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Monogeneans of the genus Gyrodactylus are well-known pathogens causing huge mortalities in wild and cultured fish. Cytokine expression is one of most important host defense mechanisms against parasite infections. In this study, the expression pattern of the key pro-inflammatory (IL-1β, IL-8, IFN-γ, TNF-α, IL-12 and iNOS) and anti-inflammatory cytokine genes (IL-10, TGFβ and IL-4) of Gyrodactylus kobayashii infected goldfish (Carassius auratus) were determined by real-time quantitative PCR analysis. Our results showed that G. kobayashii infection caused increased expression of the pro-inflammatory cytokines including IFN-γ, TNF-α and iNOS in all detected tissues throughout the infection period. Among these genes, iNOS has the highest transcript level accompanied with increased nitric oxide (NO) concentration in the serum of all infected goldfish. The mRNA level of IL-1β in the liver, spleen and head kidney was significantly up-regulated during the early stage of infection (days 2-8). While high expression level of IL-8 and IL-12 was observed during the elimination phase of infection (days 10-14). As for anti-inflammatory cytokines, the expression profiles of IL-10 were distinct from those of TGF-β and IL-4. Specifically, the mRNA level of IL-10 did not increase in the spleen and head kidney during the early stage of infection, while increased expression of TGF-β and IL-4 were likewise seen. Besides, all infected fish had significantly higher complement C3 but lower IgM levels than the non-infected fish. The results provide insights into the interaction between gyrodactylids and the fish host, and indicate that systemic cytokine responses are critical for controlling parasite infection in fish., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Many natural products from medicinal plants are small molecular weight compounds with enormous structural diversity and show various biological activities. Magnolol is a biphenol compound rich in the stem bark of Magnolia officinalis Rehd et Wils., and is able to suppress viral replication in GCRV-infected grass carp (Ctenopharyngodon idella) kidney (CIK) cells in the previous study. In this study, in vivo studies demonstrated that magnolol was efficient to restrain the replication of GCRV and repair the low level of superoxide dismutase and total antioxidant capacity in serum at the non-toxic concentration in vivo. Furthermore, magnolol inhibited CIK cell apoptosis induced by GCRV and kept the normal cellular morphological structure, reflecting in the protection of CIK cells from cell swelling, the formation of apoptotic bodies, the disappearance of cellular morphology and nuclear fragmentation. Reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that magnolol facilitated the expression of apoptosis-inhibiting gene bcl-2, while suppressed the expression of apoptosis-promoting gene bax in GCRV-infected cells. Besides, RT-qPCR and enzyme activity assays proved that magnolol suppressed the expression of caspase 3, caspase 8 and caspase 9. Moreover, interactions between magnolol and proteins were predicted by using the STITCH program, which revealed that ten proteins including caspase 3, were involved in the apoptosis pathway, p53 signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway and toll-like receptor signaling pathway. Further assays were performed to test the effect of magnolol on apoptosis pathway, which showed that magnolol dramatically inhibited the activity of caspase 3 rather than those of caspase 8 and caspase 9. Collectively, the present study revealed that magnolol heightened the resistance of grass carp against GCRV infection and refrained GCRV-induced apoptosis, which may be attributed to the direct interaction of magnolol with caspase 3. The present results make a contribution to understanding the mechanisms by which small-molecule drugs possess antiviral activities, and lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Goldfish is known to be parasitized by at least seven species of Dactylogyrus and considered as one of the most common hosts. Dactylogyrus vastator and Dactylogyrus intermedius are the dominant species living on the gills of goldfish. However, little information on morphometric characterization is available, which easily causes misidentification. The purpose of this study is to provide comprehensive morphometric and molecular characterization of D. vastator and D. intermedius collected form a fish farm in Henan, China. The characterization was presented based on the high-resolution images and standard molecular markers (18S ribosomal DNA subunit and the internal transcribed spacer region), as well as a total of 10 point-to-point morphometrics characters. In addition, a detailed comparison of morphometric and phylogenetic characterization in D. vastator versus D. intermedius was performed. The results demonstrated that all parameters measured differed significantly between these two species of Dactylogyrus, whereas molecular comparison showed subtle differences between them in nucleotide divergence and genetic distances. These findings suggested that these two species of Dactylogyrus can be distinguished more easily based on morphometric measurements than molecular data for 18S ribosomal DNA + internal transcribed spacer region (ITS-1).

Dactylogyrus formosus Kulwiec, 1927 (Monogenea: Dactylogyridae), widely distributed across Eurasia, is considered as one of the most frequently encountered ectoparasites of goldfish (Carassius auratus). In the present study, D. formosus on gills of cultured goldfish was reported in central China for the first time, using the methods of morphological characters and molecular markers. It was characterized by "Anchoratoid-Wegeneri" type anchor with elongate inner and unobvious outer root. The morphology of the opisthaptoral hard parts of D. formosus superficially resembles species of D. arcuatus Yamaguti, 1942 parasitized crucian carp, using light and scanning electron microscopy. However, the anchor total length of D. arcuatus (range 90-102) is almost double the size of D. formosus (range 43.7-57.1) which allows for their rapid discrimination from each other; on the other hand, the two species also can be easily separated by the shape of the male copulatory organ. The morphological description of D. formosus is supplemented with phylogenetic analysis of a reference DNA sequence spanning 18S rDNA and ITS1. What is more, this study also redescribes comprehensively D. formosus, providing for the first time a full ten point-to-point morphometric measurements of the hard parts, and importantly, a photographic record of the armature of the haptor and the male copulatory organ.

