The Prevalence of Cryptocaryon irritans in wild marine ornamental fish from Vietnam

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.


Introduction
Ornamental fishes are receiving an increased attention due to the growth of local and global demand and the consequent bloom of small and medium commercial aquariums and marine museums for leisure activities in Vietnam. A vast number of aquatic species have been successfully adapted to live in the aquarium. Marine ornamental fishes aremore colorful and shapely. These fish groups are, therefore, more attractive to aquarists. However, ornamental fishes kept in aquariums are susceptible to numerous diseases particularly parasitic diseases. Cryptocaryon irritans infections in fish are a significant disease problem for marine aquarists and commercial marine culture worldwide. Cryptocaryon irritans, a ciliated protozoan parasite, is one of the most devastating parasites of marine fish cultured in temperate and tropical areas.This parasite is an obligate ectoparasite that causes Cryptocaryonosis, also known as the white spot disease in marine fish [1]. Although C. irritans is commonly found in tropical, subtropical and warm temperate waters at low infection intensity [2], infection by this parasite has emerged as a major problem in confined environments, such as in marine culture and aquariums due to the buildup of the parasite and high population density of fish in these systems [3].
Cryptocaryon irritans has a direct life cycle [4]. This means infection can spread within a group of 2 1234567890 ''"" fish without the need for another animal host for development. However, the life cycle is complex. It includes 4 stages that develop on and off the fish, which is briefly described by Yanong [5] as follows: The trophont is the "feeding stage," during which the parasite is found embedded within the tissues of the fish. After the trophont leaves the fish, it becomes a protomont before encysting and transforming into a to mont, or "reproductive stage." The tomont develops and divides into numerous to mites, which eventually leave the cyst as the ronts, the free-swimming infective stages.Theronts actively seek fish hosts for a new life cycle. The length of the entire life cycle varies, depending upon a number of factors, including the strain of Cryptocaryon, temperature, and fish host [4,[6][7][8][9]. Even for a specific strain of the parasite and fish host, the life cycle may vary by weeks or months [4]. An average life cycle appears to be 1 to 2 weeks. However, life cycle durations may range from 6 days to 11 weeks, primarily because of the unpredictability of tomont development [4,9,10].
The most commonly observed stage is the trophont or "feeding" stage. The trophont is found on the fish, usually underneath the outer skin layers. Spherical or pear-shaped, with cilia all over its body, the trophont will normally be seen "rolling" or rotating slowly under the epithelium. Because the trophont is embedded within the skin, it is relatively protected from any potential treatments [5].Trophonts can range in size from about 48 x 27 μmto 452 x 360 μm. The trophont feeds on the body fluids and cells of the fish for about 3-7 days before leaving the host. Trophonts will also actively leave fish that have died, but are not immediately infective. They require additional time to develop from protomonts to tomonts, just as they would if leaving a live host [5].
Although numerous studies have been conducted on various aspects of C.irritans, information on infections related to this parasite on marine ornamental fish in Vietnam is very limited. The report aims to present the current results and updated information on the infection of C.irritans on wildcaught marine ornamental fish in Vietnam. The study was conducted at Vinpearlland Aquarium Times City,which is one of the largest commercial marine aquaria in Vietnam.

Methodology
The study was conducted from 2/2016 to 12/2016 through three sampling batches corresponding to the times of the spring, summer and autumn of the year. In total 211 fish of 15 marine ornamental fish species captured from Ha Long and Nha Trang were examined (table 1). The fish were transported by closed system oxygen packed polythene bags [11]. After transportation, the fish were held in separated recovery tanks to examine the infection of Cryptocaryon irritans according to the method described by Dogiel (1929) cited by Ha Ky and Te [12]. During this acclimation period, infected fish were also visually observed in terms ofexternal signs and behaviour changes. To inspect the presence of the parasites, mucus samplesfrom gills and skin of the fish were taken and then smeared onto clean microscope slides to observe under the microscope with magnification powers of x4, x10, x40 and x100.
Based on the collected data, the prevalence and intensity of infection by seasons and fish specieswere calculated as follows: Prevalence of infection = number of infected fish/total number of fish inspected x 100 % Infection intensity = total number of parasites on fields(4x10)/total number of fields with the presence of parasite. The data were collected and analysed on Excel 2010.

