Scanning Electron Microscopic Observation of the In Vitro Cultured Protozoan, Perkinsus olseni, Isolated from the Manila Clam, Ruditapes philippinarum

doi:10.21203/rs.3.rs-22952/v1

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Scanning Electron Microscopic Observation of the In Vitro Cultured Protozoan, Perkinsus olseni, Isolated from the Manila Clam, Ruditapes philippinarum

https://doi.org/10.21203/rs.3.rs-22952/v1

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published 03 Aug, 2020

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Background

Perkinsosis is a major disease affecting the commercially important marine mollusk Ruditapes philippinarum (Manila clam) in Asian waters. In this study, we investigated the morphological characteristics of Perkinsus olseni, the causative agent of perkinsosis, cultured under laboratory conditions at different stages of its life cycle by using a scanning electron microscope (SEM).

Results

Prezoosporangia that formed following induction with Ray’s fluid thioglycollate medium developed into zoosporangia. During this process, a discharge tube formed a porous sponge-like structure that detached before the zoospores were released; thus, this organelle operated as a bung. Liberated zoospores gradually transformed into immature trophozoites, during which detachment of the anterior flagella occurred but loss of the posterior flagella was not clearly observed in the present study. Mature trophozoites underwent schizogony by cleaving the cell forming a number of merozoites in schizonts, which were released by the rupturing of the cellular membrane of the schizont within a few days.

Conclusions

Our morphological and ultrastructural studies contribute new information on the life cycle and propagation of P. olseni.

General Microbiology

Perkinsus olseni

life cycle

Manila clam

morphology

SEM

Perkinsus olseni is a parasitic protozoan that is the causative agent of perkinsosis in the Manila clam (Ruditapes philippinarum), which is found in tidal flats and sandy beaches along the coasts of Korea, China, and Japan [1, 2, 3]. The Manila clam that is found in Korean waters is an important edible shellfish of the mariculture industry; however, recurrent mass mortalities of this species have significantly impacted mariculture production [4]. Histopathological studies have reported that severe inflammatory reactions can occur in the connective tissue of the gonad, gills, mantle, stomach, and digestive tubules due to severe P. olseni infections that could have deleterious effects on reproductive efforts and food digestion [5, 6, 7]. [8] reported that the recent summer mortality of Manila clams on the west coast of Korea was caused by the combined effects of P. olseni infection and thermal stress. In Japan, [9] demonstrated the negative effects of P. olseni on survival of wild clam populations in Ariake Bay.

Since the first occurrence of Perkinsus sp. in the Manila clam in Asia in late 1990’s, it has been identified as P. olseni by molecular studies [2, 10]. In Asian waters, Manila clam is a host of not only P. olseni but also P. honshuensis. According to [11], P. honuensis parasitizes Manila clams in Japan. In Korea, it was reported that P. honshuensis was found only in R. variegatus on Jeju Island on the southern coast of Korea after a nation wide survey of P. honshuensis in the Manila clam using (P. honshuensis specific primers to amplify the DNA of Manila clams from 23 locations [12]. Thus, the distribution of P. honshuensis appears to be limited to Juju Island, at present.

The life cycle of Perkinsus spp. involves several life stages; namely, the trophozoite, prezoosporangium, zoosporangium, and zoospore stages [13, 14, 15]. The trophozoite stage is the vegetative proliferation stage that occurs in host tissue, and has a characteristic signet-ring structure. This stage is the parasitic stage, causing inflammation, a main virulence of the pathogen, in the host [14, 16]. The prezoosporangium stage is considered to be the dormant stage in the life cycle of Perkinsus spp. This stage allows the organism to endure unfavorable conditions [17, 18]. When trophozoites are exposed to anaerobic conditions, they grow larger and transform into thick-walled cells. This phenomenon was confirmed by incubating host tissue infected with Perkinsus spp. in liquid thioglycollate medium [1, 19]. The enlargement of trophozoites has also been observed in moribund hosts [20, 21 22]. When isolated prezoosporangia are introduced to sea water or culture medium, they transform into zoosporangia, and have multiple cell stages through progressive karyokinesis and cytokinesis [13]. Finally, the zoosporangia release motile zoospores into the medium via the discharge tube [13]. Zoospores gradually lose their zoospore characteristics, such as flagella and body shape, and transform into immotile trophozoites [23]. Subsequently, they develop into schizonts, and finally release multiple daughter cells called merozoites [24].

