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Effects of High Temperature and Dissolved Oxygen Concentration on the Development of Cryptocaryon irritans (Ciliophora) with a Comment on the Autumn Outbreaks of Cryptocaryoniasis.
Abstract
The effects of high temperature on the parasitic stage (trophont) and encysted stage (tomont) of Cryptocaryon irritans were examined. The effects on trophonts were examined by rearing fish artificially infected with C. irritans at 25, 28, 31 or 34°C. The effects on tomonts were examined by incubating tomonts at the same temperatures. In these experiments, both the trophonts and tomonts showed normal developments below 31°C, whilst their development was badly damaged at 34°C. The effects of dissolved oxygen on tomont development were examined. Tomonts were incubated in hyperoxic (141%), oxic (100%), hypoxic (24%) and anoxic (0%) conditions. Under the hypoxic and hyperoxic conditions, tomont development was suppressed. Under the anoxic condition, tomonts died. However, when tomonts were transferred into an oxic condition after 2 or 4- week incubation in the hypoxic condition, development resumed, showing a rapid increase in theront excystment rates 10 to 11 days after the transfer. These results show a possibility that the supply of oxygen into water bottom along with the disappearance of thermoclines is involved in the autumn outbreaks of cryptocaryoniasis of cultured marine fish in floating net cages located in inner bays.
... Trophonts can develop well in fish hosts between 18 and 32 C, and the time needed for trophonts to fully develop and leave host fish is temperature-dependent: 11 days at 18 C and 3-4 days at 24 C and higher. 36,37 Tomont development and subsequent theront excystation also occur between 18 and 32 C, and between 25 and 50 ppt in salinity. 31,37 The excystation is asynchronous even when tomonts are incubated under identical conditions. ...
... 31,37 The excystation is asynchronous even when tomonts are incubated under identical conditions. 36 Full development of tomonts are also temperature-dependent: the mean period needed is 11 days at 18 C, 5 days at 24-26 C and 4 days at 30-32 C. 37 The numbers of theronts released from a single tomont are estimated to be around 100-870 cells. 38 However, these biological characteristics also differ depending on the isolate and host species. ...
... Tomonts can become and stay dormant, without any development, under hypoxic conditions. 36,42 This phenomenon could help the development of other preservation methods for this parasite in the future. ...
Ciliates are considered the most harmful parasites of fish, causing mass mortalities and thus substantial economic losses to the aquaculture and ornamental fish industries. To better elucidate their pathogenic mechanisms and facilitate the development of effective prevention and control strategies, it is essential to apply novel technologies to ensure the continuous and sufficient supply of parasite specimens. This review discusses the latest research progress in the artificial culture of six important parasitic ciliates affecting both freshwater and marine fish globally: Ichthyophthirius multifiliis, Cryptocaryon irritans, Chilodonella (C. hexasticha, C. piscicolaand C. uncinata), Balantidium ctenopharyngodoni, Scuticociliatida gen. spp., and Tetrahymena spp. Methods for in vitro and in vivo culture as well as cryopreservation/preservation (depending on the specific species) of these organisms are described herein. Challenges and future applications of parasite culture are also discussed.
... The lower prevalence of C. irritans infections was observed in the fish captured during the autumn (64.94 %), while the lowest value occurredon the fish captured during the summer (39.29 %). According to Yoshinaga [14], who conducted a study on the impacts of temperature and dissolved oxygen levels on the development of C. irritans, temperatures for the optimal growth of most strains of Crypto caryon are between 23 and 30 °C [10,14]. The development of the parasite was significantly damaged at the temperature above 31 0 C and no parasite was recovered from any fish reared at 34 0 C. ...
... The lower prevalence of C. irritans infections was observed in the fish captured during the autumn (64.94 %), while the lowest value occurredon the fish captured during the summer (39.29 %). According to Yoshinaga [14], who conducted a study on the impacts of temperature and dissolved oxygen levels on the development of C. irritans, temperatures for the optimal growth of most strains of Crypto caryon are between 23 and 30 °C [10,14]. The development of the parasite was significantly damaged at the temperature above 31 0 C and no parasite was recovered from any fish reared at 34 0 C. ...
