The current study documented the morphological features of the complete life cycle of P. olseni isolated from Manila clams under in vitro conditions using SEM. In the life cycle of P. olseni, our first observation of the pathogen was of the zoosporangia, and the most noticeable features of these cells were the morphology and function of the discharge tube. The term “discharge tube” was coined by  for a hollow appendage that extruded from the wall of P. marinus zoosporangia and through which mature motile zoospores exited zoosporangia, after dissolution of a plug that sealed a discharge pore in the thick zoosporangium wall. Similarly, Parvilucifera prorocentri, a tube-forming intracellular parasite of dinoflagellates, discharges zoospores through a discharge tube [27, 28]. However, unlike P. prorocentri, motile zoospores of P. olseni were released via a circular opening of the cell wall of the zoosporangium when the discharge tube disintegrated and separated from the zoosporangium, suggesting that the discharge tube functioned as a bung. The light microscopic analysis in the current study also showed the presence of an elongated rod-shape organ (discharge tube) during the early phase of zoosporulation; however, when discharging zoospores, the elongated discharge tube disappeared, and zoospores were released directly from the zoosporangium. Several reports have included similar images, with no elongated discharging device at this stage [14, 29].
In the present study, motile zoospores started to transform into trophozoites on the third day of zoospore cultivation and lost their mobility, as observed under light microscopy. It was assumed that the reduced activity of zoospores was caused by a defect in flagella movement, suggesting the loss of flagella. The loss of flagella could be caused by flagellar detachment or resorption in response to various stimuli or the cell cycle . Indeed, we were able to observe the vestige where the anterior flagellum was amputated from the body of the zoospore, and a number of detached anterior flagella were observed in the culture media by SEM. It was reported that deflagellation of Chlamydomonas sp., a green alga, occurs at the distal end of the flagella transition zone, where axoneme microtubules terminate when they is exposed to various stresses, such as pH shock, heat, alcohol treatment, and mechanical shearing [30, 31]. During deflagellation, several proteins cause the fission of the flagellar membrane and the severing of the outer doublet microtubules; these proteins include centrin and katanin [32, 33]. In contrast, flagellar resorption is seen in nature in various groups of organisms, including Hexamita, amoebae, green algae, and fungi, during transformation from flagellates into amoeba, before cell division and encystment, or in response to stress [34, 35, 36]. However, in the present study, detachment of the anterior flagella was confirmed, but the posterior’s deflagellation was doubtful yet. Thus, further TEM studies on flagellar disappear in this pathogen must be conducted. As flagella are known to play important roles in cell motility, attachment, and host cell invasion , studies on the morphological features and function of P. olseni flagellates will be useful for understanding the transmission of this pathogen.
Schizogony, asexual reproduction of protozoans by multiple fissions, is a typical method of proliferation of the trophozoite phase of Perkinsus spp. A number of studies described sibling daughter cells in vitro or in histological observation . In our study, schizogony of P. olseni from mature trophozoites to merozoites was shown in detail by SEM observation. In this process, each schizont in the early phase of schizogony consists of several long sides (polygonal shape) of merozoites, but as schizogony progresses, the merozoites become smaller and the number of sides of each polygon increases. Thus, the merozoites become circular. In the final phase of schizogony, hundreds of circular merozoites are released by rupture or abrasion of the schizont membrane. It is highly likely that schizont morphology and the number of merozoites of P. olseni induced by nutrient-enriched media in the present study might differ from those occurring in nature, as suggested by . However, our observation provides useful clues for understanding the multiple fission of P. olseni.
In conclusion, we provided a detailed description of the external morphological features of P. olseni at all stages of its life cycle. Our study showed that the life cycle of P. olseni is generally similar to that previously reported. However, we also provide a detailed description of zoosporulation, transformation, and schizogony of the pathogen, which is expected to advance our understanding of the life cycle and transmission of the pathogen.