ichthyodinium ichthyodinium ichthyodinium is a monotypic genus of dinoflagellates in the family dinophysaceae. ichthyodinium chabelardi (/ɪkθioʊˈdɪniəm/) is currently the sole described species of the genus. ichthyodinium scientific classification domain: eukaryota kingdom: protista phylum: dinophyta class: syndiniophyceae order: syndiniales family: dinophysaceae genus: ichthyodiniumhollande & j.cachon, 1952 species: i. chabelardi binomial name ichthyodinium chabelardihollande & j.cachon, 1952 i. chabelardi is a generalist parasite with a very broad geographic range. it has been found in locations such as the coasts of indonesia and vietnam, the mediterranean sea, and off the coast of portugal. the full extent of its distribution is not known. while it can be found throughout most of the year, it has been observed to infect sardine eggs with the highest rates in the winter and early spring, from december to march. it is of economic significance to the fish industry, where it depletes the numbers of many species of fin fish by acting as an endoparasite in host embryos. it was originally described in 1952 by hollande and cachon, who described it as an agent of infection amongst sardines off the coast of algeria, in the mediterranean sea. in 2006, gestal et al. proposed a reclassification of i. chabelardi into the genus perkinsoide based on its ultrastructure and rna sequence, but several papers have opposed this reclassification based on its zoospore ultrastructure, as well as its small subunit rrna and large subunit rrna sequences. recently, there has been evidence brought forth that there may be at least two distinct species of ichthyodinium, since slight genetic differences have been detected between the european and asian populations. taxonomy the genus ichthyodinium was originally described in 1952 by hollande and cachon, who named the novel species they found ichthyodinium chabelardi, and described it as an agent of infection amongst sardines off the coast of algeria, in the mediterranean sea. they published a second paper with additional life cycle observations a year later, in 1953. phylogenetic analysis of i. chabelardi conducted in 2009 found evidence to support the inclusion of ichthyodinium in marine alveolate group i (magi). with this addition, magi will consist of the genera ichthyodinium and dubosquella, both of which are parasitic alveolates, thus maintaining magi as an exclusively parasitic group. when looking at the flagella of ichthyodinium in cross section, they are spherical and thus lack the typical striated strand of a dinoflagellate transverse flagellum. the striated strand is also lacking on the flagella of zoospores of duboscquella, suggesting this may be a synapomorphy of magi. in 2006, gestal et al. published a controversial paper calling for the reclassification of i. chabelardi. gestal et al. asserted that i. chabelardi had been wrongly placed in the order syndiniales, and should instead be placed in a new genus, perkinsoide, and thus be renamed perkinsoide chabelardi. they claimed that evidence for this reclassification could be found by looking at the life cycle, schizogonic divisions, structure of schizonts inside the host, nuclei without the typical dinoflagellate appearance, presence of rhoptry-like structures, and the presence of possible pseudo-conoid and biflaglleated spores. gestal et al. went on to conduct a phylogenetic analysis of the small and large subunit ribosomal rna genes in the hope that this would provide additional evidence to support the reclassification. however, the results they found did not provide clear answers. based on their phylogenetic analysis, they were merely able to determine that the parasite of interest should be positioned somewhere within the group consisting of dinoflagellates, perkinsids, and syndiniales. several papers have since come out against this reclassification, arguing that i. chabelardi is the rightful name. one argument for this is that the life cycle of the parasite described by gestal et al. does not match those of the genus perkinsus, as it was described by perkins in 1996. the parasite in question is reported to have a different pattern of cell division from members of perkinsus and lacks a discharge tube of zoospores. it has been suggested that perhaps the early exogenous phase that gestal et al. observed was indeed i. chabelardi, but that the observations made of the later stages, including the zoosporangia and zoospores, were of a different parasitic protist, one that was quite possibly not a dinoflagellate. there is currently only one identified species in the genus ichthyodinium, named ichthyodinium chabelardi, which was first identified in the mediterranean sea, off the coast of algeria by hollande and cachon in 1952. however, there is growing evidence that at least two distinct species may belong in the genus (mori et al., 2007; skovgaard et al., 2009;). in 2007, the mode of infection, development, and ssu rdna sequence of an unknown parasite infecting leopard coral grouper (plectropomus leopardus) off japan were analyzed and compared to i. chabelardi (mori et al., 2007). although many similarities were drawn between the two, several differences were noted, causing the unknown parasite to be tentatively classified as ichthyodinium sp. pl (pl being the initials of the scientific name for leopard coral grouper) (mori et al., 2007). polymerase chain reaction (pcr) analysis using specific primers created from the ssu rdna sequence of icthyodinium sp. over several different years revealed the same species of parasite to be infecting the fish year after year (mori et al., 2007). in 2009, the sequences of the ssu rdna, its1 (internal transcribed spacer 1), its2, and 5.8s rdna of i. chabelardi isolated from bogue (boops boops) and european pilchard (sardina pilchardus) off the coast of portugal were analyzed and found to be identical to each other (skovgaard et al., 2009). this suggests that the parasites infecting both species of fish were from a single population (skovgaard et al., 2009). however, when the ssu rdna sequence from the portuguese population was compared to sequences gathered from yellowfin tuna (thunnas albacares) and leopard coral grouper (plectropomus leopardus) in asia, there was only a 97% similarity between them (skovgaard et al., 2009). no morphological differences between the european and asian populations have been identified yet (skovgaard et al., 2009). life cycle