Wild-caught ornamental marine fish from NhaTrang (Khanh Hoa) and Ha Long (Quang Ninh) in Vietnam were examined over the three sampling batches corresponding to the spring, summer and autumn times for the prevalence of Cryptocaryon irritans. Out of a total of 211 fish (15 species), 143 (67.7 %) were found to be infected with the mean intensity of 7.67 parasites per field of view (x4 magnification). The prevalence of C. irritans in fish caught during the spring (91.0 3% on average) was significantly higher than that of the fish caught during the summer (39.29 %). A wide variation in the prevalence of the parasite was shown among the fish species. The highest prevalence and intensity of the infection occurred in Plataxteira, Diodon holocanthus, Paracanthurus hepatusat 100 % of infection and density of 12 parasites/field of view (x4 magnification) while and the lowest prevalence of C. irritans appeared on Rhinecanthus aculeatus, Zancluscornutus, and Zebrasoma veliferum with less than 50% of fish infected. Clinical signs of fish infected of C. irritans showed such as tiny white spots on skin, gills, and fins; ragged fins, changes in skin colour, cloudy eyes and increase mucus production.
... Another research on C. irritans established that low temperature and low oxygen stopped C. irritans development or instigated a dormant state, but restoring a suitable external environment revived the ciliate (Watanabe et al., 2018;Yoshinaga, 2001). In this experiment, abnormal salinity did not instigate C. irritans dormancy but slowed down ciliate development. ...
Cryptocaryon irritans are the main pathogens of “white spot disease” on the body surface of marine teleosts, but the seawater environment affects pathogen development and virulence. This study investigated C. irritans tomont formation, cell division, hatching, and infectivity to Larimichthys crocea under eight salinity gradients (S0, S5, S10, S20, S30, S40, S45, and S50) to understand how C. irritans grows, develops and harms host fish in a variable natural environment. The results showed that the protomont hatching rate in S5, S10, S20 and S30 significantly increased over time, but the highest and lowest values appeared in S20 and S30, respectively; and the hatching rate in S20 is significantly higher than S30.The rate of tomont formation increased over treatment time, and the highest and lowest rates were observed in S20 and S0 groups, respectively. Seawater at extreme salinity (S0, S40, S45, and S50) significantly reduced the encystment duration to <5 min. In addition, the tomont division rate at S5, S10, S20, S30, S40 and S45 significantly increased over time, and the highest and the lowest values were recorded in S20 and S45, respectively; and the division rate at S20 is significantly higher than S45. The hatching rate in S5, S10, S20, S30, and S40 also significantly increased over time, but the highest and lowest values appeared in S20 and S5, respectively; and the hatching rate in S20 is significantly higher than S5. The tomonts treated with seawater of 5–45‰ salinity divided evidently, but most tomonts under 0 and 50‰ salinity dissolved. TEM observation showed the pellicular alveoli of tomonts in S40 and S45 were wider than S20, the chromatin was loose, and the cyst wall was separately compressed into thin-layered layers. Finally, the aspect ratios of the theronts hatched at S5, S10, S20, S30, and S40 significantly increased than those at S20, and the theronts became long and thin. The infectivity of the theronts hatched from tomonts under S5 and S40 salinity treatments were significantly lower than the control (S20) because of the changes of theront number and morphology. In conclusion, salinity changes regulate the formation and development of C. irritans tomonts. Low (S5) or high (S40) salinity inhibits the infectivity of incubated theronts.
... As the medium of life for C. irritans, the physical and chemical seawater environment greatly affects its growth and development (16). Seawater parameters such as temperature, salinity, and dissolved oxygen significantly affect the life cycle and disease occurrence of C. irritans (17)(18)(19). For example, within a certain temperature range, the time needed for trophonts to parasitize the host and the time required for tomonts to hatch will decrease with increasing temperature (17). ...
Cryptocaryon irritans is a ciliate parasite that causes “white spot disease” in marine teleosts. The disease outbreak is influenced by hosts and a range of abiotic factors, such as temperature, salinity, and pH.
... This operation can be applied to cage culture. For example, a cryptocaryoniasis outbreak can be effectively controlled by moving diseased fish reared in a floating net cage from shallow to deep areas.58,79 Additional physical methods that have been used to treat C. irritans infection include ultraviolet sterilization, ozonation, drying and heat treatment.9Chemical ...
