The protozoan parasite Perkinsus olseni actively proliferates within marine molluscan hosts and causes serious negative effects on mollusk production worldwide. Recent research revealed that one Japanese isolate requires exogenous glycine betaine (GB) for vegetative proliferation, whereas isolates from some congeneric species do not, suggesting that this requirement is a derived characteristic of this genus. In the present study, we established various isolates from Japan, Europe, and Oceania, and showed that P. olseni isolates from Japan and Europe commonly require exogenous GB, whereas Oceanian isolates do not. This suggests that Oceanian isolates may retain ancestral-like characteristics compared to Japanese and European isolates. Analyses of the non-transcribed spacer region of ribosomal DNA further support the above interpretation that the Oceanian isolates represent a more basal split, and additionally suggest that the Japanese and European isolates belong to lineages that have undergone relatively recent geographic expansion. Morphological comparisons among isolates showed that the Oceanian isolates possessed larger schizonts and trophozoites, producing fewer daughter cells, but formed larger hypnospores capable of producing a greater number of zoospores, although the association between GB requirements and morphological differences remains unknown. The present study demonstrates the existence of distinct lineages of this parasite and provides novel insights into the evolution, geographical dispersion, and shifts in life-history strategies of P. olseni.

Cryptocaryon irritans is an obligate parasitic ciliate and the causative agent of cryptocaryoniasis. This parasite is widely distributed in tropical and subtropical marine waters, and numerous outbreaks in aquaculture have been reported worldwide. Previous studies have documented variations in biological traits, such as cell size, serotype and environmental conditions for infection, including salinity and temperature, suggesting considerable intraspecific diversity. Molecular and serological variations have been reported in Japan; however, the extent of biological diversity remains unclear. Understanding this variation is critical because it may influence experimental outcomes and control strategies. In this study, C. irritans was collected from three geographically proximate coastal sites in Japan, and clonal cultures were established. Biological characteristics-including cell size, serotype, pathogenicity and development and infectivity under different salinity and temperature conditions-were compared to assess intraspecific variation. Genetic diversity was also evaluated using phylogenetic analysis. Biological characteristics differed even among isolates from nearby sites, indicating the presence of biologically distinct populations within Japan. Moreover, phenotypic comparisons and phylogenetic analyses showed that distinct traits were observed not only between genetically distant isolates but also within the same group, underscoring high intraspecific diversity. These findings may contribute to guiding future research and developing effective control strategies for cryptocaryoniasis.

Miamiensis avidus is a parasitic pathogen responsible for scuticociliatosis, a lethal infection affecting marine fish worldwide, including Japanese flounder. Immunisation with formalin-killed M. avidus has shown promise in inducing protective immunity, positioning it as a potential vaccine candidate against scuticociliatosis. However, challenges such as the high cost of producing sufficient cells and inconsistent quality due to the lack of cryopreservation methods hinder its development. In this study, we expressed M. avidus ciliary proteins in Tetrahymena, a culturable ciliate, and used these cells to immunise Japanese flounder. The immunised fish produced antibodies against M. avidus. Additionally, immunisation with two transgenic Tetrahymena strains, each expressing different ciliary proteins, induced the production of antibodies against two serotypes of the parasite. In challenge experiments, fish immunised with the transgenic Tetrahymena showed prolonged survival compared to the control group, highlighting the potential of this approach as a vaccine candidate. These findings suggest that transgenic Tetrahymena cells could be a viable platform for developing vaccines against multiple serotypes of M. avidus.

We examined the effects of light exposure on the theront excystment circadian rhythm in Cryptocaryon irritans using a newly invented apparatus, which enabled us to examine the excystment rhythms of theronts from tomonts with low labor. Using the apparatus, we examined the timings of theront excystment from tomonts exposed directly to light and from tomonts exposed to light-exposed seawater by counting the number of excysted theronts. We found that the theront excystment time changed only when tomonts were directly exposed to light, indicating that light reception is essential for circadian rhythm entrainment. When tomonts were exposed to light only once for 12 h, either on Day 1, Day 2, or Day 3 after leaving host and being encysted, the circadian rhythm was entrained according to the photoperiod given on tomonts. Tomonts exposed to a low light (1 lx) with 12L:12D photoperiod daily showed a circadian rhythm similar to that in tomonts exposed to an intense light (500 lx) under the same photoperiod. When tomonts were incubated at 22 °C, 25 °C, or 28 °C under the same photoperiod, almost the same circadian rhythm was developed, suggesting temperature has little effect on the circadian rhythm entrainment between the range, even though the date of excystment was delayed in lower temperatures. These results suggest the circadian rhythm of theront excystment can be entrained in tomonts on the seabed of inner bays where net-cage aquaculture is conducted, and be involved in the outbreaks of cryptocaryoniasis there.

