Mucosal IgM Antibody with d-Mannose Affinity in Fugu Takifugu rubripes Is Utilized by a Monogenean Parasite Heterobothrium okamotoi for Host Recognition.

How parasites recognize their definitive hosts is a mystery; however, parasitism is reportedly initiated by recognition of certain molecules on host surfaces. Fish ectoparasites make initial contact with their hosts at body surfaces, such as skin and gills, which are covered with mucosa that are similar to those of mammalian guts. Fish are among the most primitive vertebrates with immune systems that are equivalent to those in mammals, and they produce and secrete IgM into mucus. In this study, we showed that the monogenean parasite Heterobothrium okamotoi utilizes IgM to recognize its host, fugu Takifugu rubripes Oncomiracidia are infective larvae of H. okamotoi that shed their cilia and metamorphose into juveniles when exposed to purified d-mannose-binding fractions from fugu mucus. Using liquid chromatography-tandem mass spectrometry analysis, proteins contained in the fraction were identified as d-mannose-specific IgM with two d-mannose-binding lectins. However, although deciliation was significantly induced by IgM and was inhibited by d-mannose or a specific Ab against fugu IgM, other lectins had no effect, and IgM without d-mannose affinity induced deciliation to a limited degree. Subsequent immunofluorescent staining experiments showed that fugu d-mannose-specific IgM binds ciliated epidermal cells of oncomiracidium. These observations suggest that deciliation is triggered by binding of fugu IgM to cell surface Ags via Ag binding sites. Moreover, concentrations of d-mannose-binding IgM in gill mucus were sufficient to induce deciliation in vitro, indicating that H. okamotoi parasites initially use host Abs to colonize host gills.

Screening of candidate genes encoding proteins expressed in pectoral fins of fugu Takifugu rubripes, in relation to habitat site of parasitic copepod Caligus fugu, using suppression subtractive hybridization.

Caligus fugu is a parasitic copepod specific to the tetraodontid genus Takifugu including the commercially important Takifugu rubripes. Despite the rapid accumulation of knowledge on other aspects of its biology, the host and settlement-site recognition mechanisms of this parasite are not yet well understood. Since the infective copepodid stage shows preferential site selection in attaching to the fins, we considered it likely that the copepodid recognizes chemical cues released or leaking from the fins, and/or transmembrane protein present on the fins. To isolate molecules potentially related to attachment site specificity, we applied suppression subtractive hybridization (SSH) PCR by identifying genes expressed more highly in pectoral fins of T. rubripes than in the body surface skin. We sequenced plasmid DNA from 392 clones in a SSH library. The number of non-redundant sequences was 276, which included 135 sequences located on 117 annotated genes and 141 located in positions where no genes had been annotated. We characterized those annotated genes on the basis of gene ontology terms, and found that 46 of the identified genes encode secreted proteins, enzymes or membrane proteins. Among them nine showed higher expression in the pectoral fins than in the skin. These could be candidate genes for involvement in behavioral mechanisms related to the site specificity shown by the infective copepodids of C. fugu.

DNA Methylation Analysis Reveals Potential Mechanism in Takifugu rubripes Against Cryptocaryon irritans Infection.

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection.

Functional role of TrIL-1ß in Takifugu rubripes defense against Cryptocaryon irritans infection.

The Japanese puffer, Takifugu rubripes, is a commercially important fish species in China that is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. We previously found that interleukin-1ß (IL-1ß), an important cytokine with a potential role in resistance against pathogens, was one of the most significantly differentially up-regulated proteins in the gills and spleen of T. rubripes infected by the protozoan parasite Cryptocaryon irritans. In this study, we assessed the potential function of T. rubripes IL-1ß (TrIL-1ß) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1ß protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1ß may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1ß promoted the expression of endogenous IL-1ß, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1ß group had less tissue damage compared with control groups of fish. IL-1ß-small interfering RNA and IL-1ß overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1ß regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1ß also promoted the differential expression of IgM, showing that it was involved in humoral immunity of T. rubripes. The cumulative mortality experiment show that TrIL-1ß could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1ß. They also suggested that recombinant TrIL-1ß could be used as an adjuvant in a subunit vaccine against C. irritans infection, which is of profound importance for the prevention and control of parasitic diseases in T. rubripes.

l-fucoside localization in the gills of the genus Takifugu and its possible implication in the parasitism of Heterobothrium okamotoi (Monogenea: Diclidophoridae).