Cryptocaryon irritans (Brown, 1951) is an obligate parasite that mainly invades the skin, fins and gills of marine fish and causes marine ‘white spot disease’. It is common to aquarium and maricultured fish and causes high mortality due to its widespread geographical distribution, indiscriminate host specificity, short and simple life cycle, and high level of virulence. Numerous physical methods and chemical bath agents are applied to cure this disease in closed, re‐circulating aquaculture systems, but these treatments are not suitable in open environments such as cages and ponds. Fortunately, new measures, such as oral chemical drugs, oral plant components, vaccines and even biological control, are being developed as promising methods to treat and prevent cryptocaryoniasis in open aquaculture systems. Here, we provide a broad overview of C. irritans research, focussing primarily on work conducted in the last decade that may offer new insights into the treatment of disease in a practical setting.
... Congestion is generally followed by an increase in the number of eosinophil granule cells. Ressang (1984) Many papers have described a strong correlation between the quality parameters of rearing water and parasite infestation in aquaculture industries including temperature (Jansen & Bakke, 1991;Karvonen et al., 2013;Möller, 1978;Scott & Nokes, 2009;Strepparava et al., 2018), salinity (Cheung, Nigrelli, & Ruggieri, 1979;Ernst, Whittington, Corneillie, & Talbot, 2005;Möller, 1978;Rogowski & Stockwell, 2006;Soleng & Bakke, 1997), dissolved oxygen (Mikheev, Pasternak, Valtonen, & Taskinen, 2014;Molnar, 1994;Yoshinaga, 2001), pH and ammonia (Banu, Khan, & Thulin, 2004). However, to the authors' knowledge, publications concerning the correlation of Z. arugamensis infestation on groupers are still very limited, only a correlation with temperature (Kua et al., 2010(Kua et al., , 2014Mastuti & Zarman, 2018). ...
The marine leech, Zeylanicobdella arugamensis, is a major threat to aquaculture in grouper‐producing countries including Indonesia. This study aimed at investigating prevalence, intensity and histopathology of the ectoparasite in humpback and hybrid groupers cultured in different rearing systems. A total of 260 groupers (60 humpback groupers and 200 hybrid groupers) were used for samples. The marine leech was observed on skin, fins, gills and mouth, followed by histopathological assay on the skin tissue. The results showed that prevalence of the leech in both groupers was higher when they were cultured in the floating net cages compared with the hatchery, p < .05. Furthermore, humpback grouper had a higher prevalence than hybrid grouper when they were cultured in a similar system, p < .05. Meanwhile, there was no significant difference in intensity between the two groupers, p > .05. Within the hybrid groupers, the highest prevalence was obtained from hybrid groupers reared in the earthen ponds. Histopathological studies showed that the infected groupers exhibited inflammation, congestion and erosion of the epidermis layer. Hybrid grouper had more severe histopathological lesions in the skin tissues. These results suggested that species and type of aquaculture system had significantly determined the prevalence, intensity and severity of lesion in Z. arugamensis infestation.
... The transcriptome analysis carried out in the host fish in an infection dose-dependent manner demonstrated that low-concentration infection can significantly induce the complement and coagulation cascade pathway in host fish, while in higher concentration, the immunity status showed marked suppression(Yin, Gao, Tang, et al., 2016). The study based on the transcriptomes of the C. irritans tomonts under low temperature (by adopting the paired-end sequencing strategy and assembling the entire transcriptome de novo from three libraries such as tomonts at 12°C and 25°C and control), revealed the genes involved for tomont dormancy(Yin, Sun, Wang, & Gao, 2016). Another report based on the transcriptome analysis of piscidin-treated theronts and trophonts shows that most of the up-regulated genes in the treated ones are involved in cell migration and apoptosis(Chen et al., 2018). ...
This review paper deals with the immunological response in marine fish infected by Cryptocaryon irritans and discussed the practical difficulties in the development of potential molecular remedies. Browsing through the literature, it is understood that the development of molecular diagnostic tool to detect the early infection in fish by C. irritans is under progress. Once it is successfully established, the possible application of this technique could be suggested for even the non‐specific treatments such as copper‐based medications and bare‐bottomed quarantine of culture tanks. However, prior protection of healthy fish from C. irritans could be accomplished through proper immunization by infective theronts. In this case, a well‐suited in vitro culture technique is necessary to harvest theronts in large scale. Recent immunological and transcriptome studies predicted that the components related to local immune response are more effective against C. irritans than their systemic counterparts. However, in vivo experiments to prove the effect of these antiparasitic molecular components are meagre. Though molecular vaccines, developed from the well‐characterized 34 kDa immobilization antigen (iAg), responded well, these remedies fail to have a uniform impact against antigenically different strains (serotypes). More research is recommended to identify common protective epitopes from different serotypes. Also, studying the adjuvant effects of various innate responsive components like cytokines is significant. The suggested studies would be immensely helpful to develop effective vaccines against C. irritans infection and thereby optimize the aquaculture practices.