i. chabelardi exists mainly in its parasitic form, during which time it lives within and obtains energy from a host fish egg. later, it is released from its host and enters the free-living phase of its life cycle. primary schizonts the earliest life stage is the primordial schizont, which occurs inside the host egg. it is a spherical uninucleate cell with a diameter of approximately 15 μm. the nuclei of the primordial schizont undergoes multiple mitotic divisions without cytoplasmic division to become a large, multi-nucleated cell of between 100 and 140 μm in diameter. the cytoplasm then forms cylindrical projections around each nucleus, which separate to form cylindrical, uninucleate cells of approximately 30 μm in diameter. these are the secondary schizonts. secondary schizonts in the secondary schizont, lecithin begins to concentrate in the parasite cells. the shape of the cell rapidly passes from bat-shaped, to triangular, and then to a rosace form, featuring a repeating, somewhat circular pattern, seemingly as a result of successive longitudinal bipartitions. from the rosace form, secondary schizonts begin to form a long cord, extending 1–2 mm in length, in layers of successive groups of eight cells connected at their poles. at this point, the oil globule of the fish egg disappears and lipid particles can be observed inside the parasite cells. last generation schizonts elongated schizonts are released from the cord, and then enter the last generation schizont phase. the schizonts become spherical after a series of divisions and are now small and uni-nucleated, resembling the early primordial schizonts. by this stage, the energy reserves of the host embryo have been heavily depleted and the yolk sac is completely full of parasitic cells and is ready to burst. the yolk sac generally bursts immediately after the host larvae hatches, but may take as long as ten hours after hatching in some cases (mori et al., 2007). the number of parasite cells released from a single yolk sac can be immense, with the number of cells released from a yolk sac of yellowfin tuna estimated to be around 40,000. immediately following release from the host, parasite cells are spherical and non-motile (skovgaard et al., 2009). two hours after release from the host, the parasites, which are now at a diameter of 20-24 μm, begin to swim using their two flagella. as the parasites spend the next two to three days dividing, they change shape to become twisted and elongated and begin to move with more vigor (skovgaard et al., 2009). after their third division, small and highly motile zoospores appear. zoospores an in-depth study of i. chabelardi zoospores in 2016 found that it is this life stage that acts as the infectious agent. this study also provides an accurate description of zoospore ultrastructure, finding zoospores to be elongated and 8-9 μm x 4-5 μm in size. the apical end is wide and round, gradually becoming narrower as the cell extends towards the caudal end. the cell has a wide, shallow, spiral groove that forms one counter-clockwise turn around the cell when viewed from the apical end of the cell, thus making the cell asymmetric. the zoospores possess two smooth flagella, at least one of which appears to lay partly in the groove. the cytoplasm of the zoospore houses well-developed mitochondria, as well as large inclusions that most likely function to store nutrient reserves. these inclusions may resemble either lipid globules or grains of storage carbohydrates, but both types have not been observed together in a single cell. trichocysts appear as electron-dense structures of about 3 μm x 0.3 μm in size in transmission electron microscopy (tem) images. in cross-section, they appear as squares and in diagonal section, they are rhombic in shape. the trichocysts are evenly distributed at the apical end, and are oriented along the longitudinal axis of the cell in the middle and at the caudal end of the cell. no structures were observed that could be used for penetration of the host egg cell (simdyanov et al., 2016). the only observation made of something that was significantly different from a typical dinoflagellate zoospore was a large electron-transparent zone in the caudal part of the cell. variations in several studies, observations have suggested that the secondary schizont stage is absent in some host species, and instead primary schizonts become last generation schizonts directly. (gestal et al., 2006; mori et al., 2007; in the case where the secondary schizont is absent, there is no rosace or cord formed. in other cases, released schizonts transformed directly into zoospores within ten minutes after release, and no zoosporangium formation was observed (mori et al., 2007). mode of parasitism in 1994, pedersen and koie suggested that ichtyodinium was spread to adult cod and turbot from their benthic prey, and was then spread to the next generation through their gametes. however, there is overwhelming evidence that refutes this, and instead suggests that the parasite is able to infect the fish host eggs laterally from the water. evidence suggests that icthyodinium infects host fish eggs laterally from the water. (mori et al., 2007; while the mode of penetration has not yet been observed, there are no detectable signs of penetration on the surface of infected egg. it has been theorized that the parasites are able to enter the host egg through the hole intended for sperm penetration following fertilization before the hole is able to close completely. in one study, primordial schizonts were detected in mackerel eggs even before the closure of the blastopore. after an outbreak in yellowfin tuna in bali, indonesia, an investigation found the rrna sequence of ichthyodinium sp. in the fertilized eggs, the larvae, and the rearing water, but not in the gonads of the parent fish or in the fish used as feed for the tuna. a further investigation showed that if the freshly spawned eggs were immediately transferred to containers of sterilized seawater, they would not become infected. however, if the eggs were left in the rearing water for one hour or longer after spawning, they would become infected. similarly, in japan, outbreaks of ichthyodinium sp. in leopard coral grouper have been found to be avoidable if the fertilized eggs are incubated in sterile, oxidant-treated seawater (mori et al., 2007). the probability of detection of parasite infection in sardine eggs increases significantly with the age of the egg