A new strain of Cryptocaryon irritans from the cultured olive flounder Paralichthys olivaceus

An obligate parasite, Cryptocaryon irritans, which is responsible for the white spot disease of marine fish is known to develop in the temperature regime over 19 degrees C. Recently, however, we found white spot disease of olive flounder Paralichthys olivaceus during winter at water temperatures ranging between 12 and 16 degrees C in Korea. In the present study we isolated a C. irritans-like ciliate from the affected fish and investigated its reproductive characters to compare the newly found ciliate with typical C. irritans. The newly found ciliate had an additional process in the reproductive stage, characterized by a budding before palintomic division, and it showed a higher ability to carry out tomitogenesis at a low temperature (16 degrees C) than at a high temperature (24 degrees C). Nevertheless, the present ciliates still had much in common with typical C. irritans with respect to clinical, histopathological, and morphological characters, suggesting that it is a new strain of C. irritans, adapted to lower water temperature.     

Characterization of Cryptocaryon irritans,a parasite isolated from marine fishes in Taiwan

The ciliated protozoan parasite Cryptocaryon irritans infecting marine fishes in Taiwan is described. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, the entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were investigated. A total of 5 isolates was obtained from different fish-host species and localities, the majority from cultured fish species. C. irritans from Taiwan is able to shift its developmental characteristics, i.e. from non-adherent to adherent tomonts, from individualistic to aggregate-forming tomonts, from infection of the gills only to infection of the gills and body. Thus, it is not possible to classify strains of C. irritans on the basis of these parameters. Premature tomonts that developed from dead fishes were able to produce theronts that could infect fish host. Isolates from Pingtung and the USA had identical nucleotide sequences while an isolate from Malaysia was identical to an Israel isolate. Percentage variation among pairs of Taiwan isolates showed a higher degree of variation than isolate sequences listed in GenBank. Sequence analysis revealed highly aberrant isolates in Taiwan, and a phylogenetic tree distinguished a marine and a low-salinity variant. C. irritans from marine fishes in Taiwan, therefore, display some characteristics not previously reported. Since manipulation of salinity in brackishwater ponds and marine cage sites is not feasible, there is a need to develop new strategies for the control and prevention of cryptocaryoniasis.     

Protective immunity in grouper (Epinephelus coioides) following exposure to or injection with Cryptocaryon irritans

The protective immunity of grouper (Epinephelus coioides) against Cryptocaryon irritans was determined after immunisation by surface exposure or intraperitoneal injection. Specific antibody titres of immunised fish serum and skin culture supernatant were determined by enzyme-linked immunosorbent assay and immobilisation assays. Specific antibody can be detected in some immunised fish at Week 1 and in all immunised fish at Week 2, and the peaks were between Weeks 4-6. Specific antibody was still evident in the serum and skin of immunised fish at Week 8, and provided good protection against challenge with C. irritans. These findings indicated that humoral and skin mucosal immunity play important roles in fish against C. irritans infection.     

Molecular cloning of a putative agglutination/immobilization antigen located on the surface of a novel agglutination/immobilization serotype of Cryptocaryon irritans

A surface agglutination/immobilization antigen was purified from the novel agglutination/immobilization serotype (serotype G37) of the ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes. Serum from fish immunized with C. irritans theronts had agglutination/immobilization activity against theronts in vitro. However, fish and rabbit antisera raised against serotype G32 (reported previously) caused little agglutination/immobilization of serotype G37 theronts. Immunological analysis indicated that the 37 kDa theront surface membrane protein may be the agglutination/immobilization antigen of this serotype. The full-length 37 kDa antigen cDNA contained 1171 base pairs, encoding a 331-amino acid protein with hydrophobic N- and C-termini, which are characteristically found in proteins containing a C-terminal glycosylphosphatidylinositol anchor. In addition, the genetically characterized nucleotide sequences of the first internal transcribed spacer region of ribosomal DNA of these 2 serotypes were compared. The internal transcribed spacer rDNA sequence of serotype G32 was identical to that of isolates from Pingtung, Taiwan, and from the USA. On the other hand, the sequences of serotype G37 were not identical to those of any C. irritans isolate.     

