Zoonotic human liver flukes, a type 1 biocarcinogen, in freshwater fishes: genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh.

BACKGROUND: Opisthorchiid flukes, particularly Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, and Metorchis spp. are the most common fish-borne zoonotic human liver flukes (hLFs). Liver fluke infections are more prevalent in resource-deprived and underprivileged areas. We herein estimated the prevalence of the metacercariae (MC) of major hLFs in common large freshwater fishes (lFWF) marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs. METHODS: The current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018-June 2022 using conventional and multiple molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (RFLP), sequencing, and bioinformatic analyses. The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared (χ2) test. RESULTS: The MC of C. sinensis, Opisthorchis spp., and Metorchis spp. were detected in 11 species of common and popular lFWF. In lFWF, the estimated prevalence was 18.7% and the mean load was 137.4 ± 149.8 MC per 100 g of fish. The prevalence was the highest (P < 0.05) in spotted snakehead fishes (Channa punctata, 63.6%). The highest rate of infection (P < 0.05) was observed with the MC of C. sinensis (11.8%). Metacercariae were almost equally (P > 0.05) distributed between the head and body of fishes. The infection rate was slightly higher in cultured (19.6%) fishes. The MC of C. sinensis, O. felineus, O. viverrini, and Metorchis orientalis in fishes were confirmed using PCR, PCR-RFLP and bioinformatics. The cercariae of opisthorchiid (Pleurolophocercus cercariae) flukes were only recovered from Bithynia spp. (3.9%, 42 out of 1089). The ova of hLFs from dogs (4.3%, 5 out of 116) and cats (6.0%, 6 out of 100), and adult flukes (M. orientalis) from ducks (41.1% 113 out of 275) were detected. CONCLUSIONS: The MC of hLFs are highly prevalent in fresh water fishes in Bangladesh. Reservoir hosts, such as street dogs, cats, and ducks carried the patent infection, and residents of Bangladesh are at risk.

MOLECULAR PHYLOGENY OF THE LEECH GENUS PONTOBDELLA (HIRUDINIDA: PISCICOLIDAE) WITH NOTES ON PONTOBDELLA CALIFORNIANA AND PONTOBDELLA MACROTHELA.

Leech specimens of the genus Pontobdella (Hirudinida: Piscicolidae) were found off the coast of the state of Oaxaca (Pacific) as well as in Veracruz and Tabasco (Gulf of Mexico), Mexico. Based on the specimens collected in Oaxaca, a redescription of Pontobdella californiana is provided, with emphasis on the differences in the reproductive organs with the original description of the species. In addition, leech cocoons assigned to P. californiana were found attached to items hauled by gillnets and studied using scanning electron microscopy and molecular approaches. Samples of Pontobdella macrothela were found in both Pacific and Atlantic oceans, representing new geographic records. The phylogenetic position of P. californiana is investigated for the first time, and with the addition of Mexican samples of both species, the phylogenetic relationships within Pontobdella are reinvestigated. Parsimony and maximum-likelihood phylogenetic analysis were based on mitochondrial (cytochrome oxidase subunit I [COI] and 12S rRNA) and nuclear (18S rRNA and 28S rRNA) DNA sequences. Based on our results, we confirm the monophyly of Pontobdella and the pantropical distribution of P. macrothela with a new record in the Tropical Eastern Pacific.

PREVALENCE OF OPECOELID TREMATODES (TREMATODA: DIGENEA: OPECOELIDAE) IN THE WOOLLY SCULPIN CLINOCOTTUS ANALIS FROM THE PACIFIC COAST OF THE BAJA CALIFORNIA PENINSULA.

Prevalence values for 3 digenean species of the family Opecoelidae were calculated during a half-year period from 20 individuals per month of the fish species Clinocottus analis, collected from an intertidal environment of Baja California, Mexico. Trematode species recovered were Opecoelus adsphaericus, Opecoelus cameroni, and Opecoelus pacificus. Of these, only O. adsphaericus was present throughout the study, whereas O. pacificus and O. cameroni were recorded for 1 or 2 mo, respectively, exhibiting relatively low prevalence values. The decrease in prevalence of O. adsphaericus coincides with the appearance of O. pacificus and O. cameroni; these last 2 species were found only in the largest hosts, and their presence represents new records for Mexican marine fauna.