BACKGROUND: The monogenean parasite Heterobothrium okamotoi only parasitizes the gills of Takifugu rubripes. In this study, we hypothesized that the carbohydrates contribute to high host specificity of H. okamotoi. METHODS: T. rubripes, T. niphobles, T. snyderi, and T. pardalis were used for UEA I staining of the gills and an in vivo challenge test against H. okamotoi. To examine the effect of l-fucose, an in vitro detachment test was conducted using the host's gills. Additionally, fucosylated proteins were isolated from the membrane proteins of T. niphobles gills. RESULTS: The location of l-fucoside and the infection dynamics in four species were correlated to some extent; H. okamotoi detached relatively quickly from T. niphobles possessing l-fucoside both on the surface of the gills and in certain types of cells, including mucus cells, but detached slowly from T. snyderi possessing l-fucoside in only certain types of cells, including mucus cells. Under the conditions examined, H. okamotoi exhibited minimal detachment from T. rubripes and T. pardalis, and l-fucoside was not detected. The significantly higher detachment rate of H. okamotoi from the host's gills incubated in l-fucose-containing medium compared with the controls suggests that l-fucose in the non-host gills induced detachment of H. okamotoi. Four fucosylated proteins, including mucin5AC-like, were identified as potential factors for the detachment of H. okamotoi. CONCLUSIONS: Fucosylated proteins covering the surface of non-host gills might contribute to H. okamotoi detachment. GENERAL SIGNIFICANCE: This research shows the possible involvement of oligosaccharides in the host specificity of monogenean parasites.

The lunule of caligid copepods: an evolutionarily novel structure.

Nearly half of the genera of the family Caligidae possess an evolutionarily novel structure called the "lunule" on the ventral surface of the frontal plate. Lunules are paired cup-like suckers that assist in securing attachment of the copepod parasite to its host. Although present in genera such as Caligus and Pseudocaligus, lunules are absent in other caligid genera such as Lepeophtheirus as well as in more primitive caligiforms such as members of the families Trebiidae and Dissonidae. We compared the morphology and development of the anterior margin of the frontal plates between two caligids, Pseudocaligus fugu and Lepeophtheirus sekii, and a more basal caligiform, Dissonus heronensis (a dissonid), using scanning electron, transmission electron, and laser confocal microscopes. Our observations suggest that the lunules originated as a modification of the marginal membranes of the ancestral frontal plates. We also demonstrated the presence of an anlagen cell population for the lunule and marginal membrane in the developing frontal plate. These primordial cells can be detected as early as the first stage of the chalimus phase. Based on these observations, an evolutionary scenario for the lunule is proposed based on cytological evidence. This case study enhances our understanding of "evolutionary novelty," which is a main focus of contemporary evolutionary developmental biology.

Genomic selection for heterobothriosis resistance concurrent with body size in the tiger pufferfish, Takifugu rubripes.

Parasite resistance traits in aquaculture species often have moderate heritability, indicating the potential for genetic improvements by selective breeding. However, parasite resistance is often synonymous with an undesirable negative correlation with body size. In this study, we first tested the feasibility of genomic selection (GS) on resistance to heterobothriosis, caused by the monogenean parasite Heterobothrium okamotoi, which leads to huge economic losses in aquaculture of the tiger pufferfish Takifugu rubripes. Then, using a simulation study, we tested the possibility of simultaneous improvement of parasite resistance, assessed by parasite counts on host fish (HC), and standard length (SL). Each trait showed moderate heritability (square-root transformed HC: h2 = 0.308 ± 0.123, S.E.; SL: h2 = 0.405 ± 0.131). The predictive abilities of genomic prediction among 12 models, including genomic Best Linear Unbiased Predictor (GBLUP), Bayesian regressions, and machine learning procedures, were also moderate for both transformed HC (0.248‒0.344) and SL (0.340‒0.481). These results confirmed the feasibility of GS for this trait. Although an undesirable genetic correlation was suggested between transformed HC and SL (rg = 0.228), the simulation study suggested the desired gains index can help achieve simultaneous genetic improvements in both traits.