In vitro treatments for the theront stage of the ciliate protozoan Cryptocaryon irritans

The ciliate protozoan Cryptocaryon irritans Brown, 1951, the 'marine white spot', causes one of the most important parasitic fish diseases, with extensive losses every year in mariculture and in the ornamental fish industry. In the present study, we explore the in vitro use of 8 different compounds against the theront (infective) stage of C. irritans; these compounds include extracts of natural products (epigallocatechin gallate (EGCG), L-DOPA, papain), peracetic acid-based compounds (Proxitane 5:23 and 15% peracetic acid, PAA), quinine-based compounds (quinacrine hydrochloride and chloroquine diphosphate) and hydrogen peroxide. All of these compounds had an effect on theront survival; however, only EGCG caused significant theront mortality when applied in doses > or =50 mg l(-1) and over a period of 3 h; papain caused a maximum theront mortality of <50%. We discuss the type of application and potential utility of the compounds tested as part of a management control strategy for C. irritans infections in marine aquaculture and the ornamental fish industry.     

In vitro culture technique for Cryptocaryon irritans, a parasitic ciliate of marine teleosts

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.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

黄斑蓝子鱼皮肤黏液对刺激隐核虫及一些病原菌的抑杀作用

黄斑蓝子鱼(Siganus oramin)具有天然抗刺激隐核虫(Cryptocaryon irritans)能力, 并从其血清中分离纯化了一种抗虫蛋白。以黄斑蓝子鱼皮肤黏液为对象, 研究了其对刺激隐核虫(C. irritans)、多子小瓜虫(Ichthyophthirius multifiliis)、布氏锥虫(Trypanosoma brucei brucei)及一些病原菌的杀灭和抑制效果。结果显示: 黄斑蓝子鱼黏液对刺激隐核虫、多子小瓜虫和布氏锥虫均具有明显的杀虫效果, 黏液蛋白对3种寄生虫的最低杀寄生虫浓度(Minimum Parasiticidal Concentration, MPC)分别为4.0、5.0和3.0 mg/mL。显微镜观察发现, 刺激隐核虫和多子小瓜虫的幼虫在经过黄斑蓝子鱼黏液作用后, 均出现纤毛脱落、虫体肿胀、外膜破裂和内容物泄漏等现象。黏液抑菌活性实验结果表明, 除大肠杆菌(Escherichia coli)外, 黄斑蓝子鱼皮肤黏液对金黄色葡萄球菌(Staphylococcus aureus)、霍乱弧菌(Vibrio cholerae)、海豚链球菌(Streptococcus iniae)、温和气单胞菌(Aeromonas sobria)、溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌(Vibrio Parahaemolyticus)、星形诺卡氏菌(Nocadia asteroides)和美人鱼发光杆菌杀鱼亚种(Photobacterium damselae subsp.)皆有明显的抑菌效果。在最低抑菌浓度(Minimum Inhibitory Concentration, MIC)测定中, 黄斑蓝子鱼黏液对金黄色葡萄球菌、霍乱弧菌、海豚链球菌、溶藻弧菌、温和气单胞菌和副溶血弧菌的MIC 分别为0.16、0.16、0.08、0.63、0.63和0.63 mg/mL。黄斑蓝子鱼皮肤黏液对小麦赤霉菌(Gibberella saubinetii)和黑曲霉菌(Aspergillus niger)没有明显抑菌作用。       

Assessment of Epinephelus coioides (Hamilton, 1822) aquaculture systems in the Gulf of Tonkin,Vietnam, by using fish parasites

The parasite fauna of Epinephelus coioides can be used as an indicator to assess different aquaculture systems. A total of 133 E. coioides were collected from fishermen (wild fish) and marine fish farms (net cages, pond farms) in the Gulf of Tonkin (Ha Long, Cat Ba and Nghia Hung), Vietnam in 2013 and 2014, and examined for the presence of protozoan and metazoan parasites. Different patterns of parasite infections were found. The monogenean Pseudorhabdosynochus spp. was predominant in the net cage system; and the protozoans Trichodina spp. and Cryptocaryon irritans were found exclusively in pond and net cage cultivation sites in Nghia Hung and Cat Ba. These three species were assessed in addition to seven further indicator species and two dominance indices, to visualize the potential of assessing different aquaculture systems in Vietnam. This suggests the use of former shrimp ponds for grouper cultivation, reducing the pressure to further extend net cage cultivation in already highly influenced mariculture regions in Vietnam.     