... The length of the entire life cycle varies, depending upon a number of factors, including strain of Cryptocaryon, temperature, salinity, and fish host [11]. Temperatures for optimal growth of most strains of Cryptocaryon appear to be about 23-30°C [26], although active infections at 15°C have been documented [12]. A more recent study demonstrated that the Cryptocaryon parasite can be found on the fish, and survived dormant for 4-5 months at 12°C [13]. ...
... Theronts are released from day 3 to day 35 or 38 after encystment, even when the tomonts are incubated under similar conditions. The development of the tomont is affected by temperature (Wilkie and Gordin, 1969), dissolved oxygen, and salinity (Yoshinaga, 2001;Coloni, 1985;Cheung et al., 1979). Strain-specific differences in the periods required for the release of theronts have also been reported (Diggles and Adlard, 1997;Jee et al., 2000). ...
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 tro-phonts, 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.
... Such a strategy can ensure the survivability of the hatched theronts, the infective stage, which appear to be sensitive to these conditions. Yoshinaga (2001) reported that dissolved oxygen (DO) levels affected the development of C. irritans tomonts, as low DO (24%) suppressed development, which was resumed once the tomonts were moved to oxic conditions, suggesting that anoxia only suppresses but does not kill tomonts. Interestingly, cyst development was also suppressed in hyper-oxic conditions (141% DO saturation). ...
Cryptocaryon irritans, a marine protozoan fish parasite with a life cycle that includes cyst formation, constitutes a major concern in intensive recirculating aquaculture systems (RASs), due to its potential propagation in a system with high fish density and minimal water change. In this study, the survival of C. irritans in sludge collected from an up-flow anaerobic sludge blanket (UASB) reactor of a RAS system was studied using a PCR assay. The detection sensitivity of the different life stages in sludge was determined to be 50 tomonts, 5 theronts and 5 trophonts. Dead C. irritans DNA was not detectable after 24 h of inoculation. The survival of the different C. irritans life stages in anaerobic conditions, as present in the UASB reactor, was evaluated. Theronts died within 3 h post-exposure to anaerobic conditions. Protomonts encysted within a day post-exposure, but unlike in aerobic conditions, the resulting tomonts did not hatch. Excystment of theronts from tomonts was delayed in anaerobic conditions, and occurred only after transfer to an aerobic environment. However, the tomonts did not die after 96 h of such exposure and the release rate of theronts did not decrease, compared to the control. The results suggest that infective theronts cannot originate from the UASB reactor and protomonts or theronts that reach the UASB do not survive. However, tomonts appear to be able to survive the reactor's conditions, and if they are released from it with the water outflow, they could hatch in aerobic parts of the RAS.
... In South China, C. irritans has afflicted farmed fish including grouper, sea bream, yellow croaker, pompano, and the direct economic loss due to cryptocaryoniasis has amounted to over one hundred million yuan RMB (equivalent to about 16 million US dollars) just in the Guangdong Province each year (data not published). Diversified treatments are available currently for the control of cryptocaryoniasis, such as the use of chemotherapeutic agents, fresh water soaking, and the cleaning of used equipment (Cardeilhac and Whitaker, 1988; Colorni and Burgess, 1997; Matthews et al., 1993; Rigos et al., 2013; Yoshinaga, 2001). However, these methods are not suitable for extensive use because of their drawbacks, such as drug residues, time-consumption, and high cost. ...
... Despite economic losses and stock mortality caused by Cryptocaryon irritans, few effective treatments or control measures have been developed for the parasite, especially for food fish cultured in open waters, although several chemotherapeutic agents are known to be effective for ornamental fish in closed environments (Colorni & Burgess 1997, Dickerson 2006. For net cage culture situations, the transfer of net cages containing infected fish to more open waters is mainly used and indeed is the only method currently available (Yoshinaga 2001). However, difficulties in the early detection of the parasite and in keeping areas pristine prior to transfer often make the therapy ineffective and unfeasible. ...
