD index reflects a greater phylogenetic distance between both. So that, both parasitoid strains can be classified from more to less generalist as Sant Pol (2,81) and Estartit (2,74). From phylogenetic data (Fig. 6; Reñé et al., Unpublished), we can observe the most ancestral lineages (Dinovorax, Perkinsid) compared with most recent ones. Thanks to mi STD index and the phylogenetic position of Perkinsid sp. 1, the “parasite paradox” can be supported, where a specialist parasite can end up becoming a generalist one (Agosta et al., 2010), similar to Parvilucifera studies (Moll, N., 2017), being able to infect a great number of hosts. Perkinsid sp. 1 is considered less generalist than Parvilucifera spp due to its STD index, showing us their infection specificity under laboratory conditions.

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Fig. 6 Maximum likelihood phylogenetic tree of SSU rDNA gene (modified from Jeon & Park, 2019) showing the phylogenetic relationships of Parviluciferaceae members, including Perkinsid sp. 1.

Nevertheless, the specificity of infections reflects the mutual evolution of both, parasites and host(s). This relationship may vary in the environment due to changes in the behaviour of both, allowing to vary, becoming specialist parasites into generalist ones, and vice versa, due to the continuous environmental variations that takes place, i.e. salinity or temperature changes (Figueroa et al., 2008), allowing the aquatic community to adapt itself in order to survive. Most of the Parviluciferae parasites known and described to date, including (from more to less generalist) P. corolla (3,57), P. sinerae (3,36), P. infectans (3,28), P. rostrata (2,98) and D. pyriformis (2,57), considered host-generalist parasites, being able to infect a wide variety of genera of dinoflagellates in laboratory conditions (Moll, N., 2017). However, as previously discussed, both parasite and the host(s) can vary in unpredictable and changing environments. Establishing that our parasite (Perkinsid sp. 1) is a generalist parasite may allow it to adapt to changing conditions in the environment, such as marine phytoplankton community, which present a rapid specie turnover, being the specificity of these parasitoids a successful strategy in the evolution of this species.

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Cysts formation Several dinoflagellate species like Alexandrium spp and G. impudicum generate resistance forms in laboratory conditions, referred as cysts (Anderson & Wall, 1978; Bolch & Hallegraeff, 1990; Doucette et al., 1989), in response to adverse environmental conditions. Moreover, in the environment, several studies recorded the presence of cyst with a strong correlation with salinity and temperature (Vila, 2001). Through observations, differences in cyst abundance among 4 dinoflagellate species were determined, belonging to Gonyaulacales (3 genera) and Gymnodiniales (1 genera) (Table 3). Coolia tropicalis showed a high abundance of cysts in the treatment where Perkinsid sp.1 zoospores were added, reaching to differ from the control treatment up to 14.4%. Gymnodinium litoralis produced cysts in both treatments, with and without the presence of Perkinsid sp. 1. However, if zoospores were added, the cyst abundance in G. litoralis increased markedly. Resting cyst production is not rare in both marine and brackish Gymnodinium species (Reñé et al., 2011). In this work, several observations of various cysts were made, among which G. litoralis cyst abundance could be highlighted, whose pellicle cysts are frequently observed in G. litoralis cultures and sediments along coastal water blooms (Reñé et al., 2011). While, for Kryptoperidinium foliaceum, there were almost no significant differences between treatments, both showed irregularly shaped cysts, like in previous studies (Figueroa et al, 2009). Their cysts can provide useful information for the study of the dynamics of harmful events (Satta et al., 2013). Moreover, Ostreopsis ovata also did not present significant differences in cyst abundance between both treatments. Their displayed pellicle cysts are generated as a response to unfavourable conditions (Accoroni et al., 2014; Scalco et al., 2012), like the presence of Perkinsid sp 1.