Specificity and specialization of congeneric monogeneans parasitizing cyprinid fish

Patterns and likely processes connected with evolution of host specificity in congeneric monogeneans parasitizing fish species of the Cyprinidae were investigated. A total of 51 Dactylogyrus species was included. We investigated (1) the link between host specificity and parasite phylogeny; (2) the morphometric correlates of host specificity, parasite body size, and variables of attachment organs important for host specificity; (3) the evolution of morphological adaptation, that is, attachment organ; (4) the determinants of host specificity following the hypothesis of specialization on more predictable resources considering maximal body size, maximal longevity, and abundance as measures of host predictability; and (5) the potential link between host specificity and parasite diversification. Host specificity, expressed as an index of host specificity including phylogenetic and taxonomic relatedness of hosts, was partially associated with parasite phylogeny, but no significant contribution of host phylogeny was found. The mapping of host specificity into the phylogenetic tree suggests that being specialist is not a derived condition for Dactylogyrus species. The different morphometric traits of the attachment apparatus seem to be selected in connection with specialization of specialist parasites and other traits favored as adaptations in generalist parasites. Parasites widespread on several host species reach higher abundance within hosts, which supports the hypothesis of ecological specialization. When separating specialists and generalists, we confirmed the hypothesis of specialization on a predictable resource; that is, specialists with larger anchors tend to live on fish species with larger body size and greater longevity, which could be also interpreted as a mechanism for optimizing morphological adaptation. We demonstrated that ecology of host species could also be recognized as an important determinant of host specificity. The mapping of morphological characters of the attachment organ onto the parasite phylogenetic tree reveals that morphological evolution of the attachment organ is connected with host specificity in the context of fish relatedness, especially at the level of host subfamilies. Finally, we did not find that host specificity leads to parasite diversification in congeneric monogeneans.     

Expression profile of immune-related genes in Lates calcarifer infected by Cryptocaryon irritans

Cryptocaryon irritans causes Cyptocaryonosis or white spot disease in a wide range of marine fish including Lates calcarifer (Asian seabass). However, the immune response of this fish to the parasite is still poorly understood. In this study, quantitative polymerase chain reaction (qPCR) was performed to assess the expression profile of immune-related genes in L. calcarifer infected by C. irritans. A total of 21 immune-related genes encoding various functions in the fish immune system were utilized for the qPCR analysis. The experiment was initiated with the infection of juvenile fish by exposure to theronts from 200 C. irritans cysts, and non-infected juvenile fish were used as controls. Spleen, liver, gills and kidney tissues were harvested at three days post-infection from control and infected fish. In addition, organs were also harvested on day-10 post-infection from fish that had been allowed to recover from day-4 up to day-10 post-infection. L. calcarifer exhibited pathological changes on day-3 post-infection with the characteristic presence of white spots on the entire fish body, excessive mucus production and formation of a flap over the fish eye. High quality total RNA was extracted from all tissues and qPCR was performed. The qPCR analysis on the cohort of 21 immune-related genes of the various organs harvested on day-3 post-infection demonstrated that most genes were induced significantly (p < 0.05) in all tissues, particularly liver (11/21 genes) and kidney (11/21). The expression profile demonstrated that induction of the MHC Class IIα gene was the highest compared to the other genes followed by serum amyloid A, CC chemokine and hepcidin-2 precursor genes. In fish that were allowed to recover from the C. irritans infection (10 days post-infection), expression of the immune-related genes was down-regulated to levels similar to the control fish. These results provide insights into the interaction between C. irritans and L. calcarifer and suggest that the innate immune system plays an important role in early defence against parasite infection allowing the fish to eventually recover from the infection.     

Intraspecific Variation in Cryptocaryon irritans

ABSTRACT. Intraspecific variation in the ciliate Cryptocaryon irritans was examined using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA) combined with developmental and morphological characters. Amplified rDNA sequences consisting of 151 bases of the flanking 18 S and 5.8 S regions, and the entire ITS-1 region (169 or 170 bases), were determined and compared for 16 isolates of C. irritans from Australia, Israela and the USA. There was one variable base between isolates in the 18 S region nd 11 variables indicate that Australian C. irritans isolates from estuarine (Moreton Bay) and coral reef (Heron Island) environments are distinct. The Heron Island isolate was genetically closer to morphologically dissimilar isolates from Israel (1.8% divergence) and USA (2.3 % divergence) than it was to the Moreton Bay isolates. Three isolates maintained in our laboratory since February 1994 originated from the same source. During this time the sequence of the isolates from wild fish in Moreton Bay remained unchanged. These genetic differences indicate the existence of a founder effects in laboratorty populations of C. irritans . The genetic variation found here, combined with known morphological and developmental differences, is used to characterise four strains of C. irritans .     