Understanding the factors determining the host ranges of Perkinsus spp., a significant group of pathogenic protozoans affecting shellfish, is essential for preventing their spread and designing effective control measures. Considering that differences in the ability to proliferate within the host may influence the determination of host range, we first injected six Perkinsus spp. into Manila clams Ruditapes philippinarum and monitored the variations of trophozoite numbers. Although all six species were detected in the challenged clams 28 days post infection, the infection intensities varied among species, and particularly two species showed contrasting infection trends: P. mediterraneus showed a decreasing trend of infection, declining to the lowest intensity, whereas that of P. olseni continuously increased, reaching the highest intensity. In vitro exposure to Manila clam hemocytes revealed that the survival of P. mediterraneus trophozoites was suppressed, in contrast to P. olseni, which maintained their viability. Despite similar phagocytic indices for both species, the rate of phagosome acidification was significantly higher for hemocytes phagocytizing P. mediterraneus than those targeting P. olseni. Notably, phagosome acidification was significantly suppressed in hemocytes phagocytizing live P. olseni trophozoites, suggesting that P. olseni may secrete a substance that modulates phagosome acidification, and thereby evades intracellular digestion by the host’s hemocytes. Conversely, P. mediterraneus, with a lower affinity for infecting Manila clams, did not exhibit such modulation. Based on these results, we consider that the ability to modulate phagosome acidification in host hemocytes might be at least one factor in determining the host range of Perkinsus species.

Cryptocaryoniasis (marine white spot disease), caused by Cryptocaryon irritans, is a major threat to marine fish cultures in tropical and subtropical waters, and a serious nuisance to hobbyists with saltwater fish tanks. With only classical treatment schedules such as copper salts or hyposaline baths being available, control of the disease remains a challenge. In this study, we investigated the effect of Biokos, a viscosin-like lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on the external life stages of C. irritans, including theronts, protomonts and tomonts. The present study demonstrated that the compound has an antiparasitic effect on all tested external stages of the parasite. In particular, when Biokos was used at 48 mg/L, it was able to kill almost all theronts and protomonts within 1 h in in vitro experiments, and using the same concentration in an in vivo challenge experiment, the parasitic load was reduced by more than 95% compared to the control group with no Biokos. Additionally, cultured fish cells were able to proliferate, and fish showed no adverse signs at Biokos concentrations that were effective in killing the parasite. Thus, Biokos may be a promising way for preventing or reducing the burden of this parasitic disease in the future.

Cryptocaryoniasis, caused by Cryptocaryon irritans , is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila , a culturable ciliate, to develop a mimetic C. irritans . Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans , to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis , allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using “mimetic parasites”.

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a major threat to marine cage culture in tropical and subtropical waters; however, controlling the disease remains challenging. In this study, we constructed DNA vaccines encoding a cysteine protease of C. irritans (pcDNA3.1-cp2-full-myc and pcDNA3.1-cp2-partial-myc) and examined the protective efficacy of the vaccines. The results of the challenge experiment showed that the number of parasites recovered from fish immunized with the DNA vaccines was lower than that recovered from control fish (phosphate-buffered saline-injected and mock vector-injected groups); this difference was statistically significant when pcDNA3.1-cp2-full-myc was used for vaccination (p < 0.05). In addition, the cysteine protease was found to be relatively conserved among different isolates of the parasite. Thus, the protease may be a potential antigen candidate for the development of a vaccine against multiple strains of C. irritans.

Cryptocaryon irritans is an obligate parasitic ciliate of marine teleosts and is the causative agent of cryptocaryoniasis (marine white spot disease). The parasite infects the skin and gills of marine teleosts and often causes mass mortality in heavily infected fish. Therefore, this parasite is a major threat to marine aquaculture in tropical and subtropical waters, and effective control methods are required. Interestingly, it was reported that fish infected with the parasite acquired protective immunity against it, and it was suggested that a vaccine strategy could be realized. Since then, many researchers have attempted to develop vaccines against cryptocaryoniasis. This review presents the current research on vaccine development against cryptocaryoniasis to provide references for future experimental studies and propose future research directions.

In this study, we aimed to analyze the functions of highly expressed proteases at the theront (invasive stage) and trophont (parasitic stage) phases of Cryptocaryon irritans during the infection process using RNAi. We also analyzed the potential of these proteases as candidate antigens for vaccination against the parasite by infecting tiger pufferfish (Takifugu rubripes) with the recombinant proteases. Compared with the control group, RNAi against four proteases (including peptidases) genes reduced the number of parasites that infected and left the fish after complete development. The four recombinant proteases were then purified and evaluated as candidate vaccination antigens. The number of parasites recovered from fish immunized with the C. irritans recombinant proteins was lower than that recovered from control fish; this difference was statistically significant when a recombinant cysteine protease was used. These results suggest that the C. irritans proteases overexpressed at the theront and trophont stages are important for infection (including invasion, food uptake, digestion, and development) and can be targeted for the development of vaccines against cryptocaryoniasis.