Ichthyophthirius multifiliis, an external fish parasite, often causes significant economic damage to the aquaculture industry. Since the use of malachite green was banned, the search of alternative substance to control I. multifiliis infections becomes stringent. In present study, in vitro and in vivo anti-ich efficacies of isopsoralen and psoralidin, two active compounds isolated from methanol extract of Psoralea corylifolia by bioassay-guided fractionation based on the efficacy of anti-ich encysted tomonts, were evaluated. In vitro antiprotozoal efficacy of psoralidin is much better than that of isopsoralen. Psoralidin can kill all theronts at concentrations of 0.8 mg/L or more during 4 h exposure; and terminate reproduction of I. multifiliis post 6 h exposure of protomonts to 0.9 mg/L and encysted tomonts to 1.2 mg/L. In vivo trials showed that 5 h exposure of infected fish to 2.5 mg/L of psoralidin significantly reduced the number of theronts released from tomonts. Furthermore, we observed that a part of protomonts, collected from infected fish post treatment, presented characteristic morphological changes of apoptosis after staining with Annexin V-EGFP/propidium iodide, indicating the possible mechanism of psoralidin against I. multifiliis trophont in situ. On the basis of these results, psoralidin can be used as a potential lead compound for the development of commercial drug against I. multifiliis.

Goldfish, Carassius auratus, widely distributed across Eurasia, is one of the earliest fish domesticated for ornamental purposes. A series of diseases break out due to high-density culture of goldfish, causing significant economic losses. Here, we report for the first time an infection of Gyrodactylus kobayashii associated with the high mortality of goldfish from a fish farm in Anziying township of Henan province, China, following their transfer to several aquariums. In the first 2 weeks after goldfish (n = 4200, weight 10.24 ± 1.45 g) transfer to aquariums, a total of 3335 goldfish were lost, representing 79.4 % of the initial stocked biomass. The examination showed that no other pathogens were found in this batch of fishes, except for G. kobayashii. The high burdens of G. kobayashii/fish (264.7, range 100-450) were the primary reason for the high mortality, although this was not the sole cause. In addition, the isolate of G. kobayashii from goldfish (C. auratus) in Henan province of central China was described. The morphological characterization was performed using morphometric measurements and drawings of opisthaptoral hard parts of the parasites. The molecular description was performed based on phylogenetic analysis of a reference DNA sequence spanning 5.8S and ITS-2. Importantly, the present study provides for the first time a full 25 point-to-point morphometric measurements and high-resolution images of attachment organ of G. kobayashii.

Dactylogyrus spp. and Gyrodactylus spp. are helminth ectoparasites that are a significant threat to the aquaculture industry. Existing treatments could cause the threats of anthelmintic resistance, risk of residues, environmental contamination, and toxicity to fish. Importantly, there is no report on a treatment against these two parasites. This study explored the possibility of using the extracts of Santalum album to treat Dactylogyrus sp. and Gyrodactylus sp. infections in goldfish. Results showed that among the four extracts (chloroform, methanol, ethyl acetate, and water) of S. album, the chloroform extract is the most effective and 40 mg/L is a safe and the lowest effective dosage. In addition, we found that Gyrodactylus elegans is more sensitive than Dactylogyrus intermedius when exposed to the extract of the medicinal plant. Finally, it is substantiated that bath treatment with long duration and multiple administrations could eliminate a greater proportion of monogenean infections. These findings show the potential for the development of effective and safe therapy to treat Dactylogyrus sp. and Gyrodactylus sp. infections of fishes.

Ichthyophthirius multifiliis (also called "ich") is an external protozoan parasite that may infest almost all freshwater fish species and caused significant economic damage to the aquaculture industry. Since the use of malachite green was banned, there have been relatively few effective alternative strategies for controlling I. multifiliis infections. The present study was designed to screen potential antiparasitic medicinal plants based on our previous studies, and comprehensively evaluate in vitro and in vivo anti-ich activity of selected plant extracts. The screening results showed that the methanol extract of Psoralea corylifolia had the highest activity against I. multifiliis theronts. In vivo theront trials demonstrated that 1.25 mg/L or more concentrations of P. corylifolia methanol extract caused 100 % mortality during the 4-h exposure period, and the subsequent in vitro trials indicated that the minimum concentration of P. corylifolia methanol extract that prevented the initial infestation was 2.50 mg/L. Protomonts and encysted tomonts surviving trials suggested that encysted tomonts were less susceptible to P. corylifolia methanol extract than protomonts, and the methanol extract of P. corylifolia at a concentration of 5.00 mg/L could kill 100 % of protomonts and 88.89 % of encysted tomonts. It was also observed that after 12-h exposure of protomonts or encysted tomonts to 2.50 mg/L of P. corylifolia methanol extract, the theronts emerged from encysted tomonts led to more infection level than the ones in the other groups. The results suggested that whether the protomonts finish encystment is crucial to the survival, reproduction, and theronts infectivity. In addition, our results showed that long duration (24 h) and high concentration (5.00 mg/L) significantly reduced the survival and reproduction of I. multifiliis tomont exited from the fish after in-bath treatment, and it is indicated that P. corylifolia methanol extract had a potential detrimental effect on I. multifiliis trophont in situ.