A medium for the in vitro culture of Cryptocaryon irritans, which is an obligatorily parasitic ciliate of marine teleosts and causes 'white spot disease', was developed. The medium consisted of a layer of cultured fish cells (FHM), with an agarose gel layer covering the cell layer. The agarose gel contained 0.22% agarose, 10% fetal calf serum, 100 I.U. ml(-1) Penicillin G potassium and 100 microg ml(-1) streptomycin sulphate. Theronts of C. irritans transformed to trophonts and grew to 180 microm in mean length in the medium, although they gradually decreased in number. When trophonts fully developed in medium were transferred into seawater 4 d after inoculation, approximately 70% of them transformed to encysted tomonts and released theronts. When fish were challenged with theronts obtained from in vitro-raised parasites, approximately 40% of the theronts were recovered from fish, indicating comparative infectivity of in vitro-raised theronts to those of in vivo-raised theronts. This is the first report that C. irritans fully developed in vitro and its entire life cycle was completed without a host fish.
Cryptocaryoniasis remains a major parasitic disease and economic challenge for marine aquaculture. Cryptocaryoniasis in marine fish is caused by Cryptocaryon irritans (Ciliata). A theront is a motile, free‐swimming stage in the life cycle of C. irritans , which is typically the infective stage that actively seeks out a host to initiate infection. Population density and growth rate of theronts were investigated in Nile tilapia, Oreochromis niloticus fed with citric acid‐supplemented feed. The experiment involved feeding three diets with graded levels of citric acid (0, control diet, 0.5, 1 and 1.5 g kg ⁻¹ diet), to seawater‐adapted Nile tilapia ( O. niloticus ) juveniles for 21 days. The results showed that citric acid in the fish feed had an impact on the theront number of C. irritans in a manner of dose‐dependent. In the experimental cohort administered a diet supplemented with 1.5 g kg ⁻¹ citric acid, the population density of theronts was observed to be significantly reduced, measured at 29 ± 3.34, as opposed to 473.34 ± 16.48 in the control group at the culmination of the experiment. The observed population growth rate of theronts was significantly higher in the control group than in the group administered the citric acid feed ( p < .005). The growth rate (r d ⁻¹ ) was 0.12 in control, 0.05 in 0.5 g kg ⁻¹ , 0.031 in 1 g kg ⁻¹ , and − 0.031 in 1.5 g kg ⁻¹ citric acid‐supplemented groups. Fish growth and feed conversion ratio were not affected by the citric acid in the feed. In conclusion, the findings of this investigation provide a valuable addition to our understanding of the potential protective effects of citric acid supplementation for fish against the C. irritans parasite. This is evidenced by the observed reduction in theronts present in the water.
In the aquatic environment, fish are subject to different pollutants and environmental variations that favour stress on animals, making them vulnerable to pathogens. Fish parasites are excellent biological indicators of environmental quality, as their parasitic population may increase or decrease in response to changes in water parameters and/or the presence of pollutants. Ectoparasites are considered excellent indicators of environmental quality because they have a monoxenic life cycle with high reproductive rates that have been responsible for the immediate response to changes in aquatic biota. In turn, parasites with a heteroxenous life cycle are also responsive to environmental variation because, with the mortality of intermediate hosts, their population can be easily changed. However, studies with endoparasites and their relation with the environment are more numerous, as they are accumulators of pollutants, especially trace elements, either via fish or through stages of development outside the host that also contribute to the accumulation of metals. Moreover, Digenea, Acanthocephala, Nematoda and Cestoda parasites can compete for trace elements that are bioavailable in the environment, as fish with a greater abundance of parasites have a lower concentration of toxic metals in their tissues. Furthermore, they have a high affinity for trace elements that are toxic to human and fish health (arsenic, mercury, lead and cadmium). This review is a comprehensive overview of the use of fish parasites as environmental sentinels and describes their potential as bioindicators of effect and accumulation, and evaluates their application in fish farms environments.