Phylogenetic position of Ancyrocephalus (sensu lato) curtus Achmerov, 1952 (Monopisthocotylea, Dactylogyridae), a parasite of fish Perccottus glenii Dybowski, 1877(Gobiiformes: Odontobutidae).

The genus Ancyrocephalus sensu lato is a large assemblage of species of dactylogyrid monopisthocotyleans without clear taxonomic boundaries. Despite an urgent need for revision, only three representatives of this taxon have been molecularly characterised so far. We found specimens of Ancyrocephalus curtus, a previously non-genotyped species, in gills of Perccottus glenii caught in the River Syumnyur, Amur Basin, Russia. The aim of this study was to assess the phylogenetic position of this parasite using partial sequences of 28S rRNA gene. In the phylogenetic tree, A. curtus appeared as a sister taxon to the dactylogyrine genus Gobioecetes. The new molecular evidence supports the hypothesis about the non-monophyletic status of Ancyrocephalus sensu lato.

Molecular and SEM studies on Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae).

Presenting new molecular and scanning electron microscope (SEM) features, this study gives additional data to the better knowledge of Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae), a parasite of the European catfish Silurus glanis Linnaeus, 1758 (Siluriformes, Siluridae) cultured in a commercial fish farm in Hungary. In addition, notes on the early development of sclerotized anchors are also provided. The main morphological difference of T. vistulensis compared to other congeneric species is associated with the male copulatory organ, which exhibits 5-7 loops in the middle of the penis length and a long open V-shaped sclerotized accessory piece, dividing terminally into two parts, securing the terminal part of the penis tube. The present study provides for the first time molecular characterization data based on the 2694 bp long nucleotide sequence of rDNA (ITS1, 5.8S, ITS2, and flanked with partial 18S and partial 28S) submitted in GenBank with the accession number OR916383. A phylogenetic tree based on ITS1 sequences supports a well-defined clade including T. vistulensis, forming a sister group with T. siluri, a species-specific monopisthocotylan parasite to S. glanis. The morphological characterization of T. vistulensis, especially for the male copulatory organ, together with the molecular data in the present study, extends knowledge about this monopisthocotylan species and provides new information for future phylogeny studies.

Identification of Lepidapedon oregonense as the current world's deepest trematode.

The deepest recorded depth for trematodes currently stands at approximately 6200 m. This depth record was achieved solely through sequence datasets of Lepidapedon sp. obtained from a gastropod. Given that trematodes of this genus typically use fish as definitive hosts, the origin of the trematode sequence was thought to be larval stages. However, the specific species remained unclear owing to the absence of reported adult-stage sequences. In the present study, we definitively identified the deepest trematode as Lepidapedon oregonense by comparing 28S ribosomal DNA sequences from adult worms from the macrourid fish Coelorinchus gilberti with data from the gastropod in the previous study.

Pathological caudal skeleton of an ichthyodectiform fish from the Upper Cretaceous Niobrara Formation of western Kansas, USA.

A series of 12 contiguous caudal vertebrae of an ichthyodectiform fish from the Smoky Hill Chalk Member of the Niobrara Formation is described. The vertebral centra exhibit extensive overgrowth of pathological bone and there is additional pathological bone within the centra and intervertebral spaces, which together resulted in the coossification of most centra. The extent of the pathology is greatest on preural vertebrae 1-3 and decreases anteriorly, which suggests that the pathology began posteriorly and progressed anteriorly. In addition to the pathological overgrowth on bones, the specimen preserves features interpreted as calcified and/or ossified soft tissues associated with the neural and haemal canals. The pathologies are unlike previously described examples of bony pathologies in fish, and it is suggested that they resulted from combined bacterial and fungal infections. As the pathologies developed, they would have adversely impacted the fish's swimming and feeding abilities, and presumably eventually led to the fish's death.

Associating with kin selects for disease resistance and against tolerance.