Thus, the life cycle of the pathogen is well documented. However, these previous studies mainly used light microscopic analyses, rather than electron microscopic observations; consequently, the descriptions of the morphological characteristics of Perkinsus spp. are limited. Therefore, here, we performed an in-depth analysis of the surface ultrastructure of P. olseni at each of its life stages using light microscope (LM) and SEM. Our study is expected to provide new insights on the life cycle and transmission of the pathogen.

2.1. Zoosporulation of in vitro cultured P. olseni

The different morphological life stages in the progression to zoosporulation in vitro in culture media are presented in Fig. 1. On the second day of in vitro culture, prezoosporangia were spherical in shape, as observed under LM (Fig. 1A), which was consistent with the electron microscope observation showing that prezoosporangia have a smooth body wall (Fig. 1B). They ranged from 28.1 to 105.1 µm in diameter (mean, 61.1 ± 16.8 µm; n = 100). On the third day, various phases of cell division were observed with LM. SEM showed that 7% of the cells showed the development of a discharge tube that continuously elongated, although it remained plugged until apparatus had completely formed. LM showed that successive bipartition took place through karyo- and cytokinesis, giving rise to zoosporangia containing different numbers of cells inside (Fig. 1C). The external growth of the discharge apparatus was shown by SEM (Fig. 1D). When elongate initiated (Fig. 1E) it had a mesh-like structure (Fig. 1F). On the fourth day, 70% of the zoosporangia had a discharge tube (Fig. 1G). The discharge tube developed into a rod shape that had a porous sponge-like structure (Fig. 1H). The lengths of the discharge tube ranged from 9.8 to 18.6 µm (14.2 ± 2.6 µm, n = 15). The length-to-diameter (zoosporangia body) ratio was 0.4 ± 0.1 µm (n = 15). Twenty-five percent of the cells became zoosporangia, with approximately 100 zoospores being closely packed in the lumen of the zoosporangium, which ranged from 28.4 to 90.7 µm in diameter (53.9 ± 13.5 µm, n = 100). On the fifth day, most gravid zoosporangia completed zoosporulation, and the motile zoospores had been discharged into the culture medium. SEM clearly showed that the discharge tube disintegrated, leaving an opening from which the motile zoospores were emerging (Fig. 1I) and had emerged (Fig. 1J).

The released zoospores were highly motile, exhibiting circular and erratic movements. They were ellipsoidal in shape, their dorsal surface was slightly convex, while their ventral surface was flattened (Fig. 2A). Their body dimensions were 3.7 ± 0.4 µm × 2.0 ± 0.2 µm (n = 60). The anterior flagellum was crooked, while the posterior one was slightly straight, with a rough surface. The anterior flagellum was 14.0 ± 1.0 µm long (n = 50) while the posterior flagella was shorter (7.0 ± 0.8 µm) (n = 50). The distal end of the anterior flagellum tapered to a short narrow tip (apical portion, AP); however, in the case of the posterior flagellum, the distal end started to taper at about two-thirds of the length of the posterior flagella, and ended with a narrow tip. The anterior flagellum had a clear unilateral array of tinsel, distributed in-between, approximately 1 µm from the flagellum base up to the front of the AP (Fig. 2B). The tinsels were 1.3 ± 2.9 µm long and appeared at 0.3 ± 0.1-µm-long intervals,

2.2. Transformation of zoospores to trophozoites

The light microscopy and FE-SEM images, respectively, depicting zoospores transforming into trophozoites, are shown in Figs. 3 and 4. On the third day following discharge, most zoospores exhibited high motility, but some zoospores transformed into non-motile trophozoites and grew larger (Fig. 3A). During the transformation from zoospores to trophozoites, SEM showed that the anterior flagellum was lost first, while the posterior flagellum was remained (Fig. 4A). At this stage, zoospores showed weak movement. Deflagellation took place at the junction between the basal part of the anterior flagellum and the body surface (Fig. 4B). Several detached anterior flagella were observed on the bottom of culture plate well. However, the detachment of posterior flagella was not observed. The trophozoites exhibited a spherical or irregular shape on the fifth day (Fig. 3A), followed by the appearance of trophozoites containing a typical signet-ring structure with an uncommonly large vacuole during the seventh to eighth days. During the transformation period, the length of the zoospore gradually decreased from 3.7 µm on the first day to 3.3 µm on the twelfth day. The diameter of trophozoites increased from 4.8 µm on the third day to 31.5 µm on the fifteenth day, as the transformation rate increased (Table 1). It took 15 days for 87% of zoospores to transform into trophozoites.