Immune response of fishes to ciliates

Ciliates are highly evolved protists comprising a phylum of diverse species, many of which are opportunistic or obligate parasites. Ciliates parasitic to fish consist of salt and freshwater forms with endo- or ectoparasitic modes of infection. Some of the more commonly encountered genera include Chilodonella, Brooklynella, Ophryoglenina, Ichthyophthirius, Cryptocaryon, Uronema, Tetrahymena, Epistylus, and Trichodina. Species range from obligate parasites and commensals to opportunistic, facultative forms. Some parasitic ciliates are highly pathogenic and fishes in closed environments such as aquaria and farm ponds are particularly susceptible to high mortalities. Nevertheless, fish have evolved an immune system capable of mounting an effective protective response against parasite challenge. Much of the experimental research on immunity against ciliates has been carried out with Ichthyophthirius multifiliis, on obligate parasite that invades surface epithelia of virtually all freshwater fish species. Interest in the immune response against I. multifiliis stems from the fact that convalescent fish become resistant to subsequent challenge (suggesting the possibility of immunoprophylaxis), and the need to curtail severe losses caused by this parasite in intensively farmed fishes. Furthermore, I. multifiliis has proven to be a useful experimental model because it is amenable to study under laboratory conditions. In this review cellular and humoral factors involved in both innate and acquired immunity against ciliates are covered and include natural killer cells, phagocytic cells, and antibody responses. Current ideas on the mechanisms of antibody-mediated cutaneous immunity against I. multifiliis are discussed and approaches toward the development of vaccines against this and other ciliate parasites are presented.     

Communities of parasites of freshwater fish of Jersey, Channel Islands

The parasite faunas of 12 species of freshwater fish from 17 localities on the island of Jersey were examined. Comparison of the species composition, number, diversity and equitability of the parasite fauna of each species of fish in each locality revealed that community diversity was always low, that most communities were dominated by a single species of parasite, and that community similarity between host species, and often between sites, was generally low. This was not related to unavailability or distribution of potential, invertebrate, intermediate host species, but appeared to be due to chance colonization events. With two exceptions, little exchange of parasites took place between host species, despite vacant niches in the parasite communities and the absence of competitors, and normal parasite specificity was maintained. Comparison of the parasite communities with those on other oceanic islands suggests that they are poorer than would be predicted by island biogeographical theory, and that this is not a very good predictor of parasite community richness on oceanic islands.     

Host specificity of monogenean ectoparasites on fish skin and gills assessed by a metabarcoding approach

Monogeneans are highly diverse fish ectoparasites with a direct life cycle, widely distributed, and are known to generally display strict host specificity. Factors related to the hosts and the parasite have been suggested to explain this high specificity. Monogeneans have also been observed to colonise fish species not in their natural host range under experimental conditions. We developed a specific metabarcoding protocol and applied it on the Sparidae-Lamellodiscus host-parasite system, to assess parasite diversity on skin and gills of several sparid host species. We first demonstrated that the use of a metabarcoding approach provided a better understanding of the diversity of monogeneans associated with teleost skin and gills than traditional approaches based on morphological identification. We identified a high diversity of both expected and unexpected (never observed on this host species) Lamellodiscus spp. on each host species and on skin and gills. No significant difference in parasite diversity was found between skin and gills. These results suggest that the establishment of the observed host specificity in monogeneans relies on multiple levels of regulation, involving the survival capacity of the larvae and host recognition mechanisms.     