Behavioural and physiological resistance are key to slowing epidemic spread. We explore the evolutionary and epidemic consequences of their different costs for the evolution of tolerance that trades off with resistance. Behavioural resistance affects social cohesion, with associated group-level costs, while the cost of physiological resistance accrues only to the individual. Further, resistance, and the associated reduction in transmission, benefit susceptible hosts directly, whereas infected hosts only benefit indirectly, by reducing transmission to kin. We therefore model the coevolution of transmission-reducing resistance expressed in susceptible hosts with resistance expressed in infected hosts, as a function of kin association, and analyse the effect on population-level outcomes. Using parameter values for guppies, Poecilia reticulata, and their gyrodactylid parasites, we find that: (1) either susceptible or infected hosts should invest heavily in resistance, but not both; (2) kin association drives investment in physiological resistance more strongly than in behavioural resistance; and (3) even weak levels of kin association can favour altruistic infected hosts that invest heavily in resistance (versus selfish tolerance), eliminating parasites. Overall, our finding that weak kin association affects the coevolution of infected and susceptible investment in both behavioural and physiological resistance suggests that kin selection may affect disease dynamics across systems.

Ornamental fish mortality reveals an old parasite introduction: A case study of Koi carp and fish louse.

Koi carp are globally known for their colors and cultural significance. The introduction of these fish to new environments poses a threat to local biodiversity, in addition to releasing parasites, such as argulid ectoparasites. This study presents a record of Argulus japonicus infecting carp in an artificial lake in Southern Brazil using morphological and molecular methods, with a 100% prevalence (n = 3) and a mean intensity of 21.6 parasites per host, distributed over the body surface. The invasion history of hosts in the study locality indicates that the introduction of A. japonicus occurred decades before its first formal record in Brazil.

Myxobolus acanthogobii Hoshina, 1952 and Myxobolus selari n. sp. (Myxosporea: Myxobolidae) infecting brain of commercial fishes in Terengganu, Malaysia.

Myxosporean infection in marine water fishes has drawn less attention than in freshwater fishes, which resulted in a higher taxonomic variety in freshwater in Malaysia. This study aimed to address the gap by conducting a myxosporean survey on two commercially significant marine fish species, Nemipterus furcosus (Valenciennes) (Eupercaria incertae sedis: Nemipteridae) and Selar crumenophthalmus (Bloch) (Carangiformes: Carangidae), collected from the northeastern part of peninsular Malaysia. During the examination of the organs, two distinct Myxobolus Bütschli, 1882 species were discovered in the brain tissue of these fishes, despite the absence of any observable pathological signs. The two Myxobolus species were characterized through morphometry, morphology, and analysis of partial small subunit ribosomal RNA (18S rDNA) gene. As a result, Myxobolus acanthogobii Hoshina, 1952, which infects 2.3% of N. furcosus, is synonymous with a myxobolid species commonly found in Japanese waters, based on its morphological traits, tissue tropism, and molecular diagnostics. Furthermore, a novel species, Myxobolus selari n. sp., was described, infecting the brain of one (11%) individual S. crumenophthalmus. This unique species displayed distinctive features, placing it within a well-supported subclade primarily comprising brain-infecting myxobolids. Maximum likelihood analysis further revealed the close relationships among these brain-infecting myxobolids, underscoring the significance of tissue tropism and host taxonomy for myxobolids. This study represents the initial documentation of Myxobolus species within the southern South China Sea, shedding light on the potential diversity of marine myxosporean in this region. This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as urn:lsid:zoobank.org:pub:7C400E35-7CB8-4DEE-92B7-F75FF3926441.

Ectoparasites of the Critically Endangered green sawfish Pristis zijsron and sympatric elasmobranchs in Western Australia.

