Potential effects of host competence and schooling behavior on parasite transmission in a host-pathogen system: a test of the dilution effect.

High species diversity in a community may reduce the risk of infectious disease, termed the dilution effect. However, the generality of the dilution effect in different disease systems remains controversial as both host competence and behaviors of hosts may play roles in dilution or amplification of disease. Using the goldfish (Carassius auratus)-monogenean ectoparasite (Gyrodactylus kobayashii) system, effects of host competence and schooling behavior on parasite transmission were investigated while holding focal host density constant. Following competency tests of 12 fish species as potential hosts for the parasite, infection by G. kobayashii was determined on fins of goldfish mixed with each of three different species based on their level of host competence, including Prussian carp, Carassius gibelio (low competence), grass carp, Ctenopharyngodon idellus (non-competent), swordtail, Xiphophorus helleri (non-competent), and the four species combined. Compared with mean abundance (85.8 ± 25.1) on goldfish in the control group, the mean abundance on goldfish decreased significantly when paired with 10 Prussian carp (30.0 ± 16.5), but did not differ significantly when paired with 10 swordtail (70.0 ± 22.2), 10 grass carp (116.1 ± 33.2), or the multi-species of three Prussian carp, four grass carp and three swordtail (75.9 ± 30.8) during the 11-day experiment. The parasite was also found on the Prussian carp in the Prussian carp group and the multi-species group at a mean abundance of 7.1 and 10.9, respectively. Video recording showed that the school of goldfish mixed well with the Prussian carp, while they maintained separation from the grass carp and swordtail when mixed together. The distance between goldfish increased, and swimming speed and contact time decreased with the additional of other fish species for all groups. The results suggested that the presence of a low-competence host in sufficient numbers was a necessary condition for a dilution effect due to encounter reduction, and the dilution effect may also be enhanced by changes in schooling behavior of goldfish in the presence of low competence hosts. However, the presence of non-competent hosts did not result in any dilution effect owing to the specialist nature of the parasites and the lack of mixing with schools of goldfish.

Anthelmintic Efficacy of Palmarosa Oil and Curcuma Oil against the Fish Ectoparasite Gyrodactylus kobayashii (monogenean).

Monogeneans are a serious threat to the development of aquaculture due to the severe economic losses they cause. The prevention and treatment of this disease are increasingly difficult because of the environmental and health concerns caused by the use of chemical anthelmintics and the emergence of drug resistance. It is thus necessary to search for effective alternatives for the treatment of monogenean infections. In the current study, anthelmintic efficacy of 16 selected essential oils (EOs) was investigated using the goldfish (Carassius auratus)-Gyrodactylus kobayashii model. The screening experiment indicated that palmarosa oil and curcuma oil had satisfactory anthelmintic activity against G. kobayashii with EC100 values of 10 and 12 mg/L after 24-h exposure, respectively. The in vivo and in vitro assays indicated anthelmintic efficacy of palmarosa oil against G. kobayashii was in a time and dose-dependent manner. Interestingly, curcuma oil showed an anesthetic effect on G. kobayashii, and its anthelmintic activity was dose-dependent rather than time-dependent in the concentration range tested in this study. Additionally, the 24-h LC50 (50% lethal concentration) against goldfish of these two EOs was 8.19-fold and 5.54-fold higher than their corresponding EC50 (50% effective concentration) against G. kobayashii, respectively. Moreover, exposure to these two EOs at 100% effective concentration against G. kobayashii had no serious physiological and histopathological influence on goldfish. These results demonstrated a high safety for goldfish of these two EOs. Overall, palmarosa oil and curcuma oil could be potential candidates for the treatment of G. kobayashii infections in aquaculture.

Antiparasitic Efficacy of Herbal Extracts and Active Compound Against Gyrodactylus kobayashii in Carassius auratus.

Gyrodactylus spp. Nordmann, 1832 (Monogenea: Gyrodactylidae) are common ectoparasites of teleost fishes. Infection with these parasites can increase the mortality of fish and cause considerable economic losses in intensive aquaculture. To find an effective antiparasitic agent for the control of gyrodactylosis, antiparasitic efficacy of crude extracts of 36 herbal medicines was evaluated using a Carassius auratus (Cypriniformes, Cyprinidae)-Gyrodactylus kobayashii model. Among all tested medicines, methanol extract of Dioscorea collettii var. hypoglauca (Dioscoreales, Dioscoreaceae) was the most efficient, with an EC50 value of 4.17 mg/L. This extract showed 100% antiparasitic efficacy against G. kobayashii at 10 mg/L and had a therapeutic index (TI, LC50/EC50) of 5.26, which is higher than that of formaldehyde (TI = 4.58), a widely used parasiticide in aquaculture. Subsequently, the potential mechanism of antiparasitic activity of dioscin, an active compound isolated from D. collettii var. hypoglauca was investigated and the histopathological alterations in goldfish after exposure to dioscin were also studied. The in vivo trial indicated dioscin showed significant antiparasitic activity with a 24 h-EC50 value of 1.58 mg/L and it exhibited 100% antiparasitic efficacy at 0.6 mg/L. Also, G. kobayashii could be completely removed in vivo within 2 h at 0.6 mg/L dioscin. Whereas, mean survival time of this worm in vitro was 4.99 h, and some individuals even reached 12 h at the same concentration of dioscin. These results indicated that 0.6 mg/L of dioscin did not completely kill all worms within 2 h, but just temporarily remove the worms from goldfish. Scanning electron microscopy (SEM) analysis showed that most of the microvilli on the tegument surface of G. kobayashii dropped after exposure to dioscin. This might be one of the potential mechanisms of antiparasitic activity of dioscin against G. kobayashii. Furthermore, no severe histopathological alteration was observed after exposure to a high concentration of dioscin for a short time. Considering both effectiveness and safety, therapeutic baths with a high concentration of dioscin for a short time might be a more optimal choice for the treatment of gyrodactylosis in aquaculture.