Table 1

Dimensions of *P. olseni* zoospores and trophozoites during transformation in culture medium.
Days after zoospores were released to the medium	Life stage	Percentage (%)	Size (µm) ± SD	Range (µm)	N
1	Zoospore	100 0	3.72 ± 0.36	-	60
1	Trophozoite	100 0
3	Zoospore	97 3	3.70 ± 0.39	-	55
3	Trophozoite	97 3	4.8 ± 1.2	2.1–8.1	40
5	Zoospore	96 6	3.45 ± 0.32	-	70
5	Trophozoite	96 6	8.0 ± 3.2	2.3–15.4	100
7	Zoospore	71 29	3.42 ± 0.38	-	70
7	Trophozoite	71 29	11.4 ± 4.9	3.2–23.3	100
9	Zoospore	64 36	3.26 ± 0.35	-	70
9	Trophozoite	64 36	15.3 ± 7.1	3.8–32.2	100
12	Zoospore	33 66	3.29 ± 0.34	-	55
12	Trophozoite	33 66	21.2 ± 9.2	4.2–46.2	100
15	Trophozoite (Schizont)	87	31.5 ± 15.5 28.2 ± 7	5.2–61.8 11.2–46.8	100 100
15	Merozoite	87	4.9 ± 1.4	1.4–7.8	100
SD, standard deviation.

2.3. Schizogony

The early-phase trophozoites were spherical or irregularly shaped (Fig. 5A). Their size gradually increased, and they had smooth surface structures once mature (Fig. 5B). Schizonts had internal divisions and released merozoites (Fig. 3B). Trophozoites enlarged and had cleavage furrows on the cell surface, indicating the early subdivision of the trophozoites, or the beginning of schizogony (Fig. 5B–C). As cleavage progressed, the number of daughter cells increased and progressed to the morula stage, with approximately 100 irregular-shaped merozoites being detected on the thirteenth day (Fig. 5D). When schizonts had fully developed, they released hundreds of merozoites, ranging in size from 1.4 to 7.8 µm (mean, 4.9 ± 1.4 µm; n = 100), following the abrasion or rupture of the schizont cellular membrane on the fifteenth day (Fig. 5E–F).

3.1. Clam sampling

Manila clams (R. philippinarum) were collected from intertidal flats of the Hongseong Region in the west coast of Korea, where heavy P. olseni infection has been recorded (unpublished data). Clams were collected in January 2011, brought to the laboratory, and reared in aquaria with recirculating sea water (temperature, 15–18 °C; salinity, 34 psu).

3.2. Isolation of prezoosporangia

The gill tissues of six clams were incubated in 40 mL Ray’s fluid thioglycollate medium (Sigma, USA) supplemented with 800 µL penicillin-streptomycin (10,000 units/mL; Gibco, USA) and 10 µl Nystatin-Chlorampenicol [0.1 g Nystatin (Sigma, USA) in 10 ml Chloramphenicol (Sigma, USA)] in a 50 mL conical tube and incubated in dark at 23 °C. After 60-h incubation, tissues were rinsed twice with sterile artificial sea water (SASW; 35 psu, filtered through a 0.2-µm filter and autoclaved), and were finely chopped using a sterile razor blade under aseptic conditions. The finely chopped tissues were suspended in 50-mL SASW, and prezoosporangia were separated from tissue particles by filtering the mixture through a sieve series of 100 µm, 50 µm, and 30 µm. The prezoosporangia retained on 50-µm and 30-µm sieves were thoroughly washed with SASW and mixed together in a 15-mL conical tube. They were then further purified by centrifugation (73 × g, 3 min). Purified prezoosporangia were identified as P. olseni by real-time PCR performed using a P. olseni-specific primer pair. In the experiment, we used the DNA of P. marinus and P. honshuensis as negative controls (data not shown).