This study reports the metazoan ectoparasite fauna of juvenile Critically Endangered green sawfish, Pristis zijsron, and sympatric elasmobranchs in Western Australia. Five parasite taxa were found on 76 screened P. zijsron: Caligus furcisetifer (Copepoda: Caligidae), Dermopristis pterophila (Monogenea: Microbothriidae), Branchellion plicobranchus and Stibarobdella macrothela (Hirudinea: Piscicolidae), and praniza larvae of an unidentified gnathiid isopod. Only C. furcisetifer and D. pterophila were common, exhibiting discrepant site-specificity, with C. furcisetifer occurring mostly on the head and rostrum, and D. pterophila around the pectoral and pelvic fins. Intensity of infection for C. furcisetifer and D. pterophila increased with host total length and was influenced by host sex, but in opposite directions; intensity of C. furcisetifer was greater on female P. zijsron, whereas intensity of D. pterophila was greater on males. In the Ashburton River, likelihood of infection for C. furcisetifer and D. pterophila on P. zijsron increased with time since substantial freshwater discharge events, suggesting decreased salinity impacts both taxa. In addition to P. zijsron, five other sympatric elasmobranch species were opportunistically screened for ectoparasites in the study area: the giant shovelnose ray, Glaucostegus typus, the eyebrow wedgefish, Rhynchobatus palpebratus, the nervous shark, Carcharhinus cautus, the lemon shark, Negaprion acutidens, and the graceful shark, Carcharhinus amblyrhynchoides. Caligus furcisetifer was found on R. palpebratus; no other parasites of P. zijsron were found on other sympatric elasmobranch species. Conversely, Perissopus dentatus (Copepoda: Pandaridae) was found on all three carcharhinids but not on batoid rays (P. zijsron, G. typus or R. palpebratus).

Two proteocephalid cestodes in the fish Malapterurus electricus and Heterobranchus bidorsalis from Lake Nasser, Egypt: a morphological, molecular, and histopathological study.

Despite the importance of the electric catfish (Malapterurus electricus) and the African giant catfish (Heterobranchus bidorsalis) in the foodweb of Lake Nasser, Egypt, little is known about their diseases and parasitic fauna. This work describes, for the first time, cestodiasis in M. electricus and H. bidorsalis. Corallobothrium solidum and Proteocephalus sp. were identified morphologically and molecularly from M. electricus and H. bidorsalis, respectively. Using PCR, sequencing, and phylogenetic analysis, the two cestodes shared rRNA gene sequence similarities yet were unique and the two new sequences for the proteocephalid genera were submitted to the GenBank database. The prevalence of infection was 75% and 40% for the two fish species, respectively. Infections significantly increased in the summer and spring and were higher in female fish than in male fish. The intestine was the preferred site of the two adult cestodes. However, in the case of C. solidum some larval cestodes were found outside the intestine in between the skin and abdominal musculature, attached to the mesentery, and within intestinal tunica muscularis. Desquamation of the intestinal epithelium and inflammation at the site of infection in addition to congestion of the intestinal wall of the tapeworm infected fish were evident, indicating that C. solidum and Proteocephalus sp. impacted the infected fish. The larval stages of C. solidum attempted to penetrate the intestine and sometimes they were encircled within fibrous layers infiltrated with inflammatory cells. The infected fish's musculature was free of cestode infections. Preventive measures should be implemented to prevent the spread of infections.

The myxozoans Myxobolus cerebralis and Tetracapsuloides bryosalmonae modulate rainbow trout immune responses: quantitative shotgun proteomics at the portals of entry after single and co-infections.

Introduction: Little is known about the proteomic changes at the portals of entry in rainbow trout after infection with the myxozoan parasites, Myxobolus cerebralis, and Tetracapsuloides bryosalmonae. Whirling disease (WD) is a severe disease of salmonids, caused by the myxosporean M. cerebralis, while, proliferative kidney disease (PKD) is caused by T. bryosalmonae, which instead belongs to the class Malacosporea. Climate change is providing more suitable conditions for myxozoan parasites lifecycle, posing a high risk to salmonid aquaculture and contributing to the decline of wild trout populations in North America and Europe. Therefore, the aim of this study was to provide the first proteomic profiles of the host in the search for evasion strategies during single and coinfection with M. cerebralis and T. bryosalmonae. Methods: One group of fish was initially infected with M. cerebralis and another group with T. bryosalmonae. After 30 days, half of the fish in each group were co-infected with the other parasite. Using a quantitative proteomic approach, we investigated proteomic changes in the caudal fins and gills of rainbow trout before and after co-infection. Results: In the caudal fins, 16 proteins were differentially regulated post exposure to M. cerebralis, whereas 27 proteins were differentially modulated in the gills of the infected rainbow trout post exposure to T. bryosalmonae. After co-infection, 4 proteins involved in parasite recognition and the regulation of host immune responses were differentially modulated between the groups in the caudal fin. In the gills, 11 proteins involved in parasite recognition and host immunity, including 4 myxozoan proteins predicted to be virulence factors, were differentially modulated. Discussion: The results of this study increase our knowledge on rainbow trout co-infections by myxozoan parasites and rainbow trout immune responses against myxozoans at the portals of entry, supporting a better understanding of these host-parasite interactions.