Results and discussion
In the study, a total of 211 marine ornamental fish of 15 species were sampled at 2 -3 sampling batches corresponding to the spring, summer and autumn time. Three to ten fish individuals from each fish species were sampled for each sampling batch. Most of the fish used in the study were captured from NhaTrang -KhanhHoa (almost 90 %); other small numbers (10 %) were sourced from Ha Long -QuangNinh (table 1). The prevalence of Cryptocaryon irritans infections ranged between 33.3 and 100 % for the 15 fish species examined with the overall prevalence of 66.8 % (table 2).Previousresearch by Diggles and Lester [7] has reported the similar prevalence (66.7%) of C. irritans infection on marine wild caught fish in Australia. However, a much lower prevalence (22.44 %) of freshwater "Ich" infecting on juvenile grass carp has been found by Van   Wide variationsof the prevalence of C. irritans infections in fish were shown among the sampling batches for each fish species (Table 3). Fish caught in the spring infected by C.irritansare at the highest prevalence of 91.03% on average with various fish species infected at 100% of prevalence such as C. adiergastos, P. hepatus, A. frenatus, P. volitans. 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. These could be the reasonsrelated to the variation of the higher prevalence of parasite infections on fish captured in the spring compared to the summer. A similar trend of growth was also reported on I. multifiis, a freshwater parasite by Van According to Duijn [15], and on Centrocestus formosanus, a trematode parasite infectingcommon carp [11].
There was a significant difference in the prevalence of C. irritans infection among the fish species from the same sampling batch. The extremely high prevalences of infection on the fish groups of lionfish and long-spine porcupinefish were observed at 85.56 -100% while the other groups of fish such as Rhinecanthus aculeatus, Zebrasoma veliferum and Zanclus cornutus were infected withC. irritansat less than 50 % of prevalence. The variation of the prevalence of infections among species could be due to the difference in susceptibility of the fish to the parasites [4,6,16,]. The same results were also reported by Diggles and Lester [7] when these authors examined the infection of this parasite on wild-caught fish from Australia. Differences in the susceptibility of wild hosts may be due to unequal exposure of hosts to infective stages because of differences in host behaviour or ecological niche [7].
Regarding infection intensity, the present results show the mean intensity of C. irritans on ornamental fish, which is 7.64 parasites per field of view (4x10 magnification) (  [7] reported a much lower intensity of C.irritans at 12.9 parasites per infected fish on wild fish. Another study conducted by Van et al. Accprding to Van et al. [13], on infection of Ichthyophthirius multifiliison juvenile of grass carp informed an infection intensity of6.68 parasites per field of view. The infected fish with a mean intensity higher than 10 parasites per field of view showed clear white spots on the skin, gills and eyes (figure 1). At the advanced stages of infection, these white spots were visible to naked eyes. Other typical signs were pale fills, ragged fins, changes in skin colour, cloudy eyes and increased mucus production. The behaviours of the infected fish also changed including hanging at the surface or at the sites of incoming water, acting lethargic, scratching (flashing) or swimming abnormally, and rapidly breathing due to the presence of the parasites on gills leading to damages of gill tissue. At this stage, sporadic to mass mortalityof infected fish occurred. Similar typical signs of infected fish were described in various studies on infection of C.irritans [4,17].
Under the microscopes, the parasites appeared in a round, spherical or oval shapes, measuring from 180 to 700 µm, depending upon the age of infection and stage of maturity (figure 2). Throughout the body of the infected fish, the parasite emerged within the epidermis of skin and also in the epithelia of gills. Invasions of subepidermal tissues have previously been attributed to advanced stages of the disease when underlying tissues are exposed by erosion and peeling of the epithelium.

Conclusion
It is clear that Cryptocaryon irritans is common all yearround in ornamental marine fish sampled in Vietnam. However, the prevalence and intensity of the infection vary widely with the fish host and captured seasons. Some species are very susceptible to the parasite infection (up to 100 % infected) while others are infected at a much lower rate (less than 50 % infected). A seasonal variation in the prevalence and intensity of C. irritans infectionwas also clearly presented. The highest prevalence of infection occurred during the spring and the lower values were shown in the summer. These findings provide important information which can be employed by aquarists in keeping and treating this parasitefor ornamental fish in a confined condition.