We established a laboratory propagation method of Cryptocaryon irritans, a parasitic ciliate of marine fishes, with black molly Poecilia sp. as host fish, using small plastic aquaria. One cycle of the propagation usually takes one week. With this method, 1500–3000 protomonts are obtained from five challenged mollies every week, from which more than 100,000–200,000 theronts are obtained. Using this method, an isolate of C. irritans has been successfully maintained more than three years. This propagation method reduces labor for maintaining and propagating the parasite and will much contribute to researches on cryptocaryoniasis.•The method is a laboratory propagation technique of Cryptocaryon irritans.•Using small plastic aquaria and black molly as a host, the parasites can be stably propagated and maintained.•An isolate of C. irritans has been successfully maintained more than three years.

Cryptocaryon irritans has clear daily rhythms in protomont detachment from fish and theront excystment from tomonts. While the rhythms seem to be regulated by photoperiods, it has not been confirmed. We investigated whether the daily rhythms of the parasite would be controlled by giving different photoperiods of 12 h light and 12 h dark (06:00-18:00 L and 18:00-06:00 D; 15:00-03:00 L and 03:00-15:00 D) to infected fish and tomonts. Protomont detachment and theronts excystment mostly occurred in the last 3 h of the dark period and from 6 h to 3 h before the end of dark period, respectively, indicating that the rhythms are controlled by photoperiods.

Cryptocaryoniasis of marine teleosts is caused by Cryptocaryon irritans, an obligatorily parasitic ciliate. Proteases may play a crucial role in the infection and development of trophonts, the parasitic stage of C. irritans, and are therefore potential targets for chemotherapy and vaccines against the parasite. However, proteases involved in the infection of C. irritans have yet to be clarified or characterized. In this study, we examined the activity of trophont proteases by zymography and the effect of protease inhibitors on the infection, survival, and growth of the parasite in vitro and in vivo. In the zymography, serine and cysteine proteases showed high activities in trophont extract. When inhibitors against serine and cysteine proteases were added into the medium of the in vitro cultures of the parasite, survival declined and growth was delayed. In the in vivo experiment, when fish were challenged with the theront (invasive) stage of the parasite, which precedes the trophont stage, in seawater supplemented with a cysteine protease inhibitor E-64 group, the number of parasites recovered from fish were significantly lower than those from the negative control. These results suggest that serine and cysteine proteases are important for the parasitic stage of C. irritans. The findings of this study may contribute to the development of new chemotherapeutic drugs or vaccines for cryptocaryoniasis.

We incubated tomonts of Cryptocaryon irritans in a hypoxic seawater environment (1.4–1.7mg/L O2) (low dissolved oxygen; DO) and examined their development using a acetocarmine whole-mount staining method we developed for nuclear staining. They showed little development and stayed in the dormant phase in the hypoxic environment. When transferred into the hypoxic environment after incubation in an oxic environment (air-saturated, 8.7–8.9mg/L O2) for 1–4days, their development stopped in 1day. However, when dormant tomonts generated in the hypoxic environment were transferred to the oxic environment, they resumed development and released theronts. These results indicate that tomonts can become dormant when exposed to a hypoxic environment, but can resume development when exposed to an oxic environment at any developmental stage. When exposed to the oxic environment, tomonts recovered from 1-month dormancy and released as many theronts as control tomonts constantly incubated in the oxic environment. The infectivity of theronts from the recovered tomonts was similar to the control tomonts. Thermoclines prevent oxygen-rich surface seawater from reaching the bottom of water column and create a hypoxic sea floor environment in summer; these thermoclines are broken down in autumn or after typhoons. The long-term viability of dormant tomonts in hypoxic environments may be a key factor in the autumn outbreaks of cryptocaryoniasis in floating net cages in temperate waters.

We investigated the development of the macronucleus of Cryptocaryon irritans, and the ingestion and digestion of host cells by the parasite. All developmental stages of the parasite except the theront (trophont, protomont and tomont) were examined with histological staining and/or whole mount staining. The sections were also subjected to in situ hybridization targeting the 18S rRNA gene of the host fish. The macronucleus developed negligibly, maintaining its four-segmented shape in trophonts, elongated and formed a massive nucleus in tomonts before cell division, and subsequently underwent repeated divisions to generate theronts. Denatured host cells, examined in situ hybridization, were accumulated in trophonts, and disappeared in tomonts by the beginning of cell division. Denatured host cells were also observed in the host tissue surrounding trophonts. They had condensed nuclei. Protomonts filled with denatured host cells presented a 180-bp DNA ladder in gel electrophoresis, suggesting apoptosis of the host cells. These results indicate that DNA synthesis occurs exclusively in the early stage of the tomont and that host cells are fed and accumulated, probably as apoptotic cells, in the trophont and are digested in the early tomont stage.