Endoparasitic helminth fauna and diet of Geophagus sveni (Pisces) in Upper Paraná River basin.

In freshwater ecosystems, parasite infection patterns are influenced by factors including spatial-temporal variations, host diet, and habitat. Fish often change diets, affecting their parasite communities. This study focused on non-native host fish Geophagus sveni, aiming to characterize diet and endoparasitic helminth fauna patterns in the invaded area, investigating spatial and seasonal possible differences of endoparasite infections and correlating with host diet, in São José dos Dourados River and Tietê River areas. The host fish were collected in these areas during the dry and rainy season using gillnets. The endoparasites were collected and preserved in alcohol and identified using taxonomic methods, and stomach contents were examined for diet analysis. Parasitism descriptors were calculated and evaluated spatially and seasonally by ANOVA and the Kruskal-Wallis tests. PERMANOVA assessed G. sveni diet differences, and RDA correlated the endohelminth abundance with the host diet. Two endoparasites were recorded: metacercariae of Austrodiplostomum compactum (Trematoda) and larvae and adults of Raphidascaris (Sprentascaris) lanfrediae (Nematoda). Spatial differences were observed for the mean abundance and prevalence of R. (S.) lanfrediae and A. compactum prevalence. Seasonal variations of parasitic descriptors occurred for the nematode in the Tietê River area. The detritus and aquatic insects were the most consumed items by G. sveni. Detritus consumption positively correlates with nematode abundance. The findings indicate that factors such as artificial channels and rainfall, which can influence resource availability, may affect the fish's diet and potentially influence the structure of its endoparasite community. The study emphasizes the importance of understanding trophic chain-transmitted parasites and calls for further research in Neotropical environments.

White spots amidst the gold: ultrastructural and histological aspects of the chronic inflammatory response of goldfish with ichthyophthiriasis.

Ichthyophthirius multifiliis, the causative agent of white spot disease, is a ciliated protozoan parasite that infects freshwater fish and induces high mortality. Outbreaks occur both in natural and production sites. The aim of the present study was to describe the lesions caused by chronic infection by I. multifiliis in goldfish (Carassius auratus) from an ornamental fish farm, highlighting important ultrastructural aspects of this protozoan. Damaged skin and gills, collected from fish with white or ulcerative skin lesions, were routinely processed for histological analysis and transmission electron microscopy. The parasitic forms present in the skin were associated with an inflammatory infiltrate consisting of macrophages, lymphocytes and other polymorphonuclear cells. The lesions associated with the presence of the parasite were organized in the form of granulomas, with macrophages in the layers closest to the parasites. A trophont-thickened membrane and induction of granulomatous inflammation were identified in this study as mechanisms for evasion of the immune response. We concluded that the presence of I. multifiliis trophonts resulted in the formation of granulomatous inflammation, whether associated or not with pathogen lysis, suggesting that the parasite can use an inflammatory response to evade the immune response.

Understanding the host response of farmed fish to blood flukes (Trematoda: Aporocotylidae) for developing new treatment strategies.

Aporocotylids (Trematoda: Digenea), also known as fish blood flukes infect the circulatory system of fish leading to serious health problems and mortality. Aporocotylids are a particular concern for farmed fish as infection intensity can increase within the farming environment and lead to mortalities. In the context of managing these infections, one of the most crucial aspects to consider is the host response of the infected fish against these blood flukes. Understanding the response is essential to improving current treatment strategies that are largely based on the use of anthelmintic praziquantel to manage infections in aquaculture. This review focuses on the current knowledge of farmed fish host responses against the different life stages of aporocotylids. New treatment strategies that are able to provide protection against reinfections should be a long-term goal and is not possible without understanding the fish response to infection and the interactions between host and parasite.

Season, size, and sex: factors influencing monogenean prevalence and intensity on Gambusia affinis in New Zealand.