D-mannose-specific Immunoglobulin M in Grass Puffer (Takifugu niphobles), a Nonhost Fish of a Monogenean Ectoparasite Heterobothrium okamotoi, Can Act as a Trigger for its Parasitism.

Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.

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.

Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae).

Neobenedenia girellae, a monogenean skin parasite, shows low host specificity. N. girellae is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. To clarify the mechanisms of host specificity, we purified and identified the attachment-inducing substances of oncomiracidia from tiger puffer fish. The attachment-inducing substances were mainly included in skin mucous extract. Skin mucous extract lost its ability to induce attachment after boiling and/or exposure to the reducing agent dithiothreitol, suggesting that attachment-inducing substances are of a proteinaceous nature. Since lectins such as Con A, WGA, PHA-L, and PSA inhibited the induction of attachment, attachment-inducing proteins were suspected to be glycoproteins. Glycoproteins specifically interacting with Con A were collected and purified by anion exchange chromatography, resulting in two active peaks (peaks 3-A and 6). The active component in peak 3-A was identified as Wap 65-2 by N-terminal amino acid sequencing, while the glycoprotein in peak 6 could not be identified. These results suggested that oncomiracidia recognised Wap 65-2 and another glycoprotein of their host.

Involvement of host recognition by oncomiracidia and post-larval survivability in the host specificity of Heterobothrium okamotoi (Monogenea: Diclidophoridae).

Heterobothrium okamotoi, a monogenean gill parasite, shows high host specificity for tiger puffer, Takifugu rubripes. In the present study, in vivo and in vitro experimental infections were conducted using various fish species, including T. rubripes, to understand the mechanisms of specificity. In in vivo experiments, T. rubripes, grass puffer, Takifugu niphobles, olive flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, were exposed to oncomiracidia of H. okamotoi labelled with a fluorescent dye, 5- (and -6) carboxyfluorescein diacetate succinimidyl ester, and the numbers of parasites on the gills and skin were recorded at intervals. Oncomiracidia were attached to gills and skin of all the experimental fish species immediately after exposure, and the infection intensity on T. rubripes was higher than that on T. niphobles and much higher than those on the other two species. After 2 days, the attached parasites remained on the gills of T. rubripes, but disappeared from the other hosts. During in vitro experiments, gill filaments excised from seven different fish species (four fish species used in the in vivo experiments and panther puffer, Takifugu pardalis, southern flounder, Paralichthys lethostigma and spotted halibut, Verasper variegates) were exposed to oncomiracidia and the attachment to each fish species and subsequent larval behaviour was observed. The percentage of post-larvae that attached to T. rubripes was slightly higher than those which attached to congeneric fish species and much higher than those of non-tetraodontid fish species. In vivo and in vitro experiments demonstrated that oncomiracidia of H. okamotoi have an affinity for their natural host, T. rubripes, and congeneric fish species. The disappearance of attached post-larvae from 'alien' hosts within 2 days during in vivo experiments suggested that host recognition by oncomiracidia and subsequent post-larval survivability are involved in the host specificity of H. okamotoi.

Isolation and characterization of bacteria from the copepod Pseudocaligus fugu ectoparasitic on the panther puffer Takifugu pardalis with the emphasis on TTX.

A total of 50 bacterial isolates was obtained from the copepod Pseudocaligus fugu, which is a common parasite, collected from the body surface of the panther puffer Takifugu pardalis. On the basis of colony characteristics, these bacterial isolates were grouped into six types, of which only two (Types-I and -II) showed a high affinity for adhesion to the carapace of the banana shrimp Penaeus merguiensis. These two types of adhesive bacteria were identified through 16S rRNA sequence analysis as Shewanella woodyi (Type-I) and Roseobacter sp. (Type-II). Representative isolates of these two adhesive bacteria were examined for tetrodotoxin (TTX) production by high-performance liquid chromatography (HPLC)-fluorometric system, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). It was rather unexpectedly revealed that TTX and anhydroTTX were present in the supernatant of culture of the Type-II isolate Roseobacter sp.