3.3. Induction of in vitro zoosporulation

Induction of zoosporulation of P. olseni was conducted by introducing purified prezoosporangia into the culture media. The culture medium was prepared, as reported by Gauthier and Vasta [25], for in vitro zoosporulation, with minor adjustments to the ingredients. In brief, Dulbecco’s Modified Eagle Medium (Sigma, USA) and Ham’s F-12 (Gibco, USA) were mixed in a 1:2 ratio and added to an oceanic natural sea salt mix (Oceanic Systems, USA) dissolved in autoclaved distilled water. The mixture was buffered with 5 mM HEPES (Gibco, USA) and 3.5 mM sodium bicarbonate (Sigma, USA) and supplemented with 5% fetal bovine serum (FBS, Sigma, USA). The medium was further sterilized by filtration through a 0.2-µm syringe filter and was then stored at 4 °C until use (salinity, 35 psu; pH 7.2–7.4). The culture medium was fortified with antibiotics as mentioned as previously. In vitro zoosporulation was performed in 12-well plates by suspending prezoosporangia in the culture medium at 15,000 cells/mL, followed by incubation at 26 °C.

3.4. Transformation of zoospores to trophozoites and schizonts

Following the release of motile zoospores from mature zoosporangia, motile zoospores were aspirated with supernatant culture medium and transferred to a new 12-well culture plate. Transformation was observed at 26 °C. The percentage of zoospores that transformed to trophozoites was calculated by counting the number of trophozoites and zoospores in 10 random photographic images of culture plates that were taken every 24 h over 15 d by using LM.

3.5. LM and SEM

The in vitro cultured cells were observed under a LM and a SEM at the different life stages, including zoosporulation, transformation of trophozoites, and schizogony. For LM, a culture plate was observed using an inverted light microscope. Images were taken at different magnifications using a digital camera (Moiticam 2300, China) mounted on the microscope. For SEM, samples were obtained periodically after introducing prezoosporangia to the culture medium. Samples were fixed in 2.5% glutaraldehyde (Sigma, USA), incubated for 1 h at room temperature, and post-fixed for another 1 h at room temperature in 2% OsO₄ (Fluka, electron microscopy grade, USA). After incubation, the fixed samples were transferred to a 0.45-µm polycarbonate membrane filter (Advantec MFS, USA) that was mounted on a 25-mm vacuum filter holder, with a glass funnel and glass filter flask. The fixatives were rinsed by aspiration using 30- and 15-psu filtered artificial seawater, followed by rinsing with distilled water. Samples for Fe-SEM analysis of deflagellation of flagella were attached to a glass cover slip for 2 h. The other samples remained in the membrane filter. After washing, parasite cells were dehydrated with a graded series of ethanol by a routine method. The cells were critical point dried with CO₂ using a critical point dryer (Leica EM CPD030, Germany). Dried filters and cover slips containing cells were coated with platinum and examined under FE-SEM (Hitachi S4800, Japan) with an appropriate accelerated voltage. Cells were measured using Motic Images Plus 2.0 mL multi-media software (Motic, China). All measurements were expressed as mean $\pm$ SD (standard deviation).

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 [26] 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 [30]. 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 [37], 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 [14]. 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 [23]. 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.

Funding

This study is supported by the National Research Foundation of Korea; grant 2017R1D1A1B03028095, and the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (No.20183010025200).

Contributions

The research was designed by KIP, and DG and SHK performed the experiments. DG, KSC, CA and KIP wrote the manuscript. All authors read and approved the final manuscript.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Scanning Electron Microscopic Observation of the In Vitro Cultured Protozoan, Perkinsus olseni, Isolated from the Manila Clam, Ruditapes philippinarum

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Abstract

Background

Results

Conclusions

Figures

1. Introduction

2. Results

2.1. Zoosporulation of in vitro cultured P. olseni

2.2. Transformation of zoospores to trophozoites

2.3. Schizogony

3. Materials And Methods

3.1. Clam sampling

3.2. Isolation of prezoosporangia

3.3. Induction of in vitro zoosporulation

3.4. Transformation of zoospores to trophozoites and schizonts

3.5. LM and SEM

4. Discussion

Declarations

References

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