A number of studies have been conducted on monogenean seasonality, though primarily in continental regions with wide annual temperatures ranges. We investigated seasonal changes in the prevalence and intensity of Salsuginus seculus infesting sexually dimorphic western mosquitofish (Gambusia affinis) in New Zealand. This represents the first examination of seasonality for this species globally, and the first seasonal assessment of any monogenean population in New Zealand, a temperate country with a mild oceanic climate. Prevalence and intensity of S. seculus with respect to fish size and sex was also examined. Prevalence of S. seculus changed temporally, peaking in summer, and was strongly positively correlated with algal concentrations. This relationship may be associated with increasing food levels, leading to an increase in fish courting and mating, resulting in high numbers and close physical associations of G. affinis individuals, facilitating transmission of the monogeneans. Thus, biotic factors may be important in determining temporal changes in S. seculus prevalence in New Zealand. Female G. affinis had a significantly higher prevalence and mean intensity of S. seculus than males. Longer fish had a higher mean intensity and prevalence of S. seculus. Female G. affinis likely host disproportionately more monogeneans as they are larger than males. Alternatively, females may have a compromised immune response during reproductive periods. Overall, seasonal change was observed in S. seculus prevalence and intensity under New Zealand's mild climatic conditions, and the larger female G. affinis in this dimorphic species supported a greater prevalence and intensity of infestation than males.

Genetic relatedness and diversity of Capillaria species infecting bayad (Bagrus bajad) in upper Egypt.

BACKGROUND: This study investigates the genetic characteristics of Capillaria isolates from the infected fish, Bagrus bajad, and their relation to human Capillaria philippinensis using Random Amplified Polymorphic DNA (RAPD-PCR) analysis. Fifteen fish Capillaria were isolated and compared to identified human C. philippinensis using six primers: M-are, M-1, G-7, G-11, G-15, and G-18. RESULTS: All six primers successfully amplified DNA, highlighting their efficacy in distinguishing between human and fish Capillaria isolates. The analysis revealed distinctive banding patterns between fish and human isolates, with variations in size and number of DNA fragments. Additionally, genetic similarity analysis showed intriguing patterns of relatedness, with certain pairs exhibiting high similarity percentages. Comparative assessment of RAPD polymorphism demonstrated consistent findings of 100% polymorphism across all primers. The Unweighted Pair Group Method with Arithmetic Mean Algorithm (UPGMA) evaluated the closest relationship between human and fish isolates. These results underscore the utility of RAPD analysis in delineating the genetic diversity among Capillaria isolates from different hosts. CONCLUSION: Overall, this study contributes to our understanding of the genetic variability and relatedness among Capillaria isolates, shedding light on their evolutionary dynamics and zoonotic potential.

A novel neurotropic microsporidium from the swamp guppy Micropoecilia picta from Grenada, West Indies.

A novel microsporidium was observed in wild swamp guppies Micropoecilia picta from Levera Pond within Levera National Park Grenada, West Indies. Initial observations indicated similarity with Pseudoloma neurophilia, an important pathogen in zebrafish Danio rerio. P. neurophilia exhibit broad host specifity, including members of the family Poecillidae, and both parasites infect the central nervous system. However, spore morphology and molecular phylogeny based on rDNA showed that the swamp guppy microsporidium (SGM) is distinct from P. neurophilia and related microsporidia (Microsporidium cerebralis and M. luceopercae). Spores of the SGM were smaller than others in the clade (3.6 µm long). Differences were also noted in histology; the SGM formed large aggregates of spores within neural tissues along with a high incidence of numerous smaller aggregates and single spores within the surface tissue along the ventricular spaces that extended submeninx, whereas P. neurophilia and M. cerebralis infect deep into the neuropile and cause associated lesions. Analysis of small subunit ribosomal DNA sequences showed that the SGM was <93% similar to these related microsporidia. Nevertheless, one of 2 commonly used PCR tests for P. neurophilia cross reacted with tissues infected with SGM. These data suggest that there could be other related microsporidia capable of infecting zebrafish and other laboratory fishes that are not being detected by these highly specific assays. Consequently, exclusive use of these PCR tests may not accurately diagnose other related microsporidia infecting animals in laboratory and ornamental fish facilities.

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.