Anthelmintic efficacy of natural saponins against Gyrodactylus kobayashii in goldfish (Carassius auratus) and their 3D-QSAR analysis.

Gyrodactylus spp. are common monogenean ectoparasites that may lead to significant fish mortality. To find effective anthelmintic agents with lower toxicity, a series of natural saponins were obtained and evaluated for their anthelmintic activity against Gyrodactylus kobayashii and acute toxicity to goldfish (Carassius auratus). Among all tested compounds, six compounds (1, 2, 3, 8, 10, and 13) shown higher anthelmintic activity and safety than widely used formaldehyde-based parasiticides, especially compound 1 having 100% anthelmintic efficacy against G. kobayashii at 0.3 mg/L and a therapeutic index of 16.6. Also, the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of these saponins have been performed to explore the structural features reasonable for the anthelmintic activity against G. kobayashii. These models demonstrated that the hydroxyl group at C-17 position and the sugar moieties at C-3 position, especially the hydroxyl groups of the sugar moieties, were critical to the anthelmintic activity. The QSAR studies could provide useful information for further rational design and optimization of novel saponins for the control of gyrodactylosis.

Transcriptome analysis of goldfish (Carassius auratus) in response to Gyrodactylus kobayashii infection.

Gyrodactylid monogeneans are widespread parasites of teleost fishes, and infection with these parasites results in high host morbidity and mortality in aquaculture. To comprehensively elucidate the immune mechanisms against Gyrodactylus kobayashii, the transcriptome profiles of goldfish (Carassius auratus) skin after challenge with G. kobayashii were first investigated using next-generation sequencing. Approximately 21 million clean reads per library were obtained, and the average percentage of these clean reads mapped to the reference genome was 82.25%. A total of 556 differentially expressed genes (DEGs), including 344 upregulated and 212 downregulated genes, were identified, and 380 DEGs were successfully annotated and assigned to 95 signaling pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, 14 pathways associated with immune response were identified mainly including mTOR signaling pathway, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, toll-like receptor signaling pathway, and phagosome. Twelve genes were selected and validated using qRT-PCR. A similar trend of these genes between RNA-Seq and qRT-PCR was observed, indicating that RNA-Seq data was reliable. Besides, the ALP activity and NO content in serum were significantly higher in the infected goldfish compared with the non-infected goldfish. In summary, this study provides better understandings of immune defense mechanisms of goldfish against G. kobayashii, which will support future molecular research on gyrodactylids and facilitate the prevention and treatment of gyrodactylosis in aquaculture.

Expression analysis of immune genes in goldfish (Carassius auratus) infected with the monogenean parasite Gyrodactylus kobayashii.

Heavy infection with the ectoparasite Gyrodactylus kobayashii commonly leads to high mortality of ornamental goldfish Carassius auratus. To understand the underlying mechanisms of goldfish against infection of gyrodactylids, transcription responses of immune-related genes including IL-1ß2, TNFα1, TNFα2, IFN-γ, TGFß, MHC II ß, TCRß1 and complement factor C3 were studied by real-time quantitative PCR analysis. Significant increases of expression of inflammatory genes such as IL-1ß2, TNFα1, TNFα2 and TGFß were detected at days 7 and 14 post-infection (pi). No significant differences of G. kobayashii load were observed in response to primary infection and re-infection. In addition, the transcript levels of genes involved in adaptive immunity such as MHC II ß and TCRß1 remained unchanged. Curiously, fish moderately infected with gyrodactylid showed elevated expression IL-1ß2, TNFα1 and TNFα2. In all the gyrodactylids-infected fish, expression of complement factor C3 was consistently inhibited. The results extend current knowledge to the understanding of gyrodactylid infection in fish and support the previous findings that innate immunity is indispensable for controlling parasite infection.

The complete mitochondrial genome of Gyrodactylus kobayashii (Platyhelminthes: Monogenea).

The complete mitochondrial genome of Gyrodactylus kobayashii was 14 786 bp in length, containing 12 protein-coding genes (lacking Atp8), 22 tRNA genes, two rRNA genes and two major non-coding regions (NC1 and NC2). The overall A + T content of mitochondrial genome was 71.6%. A close relationship between G. kobayashii and the three Gyrodactylus species (G. salaris, G. thymalli and G. derjavinoides) was uncovered in the phylogenetic tree based on amino acid sequences.