Cryptocaryoniasis is a severe disease of marine cage culture fish, caused by the protozoan Cryptocaryon irritans. However, the sources of C. irritans infection in cage culture farms are still unclear. Previous studies confirmed many cases of biological fouling on the nets of the culture cages. Therefore, we speculate that fouling may be a key infection source of the cryptocaryoniosis outbreaks. This study evaluated whether tomonts develop the dormant stage at different seawater depths and in the sludge. The results showed that the seabed and sludge tomonts exhibited low division rates on the first 3 days after entering the reproductive stage. However, no significant differences in the hatching rate of seabed and sludge tomonts were observed in comparison to the shallow water layer on the 6th day after entering the reproductive stage. The nets with different biological fouling biomass were placed at the base of a tank containing diseased fish. We established that the tomonts could adhere to the net clothing and that the adhesion rate positively correlated with the fouling biomass. In addition, we determined the number of theronts in the seawater at the base of the net cages containing diseased fish in a cage culture farm by a qPCR to detect the DNA of theronts. The number of theronts in the seawater near the bottom of cages with diseased fish was significantly higher than that in other areas. In general, net biofouling is a vital source of C. irritans infection in cage cultures. Our findings provide a theoretical basis for the prevention and control cryptocaryoniasis in the cage culture farms.
The adoption of new investigative strategies based on environmental DNA (eDNA) can be used to monitor parasites, associated bacterial microbiomes, and physical-chemical parameters in fish farms. In this study, we used the economically important and globally distributed fish ciliate parasite Cryptocaryon irritans as a model to understand the parasite abundance and potential drivers of its presence in marine fish farms. Environmental (rainfall) and physical-chemical (temperature, oxygen, salinity, pH) data collected from a marine fish farm in Hong Kong were analyzed together with the eDNA approach targeting C. irritans abundance based on digital droplet PCR and 16S metagenomics to determine associations and triggers between parasites and specific bacterial groups. Rainfall and temperature demonstrated positive associations with high abundance of C. irritans (eDNA) at the studied marine fish cage farm. However, rainfall was the only parameter tested that demonstrated a significant association with parasite eDNA, indicating that the raining season is a risky period for fish farmers in Hong Kong. Coraliomargarita was the bacterial genus with the most significant relationship with low abundance of C. irritans in water. Understanding the environmental triggers of ciliate parasites propagation and associated bacterial microbiome could elucidate new insights into environmental control, microbial management, and promote the reduction of chemical use in marine fish farms.
Parasitic infections are a severe issue in many regions of the world. We assume that if a chemical can destroy a DNA barcode sequence, then this chemical could be developed as a species-specific parasiticidal agent. To test this hypothesis, we designed sgRNAs that target the sequences of both a DNA barcode (ITS-2) and a control (5.8S rDNA) in Cryptocaryon irritans. In in vivo tests, we found that exposure to Cas9 mRNA mixed with sgRNAs was able to significantly reduce the hatching rate of tomont and the survival rate of theront. Quantitative Real-time PCR demonstrated that the DNAs of tomont and theront exposed to sgRNAs and Cas9 mRNA were significantly disrupted, no matter whether they were exposed to a single sgRNA or a mixture of two sgRNAs. DNA sequencing also suggested the test group that was exposed to a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments and the test group exposed to two sgRNAs combined with Cas9-induced deletion of large pieces. The findings and principles provided by this study contribute to the development of novel nucleic acid therapeutic drugs for cryptocaryoniasis and other parasitic diseases and provide insight into the development of species-specific parasiticidal agents.
Aquatic parasites may respond to various attractants and cues to find and infect a host. Traps that use these attractants as ‘bait’ have potential to reduce the number of pathogenic agents in aquaculture environments. This study examined four potential attractants (i.e., urea, host mucus, parasite conspecifics and light) and the response of two problematic marine parasite species, to identify the most suitable bait for trap development in finfish aquaculture. Two globally distributed parasite species (i.e., Neobenedenia girellae, (Hargis, 1955); and Cryptocaryon irritans, Brown, 1951) were chosen as models. A chemotaxis experiment was used to compare the attractiveness of each species' infectious life stage to urea, host mucus, parasite conspecifics and a seawater control, while a phototaxis experiment was used to identify phototactic responses of the parasites to light or dark. We found that urea and light attracted more than twice the number of infective protozoans and flukes (monogeneans), respectively, compared to other attractants/controls. Cryptocaryon irritans theronts were positively chemotactic to urea (Beta Regression Analysis; Odds Ratio (OR) 2.69, p = 0.00017), while Neobendenia girellae was positively phototactic to light (Mixed Effect Logistic Regression; OR 2.5, p = 0.0014). A final experiment examined the emergence of C. irritans over a 24-hour period and identified that the vast majority excysted at night (ANOVA; p-value <0.001). In contrast, previous studies have shown that the majority of N. girellae oncomiracidia hatch in the morning. This indicated that the best time to deploy traps to capture infective C. irritans theronts and N. girellae oncomiracidia would be prior to sunset and sunrise, respectively. The manipulation of urea and light and other potential attractants combined with strategic deployment of traps to coincide with the emergence of infectious life stages may prove useful in aquaculture where parasite epidemics can compromise production and animal welfare.