斜带石斑鱼L-氨基酸氧化酶基因克隆及表达分析

鱼类的L-氨基酸氧化酶(L-amino acid oxidase, LAAO)具有广泛的抑菌杀虫效果, 为了解斜带石斑鱼(Epinephelus coioides)LAAO基因序列特征及其在刺激隐核虫(Cryptocaryon irritans)感染后的表达变化, 该试验克隆得到2个石斑鱼LAAO基因: EcLAAO-1和EcLAAO-2, 它们的ORF长度分别为1536和1569 bp, 编码511和522个氨基酸, 均含有氨基酸氧化酶(Amino_oxidase)结构域以及LAAO保守序列: DBM和GG motif。多重序列比对显示石斑鱼LAAO与其他鱼类LAAO具有较高的相似性。系统进化树分析表明, 斜带石斑鱼的LAAO与硬骨鱼类亲缘关系较近。实时荧光定量PCR检测结果显示EcLAAO-1和EcLAAO-2在石斑鱼各组织均有表达, 其中皮肤、鳃、胸腺、肝脏和肌肉中含量较丰富; 在感染刺激隐核虫后, 鳃和脾脏EcLAAO-1, EcLAAO-2表达量显著升高(P<0.05), 这些结果暗示了石斑鱼LAAO参与先天性免疫, 并在抗御刺激隐核虫感染中发挥重要作用。     

Nestedness in assemblages of gyrodactylids (Monogenea: Gyrodactylidea) parasitising two species of cyprinid--with reference to generalists and specialists

The structure of gyrodactylid assemblages in individual fishes of two species of cyprinid was determined. A total of 100 specimens of minnow, Phoxinus phoxinus, and 137 specimens of roach, Rutilus rutilus, were investigated for presence of gyrodactylids. Host specificity, specialists vs. generalists, was noted in each host fish. A nested pattern was recorded in parasite assemblages of minnow, the host with a dominant number of specialist gyrodactylids. A non-nested pattern was observed in parasite assemblages of roach, the host with a dominant number of generalist gyrodactylids. The host specificity appears to be a meaningful factor that determines the pattern of gyrodactylid assemblages of both fish hosts.     

Experimental infections of the monogenean Gyrodactylus turnbulli indicate that it is not a strict specialist

Parasites represent a threat to endangered fish species, particularly when the parasite can host switch and the new host is vulnerable. If the parasite is highly host specific then successful host switching should be a rare occurrence; however, the host range of many parasites which are assumed to be specialists has never been tested. This includes the monogenean Gyrodactylus turnbulli, a well-studied ectoparasite found caudally on its known host, the guppy, Poecilia reticulata. In this study, we monitored parasite establishment and reproduction on a range of poeciliids and more distantly related fish. Individually maintained fish were experimentally infected with a single parasite and monitored daily to establish whether G. turnbulli could survive and reproduce on other fish species. Gyrodactylus turnbulli can infect a wider range of hosts than previously considered, highlighting the fact that host specificity can never be assumed unless experimentally tested. Our findings also have significant implications for parasite transmission to novel hosts and provide further insight into the evolutionary origins of this ubiquitous group of fish pathogens. Previous molecular evidence indicates that host switching is the key mechanism for speciation within the genus Gyrodactylus. Until recently, most Gyrodactylus spp. were assumed to be narrowly host specific. However, our findings suggest that even so-called specialist species, such as G. turnbulli, may represent a threat to vulnerable fish stocks. In view of the potential importance of host switching under artificial conditions, we propose to describe this as 'artificial ecological transfer' as opposed to 'natural ecological transfer', host switching under natural conditions.     

Experimental infections with the tropical monogenean,Gyrodactylus bullatarudis: Potential invader or experimental fluke?

Introduced exotic species have the potential to spread their associated parasites to native species which can be catastrophic if these hosts are immunologically naïve to the novel parasite. The guppy (Poecilia reticulata) has been disseminated worldwide outside of its native habitat and therefore could be an important source of infection to native fish species. Its parasite fauna is dominated by the ectoparasitic monogeneans, Gyrodactylus turnbulli and Gyrodactylus bullatarudis. The current study tested the host specificity of G. bullatarudis by experimentally infecting a range of isolated fish hosts, including temperate species. Surprisingly, the parasite was capable of establishing and reproducing, for several days, on the three-spined stickleback when transferred directly to this host. We also established that G. bullatarudis could be transmitted under aquarium conditions at both 25 °C and 15 °C. At the higher temperature, the parasite was even capable of reproducing on this atypical host. The implications of these findings are discussed in terms of host specificity, host switching and climate change.