Detection of tetrodotoxin (TTX) from two copepods infecting the grass puffer Takifugu niphobles: TTX attracting the parasites?

In May 2002, two parasitic copepods, Pseudocaligus fugu and Taeniacanthus sp., were collected from the body surface and gill of the grass puffer Takifugu niphobles, respectively, in Takehara city, Hiroshima Prefecture, faced with Seto Inland Sea located in the western part of Japan. To them was added 5 ml of 0.1% acetic acid, then the suspension was subjected to ultrasonic disruption with an ultrasonicator for 10 min. The resulting mixture was heated in a boiling water bath for 10 min, and then centrifuged. The supernatant was concentrated under reduced pressure, and loaded on to a Sep-Pak plus C18 Environmental Cartridge (Waters). The unbound fraction was analyzed by HPLC and gas chromatography-mass spectrometry (GC-MS) for tetrodotoxin (TTX). It was rather unexpectedly revealed from these results that this fraction was comprised of TTX and its analogues. As far as we know, this is the first record to show the existence of TTX in the copepods. In addition, relationships between the more and less than the average number of the two parasites and the toxicity of its skin mucus of the host were examined by student's t-test. In P. fugu, the average number per host was 13.9, and those are 520.7 (n=9) and 269.0 MU/g (n=22), respectively. A highly significant difference between them was detected at p-value 0.0011. In contrast, as for Taeniacanthus sp., the average number was 2.7, and those were 338.0 (n=14) and 345.5 MU/g (n=17), respectively. No significant difference was detected in Taeniacanthus sp. The high host-specificity of P. fugu on the toxic puffer and the present bioassay of its skin mucus suggest a possibility that TTXs may attract the parasite.

Identification and characterization of pufflectin from the grass pufferfish Takifugu niphobles and comparison of its expression with that of Takifugu rubripes.

Pufflectin found in Takifugu rubripes (Tr pufflectin) is the first animal lectin reported to show sequence similarity to monocotyledonous plant lectins. In the present study, we identified and characterized an orthologous lectin from Takifugu niphobles (Tn pufflectin), a species closely related to T. rubripes. Tn pufflectin exhibits 86% identity to Tr pufflectin with two conserved mannose-binding domains. Tn pufflectin was mainly expressed in the skin, gills, brain, and muscles; however, it was expressed at a lower level in the other examined tissues. Recombinant Tn pufflectin, expressed by Escherichia coli, exhibited binding activity specific for d-mannose. The expression of pufflectin in the gills was much lower in T. niphobles than in T. rubripes; notably, the former and latter are resistant and susceptible, respectively, to the monogenean parasite Heterobothrium okamotoi, which parasitizes gills. This suggests that pufflectin might be utilized by the parasite for host recognition.

Two new species of lepocreadiid trematodes of marine fishes from the Taiwan Straits, China.

Two new species of lepocreadiid trematodes are reported from marine fishes from the Taiwan Straits, China: Diploproctodaeum spinosus n. sp. from the intestine of Takifugu oblongus (= Fugu oblongus) and Preptetos parapristipomae n. sp. from Parapristipoma trilineatum. Diploproctodaeum spinosus differs from the known species of Diploproctodaeum, with 1 spine at the base of the ejaculatory duct. Preptetos parapristipomae is most similar to Preptetos rotto in having an entire ovary but differs from it in the sucker ratio, distribution of the uterus, position of the cirrus sac, and locality.

Variation in the small subunit ribosomal DNA confirms that Udonella (Monogenea: Udonellidae) is a species-rich group.