We incubated tomonts of Cryptocaryon irritans in a hypoxic seawater environment (1.4–1.7 mg/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, 8.7–8.9 mg/L O2) for 1–4 days, their development stopped in 1 day. 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.
Cryptocaryon irritans is a parasitic ciliate that causes major economic losses in marine fish aquaculture globally. Despite the wide array of treatment methods, control of cryptocaryoniasis is still very challenging, especially in food fish culture. Thus, additional control methods against this parasite might be required to further reduce the occurrence of this disease. In this study, the swimming ability, excystment sequence, and distribution of theronts, the infective stage of C. irritans, were investigated in our effort to develop a physical control strategy. A video analysis for assessing the swimming ability of theronts showed diminishing mobility over time. The excystment of theronts primarily occurred during the dark period. Examination of the vertical distribution of theronts showed that they were mostly distributed at the 5 cm sampling point measured from the substrate, indicating that they have low upward swimming ability. From these results, we conclude that theronts possess limited mobility. Theronts also displayed an excystment pattern that might be influenced by photoperiod, since most theronts were released during the dark period of the day. Control strategies can be developed from these properties, such as increasing the water flow in a culture tank during the release period. Further, combined treatment methods against multiple stages of C. irritans can help minimize the occurrence of cryptocaryoniasis in culture facilities.
We developed a quantitative PCR assay for detecting the parasitic ciliate Cryptocaryon irritans, which causes “white spot disease” in marine fishes, from the natural environment. A specific primer set for C. irritans was designed and its high specificity was confirmed in silico: almost all of the sequences deposited in the GenBank nucleotide
database were covered, 22/23 for the forward primer and 7/7 for the reverse primer. We estimated that there were 3,415.9 rRNA
gene copies per genome of C. irritans. In artificial mixture experiments to validate whether the qPCR assay is applicable to natural samples, the estimated copy
numbers showed significantly positive correlations with the number of theronts added (p<0.001). When we applied this qPCR assay to natural samples collected bimonthly from surface and bottom seawaters at an
aquaculture site (water depth, 10m) from May 2009 to March 2010, we only detected C. irritans (112.0±6.3cells/l) in the surface seawater sample in November. This qPCR assay is a useful tool for detecting C. irritans rapidly and quantitatively in natural environments; it could also help advance our understanding of the ecology of C. irritans, as well as facilitate the diagnosis of the disease.
Keywords
Cryptocaryon irritans
–Quantitative PCR (qPCR)–Natural aquaculture environment
A method for the propagation of the parasitic ciliate Cryptocaryon irritans, with black mollies Poecilia latipinna as the host fish, is described. A stable infection has been maintained for 2 years in this system. Parasites remained on fish for at least 3 d before leaving and secreting cysts. Parasites divided within cysts to form infective theronts, which began exiting on day 4. All theronts left the cysts during the same time of day: 0200–0900 hours. When fish were exposed to theronts that had been outside the cyst for less than 3.5 h, 67.2% of the parasites could be recovered from the fish. The recovery rate was less than 4% when fish were exposed to theronts older than 6 h.