The ability of the dinoflagellates to produce cysts is of very fundamental ecological importance as, the ensure of their survival in adverse environmental conditions like parasite presence, in addition to facilitating the recurrence of blooms and, promoting dispersion (Anderson & Wall, 1978; Anglès et al., 2012; Dale, 1983; Dale et al, 1978 Lambrecht et al., 2015), despite the fact of losing swimming capability. Cyst-forming species, in the environment, are mostly common in Peridiniales, Gonyaulacales and Gymnodiniales, and quite rare for the Dinophysiales and Prorocentrales (Head, 1996). Our study supports the formation of resistance cysts in the dinoflagellates in order to avoid infections by the parasite, or not, although many of these species of dinoflagellates already generate cysts by themselves in the face of adverse conditions in the environment. In addition, thanks to the addition of Perkinsid sp. 1 zoospores in the cultures, it was possible to observe the cyst formation among the 4 dinoflagellate

22 species explained before. Cyst observations was characterized by providing resistance to the host against Perkinsid sp. 1 presence by the creation of a resistant wall, called pellicle cysts, which cause their settling on the bottom of the well-plate, similar to environmental studies carried out along the Gulf of Naples, Mediterranean coast (Montresor et al., 1998).

Table 3. Summary of dinoflagellate species infected by both Perkinsid sp.1 strains (Estartit and Sant Pol). The number indicates how many species of each respective genera were infected by each Perkinsid sp.1 strain.

Order Family Genus Nº of species infected by Estartit Sant Pol Gonyaulacales Goniodomataceae Gambierdiscus 0 1 Gonyaulacaceae Alexandrium 9 9 Ostreopsidaceae Coolia 2 2 Peridiniales Peridiniaceae clade I Heterocapsa 1 1

Peridiniaceae clade III Kryptoperidinium 1 1

Peridiniaceae clade V Scrippsiella 1 1

Prorocentrales Prorocentraceae clade II Prorocentrum 1 1

Conclusion In conclusion, both strains of Perkinsid sp. 1 examined in this study share many characters in common with other members of Parviluciferae, including the use of dinoflagellates as hosts (Table 1), the various stages of the life cycle and, their development (feeding and growth) within the host. The dinoflagellates endoparasite Perkinsid sp.1 is defined as generalist, being able to infect a diverse community of dinoflagellates and allowing us to determine an intraspecific variability between the strains. Perkinsid sp.1 can infect a smaller number of dinoflagellates compared with other Parviluciferaceae species (P. sinerae or P. infectans). The susceptibility of different host to be infected by the parasitoid was studied, and the results showed important information about the life-cycle and host-range, unknown until the date. The life-cycle of this parasitoid is summarized in three stages where the free-living zoospores penetrate a healthy host and transform into the feeding stage, called

23 trophont. Then, the zoospores feed from the internal cellular material from the host, generating the sporocyst, producing numerous new zoospores which will be released, through a break in the cell membrane, into the environment.

The parasitic interactions (host and parasitoid) can become an important factor in the bloom’s dynamics due to the increment of the parasite density, which can affect total density of the dinoflagellate population. It is necessary to carry out additional studies in the field in order to better understand the biological interactions along HABs development, and the distribution of Perkinsid sp. 1 along the Mediterranean Sea, especially in ports and estuaries. The most susceptible groups of dinoflagellates infected by Perkinsid sp.1 strains are grouped in two orders, Gonyaulacales and Peridiniales.

The presence of resting cysts in several dinoflagellates allowed us to study the dinoflagellate response to the parasitoid presence. These non-motile cysts give resistance to G. litoralis and C. tropicalis i.e., against reverse conditions like the presence of parasites in the environment, allowing a possible “top-down” bloom control.

Acknowledgements I would like to thank R. Gallisai and A. Reñé as directors of the project and their useful contributions to the analysis and interpretations of the results, including their continuous development as tutors of this master project; E. Garcés, as the director of the investigation project, allowing me to join to their scientific group; Institut de Ciències del Mar (ICM-CSIC) for allowing me to do my master's work in their facilities; and many thanks to my girlfriend and family, who have been a fundamental support throughout this year.

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