Numerous global reports of the species Udonella caligorum, currently thought to be a species complex, suggests that the group may be species-rich. Herein we describe Udonella fugu n. sp., previously described as U. caligorum, found on the parasitic copepod Pseudocaligus fugu infecting Takifugu spp. from Japan. Using morphological data U. fugu can be distinguished from the current valid species by at least one of the traditionally used characters in udonellid taxonomy, and phylogenetic analyses of ssrDNA sequence data for U. fugu and other udonellids confirm that U. fugu forms a distinct clade from other udonellids including U. caligorum. Variable regions in the ssrDNA demonstrated a range of between 2.75 and 5.5% difference between currently recognized species of Udonella. These differences in ssrDNA sequences are phylogenetically useful when distinguishing between morphologically similar udonellids and can be used in conjunction with other data (morphology, phylogeography and fish host) to help clarify udonellid systematics. Udonella fugu was also found to cause significant damage to farmed tiger puffers through their feeding activities. Individual skin lesions were round in shape but merged with adjoining lesions to form more extensive lacerations. In some of the specimens from P. fugu infecting Takifugu niphobles, the protozoan ciliate Trichodina was found on the udonellid body surface and in their intestinal contents. We conclude that the udonellids are a more species-rich group than currently recognized, that early descriptions of new species may have been synonymized with U. caligorum in error and that the frequent global reports of U. caligorum may actually represent new species. This has led to a wide range of morphological descriptions for U. caligorum, blurring the usefulness of morphological data for the group.

Immobilization antibodies of tiger puffer Takifugu rubripes induced by i.p. injection against monogenean Heterobothrium okamotoi oncomiracidia do not prevent the infection.

We examined whether infection by the monogenean Heterobothrium okamotoi induces production of specific antibodies against oncomiracidia and their cilia, larvae on the gills, and adults on the branchial cavity wall of tiger puffer Takifugu rubripes. We also investigated whether specific antibody production participates in acquired protection against H. okamotoi. Sera from persistently infected fish immobilized H. okamotoi oncomiracidia 89 days after exposure and antibody levels (measured by enzyme-linked immunosorbent assays) in the sera against oncomiracidia and their cilia increased compared with sera from control (naïve) fish. Antibody levels in these sera against the larvae and adult stages did not increase. The number of H. okamotoi on persistently infected fish was significantly lower than for control fish (P<0.05) when persistently infected fish and control fish were exposed to oncomiracidia in the same tank. Thus tiger puffer produced specific antibodies against oncomiracidia and their cilia, and acquired partial protection against H. okamotoi. Intraperitoneal injection of proteins of sonicated oncomiracidia or their cilia with an adjuvant also produced oncomiracidium agglutination antibodies in sera from tiger puffer; the antibody levels in these sera against oncomiracidia and their cilia increased compared with sera from control fish (injection of BSA with an adjuvant) at 14, 44, and 75 days after the booster immunization. However, in a parasite challenge at 54-58 days after the booster immunization, the infection levels of fish immunized with parasites of sonicated oncomiracidia or their cilia were the same as the control fish. Western blot showed that sera from persistently infected fish and fish immunized with sonicated oncomiracidia or their cilia recognized similar antigenic bands, suggesting that tiger puffer tends to react against these antigens compared with other antigens. These results indicated that specific antibodies against these cilia and oncomiracidia induced by i.p. injection do not prevent H. okamotoi infection.

Temperature effects on the development of Enteromyxum spp. (Myxozoa) in experimentally infected tiger puffer, Takifugu rubripes (Temminck & Schlegel).

The effects of water temperature on the development of the enteric myxosporeans, Enteromyxum fugu and Enteromyxum leei, were investigated in experimentally infected tiger puffer, Takifugu rubripes. After naïve tiger puffer were fed gut tissue infected with both E. fugu and E. leei, they were divided into separate tanks and kept at different constant temperature regimes between 10 and 25 degrees C. Regardless of the water temperature tested, E. fugu was consistently detected with a high prevalence of infection (60-100%), although no sporulation occurred at 10 and 15 degrees C. Development of E. leei and the onset of disease were suppressed by low water temperatures (<15 degrees C). However, a temperature increase to 20 degrees C promoted the development of E. leei, followed by an increase of disease rate in the fish. The present study demonstrates that water temperatures below 15 degrees C have an inhibitory effect on the development of E. fugu and E. leei, resulting in suppression of enteromyxosis at low temperatures.