The parasitic, reproductive, and free living phases of Cryptocaryon irritans Brown 1951, a ciliate parasite of marine fish, were studied by means of transmission and scanning electron microscopy. The ciliature of this protozoan is arranged in 78-80 monokinetid meridians which run lengthwise converging at the oral cavity and at the posterior pole of the cell. In the trophont, a crown of pointed ciliar triplets fused at the tip delimits a small cytostome whose radially ridged walls lead to a shallow cytopharynx. The trophont feeds on whole host cells and tissue debris. An electron-dense, foam-like, PAS-positive substance fills the pellicular alveoli of the growing trophont. The mechanism of its formation is yet to be determined and several possible functions for it are hypothesized. The macronucleus in the young trophont consists of four linked bead-like segments twisted in a crescent-shaped alignment; up to five micronuclei are adjacently located. At this stage, the macronucleus is homeomeric. Along with trophont growth, the macronucleus increases in volume and its coarse network of chromatin expands. As the trophont leaves the host, development proceeds onto the protomont and tomont stages, during which a substantial reorganization occurs in the cell. The dense chromatin clumps apparently coalesce while the electron-lucent matter expands and the four macronuclear segments fuse into one thick, elongated strand which coils throughout the protoplasm. The micronuclei are no longer detectable in the protomont. The tomont then begins to undergo palintomic division, yielding scores of tomites. In the tomite, the macronuclear chromatin bundles are thin and abundant within the electron-lucent matrix. The micronuclei reappear. Following excystment, the emerging infective theront actively seeks out its host. At this stage its oral apparatus appears as a narrow slit surrounded by cilia shorter than the somatic ones, and is presumably not yet functional. The macronucleus is homeomeric again, has assumed its characteristic quadripartite shape with adjacent micronuclei.
The course of infection of the parasitic ciliate Cryptocaryon irritans was followed on Lates calcarifer and Macquaria novemaculeata at 20 and 25 C. The parasite was originally isolated from locally caught Acanthopagrus australis. At 20 C trophonts stayed on the fish longer, tomonts took longer to excyst, and the resulting theronts were larger than at 25 C. On L. calcarifer at 20 C, trophonts grew slowly at first but eventually became significantly larger (mean tomont diameter 466 x 400 microns) than at 25 C (mean diameter 373 x 320 microns). On M. novemaculeata, trophonts never grew as large as on L. calcarifer and at 20 C they grew poorly. The number of theronts produced per tomont was directly related to the size of the tomont but was not influenced by incubation temperature. The tomont incubation period was not related to the diameter of the tomont but was significantly influenced by the host origin of the tomont. Theront size was also significantly affected by the host origin of the tomont but not the diameter of the tomont. These results show that C. irritans exhibits variability in morphometrics on different hosts and under different temperature conditions. This variability needs to be taken into account if utilizing morphometric data for separating strains of C. irritans.
White spot disease by Cryptocaryon irritans broke out among cultured Japanese flounder (Paralichehys olvivaceus) in October 1983, in Mie Prefecture. About 1000 2-years old fish died of this disease within three days. Multiple white spots with a pin point size were observed in the gills. The histopathological examination revealed that many parasites penetrated under the epithelia of gill filament and lamellae causing hyperplasia of epithelial cells and mucous cells at the parasitic areas. © 1985, The Japanese Society of Fish Pathology. All rights reserved.
Epizootics of the holotrichous ciliate, Cryptocaryon irritans, have impeded monoculture of red snapper, Lutjanus campechanus, at the Florida Department of Natural Resources facilities in St. Petersburg, Florida. Cryptocaryoniasis was associated with most mortalities and was controlled only in one group of red snapper being conditioned for induced spawning. An effective treatment regime which has prevented recurring Cryptocaryon infections included hypersalinity, quinine hydrochloride, chloroquine, and minimal handling.
The life cycle and halotolerance of Cryptocaryon im-tans were studied. Trophonts completed their growth phase on the host in 3 to 7 d. The reproductive process of the tomonts yielded tomites as early as 3 and as late as 28 d. Tomite life span was 24 to 48 h. Trophonts embedded in the fish skin withstood 18 h exposure of the host to fresh water. Tomonts withstood salinities ranging from 20 to 70 %O for 48 h without losing viability. Tomites were seen emerging only from tomonts kept in constant salinities ranging from 25 to 50 %o. All tomonts degenerated when immersed in 0 to 10 %O for 3 h. Eradication of the parasite based on these findings was achieved. Four consecutive treatments of 3 h each in 10 %O salinity 3 d apart destroyed all tomonts before tomites could excyst and were well tolerated by the fish. C. imitans life cycle was similarly interrupted by 4 consecutive transfers of fish into clean tanks at 3 d intervals. A certain degree of resistance to C. irritans invasion was observed in fish that had survived several infections.