Integration of transcriptomic and metabolomic profiling of encystation in Cryptocaryon irritans regulated by rapamycin.

Encystation in Cryptocaryon irritans is a fundamental process for environmental resistance and development. Autophagy participates in the encystation of ciliates, and rapamycin can induce autophagy in the cells. A set of genes and metabolites related to autophagy and encystation are highly elaborative. The existence of these genes and metabolites and their role are well characterized. However, little is known about their role in protozoans such as ciliates. The newly produced C. irritans protomonts were exposed to an optimal concentration of rapamycin (1400 nM), and the survival, encystation, microstructure/ultrastructure, transcriptomic and metabolomic profile in treated and control protomonts were investigated. The results showed that exposure of protomonts to rapamycin at 4 h significantly lowered the survival and encystation rates to 91.62 % and 98.44 % compared to the control group (100 %, p ≤ 0.05). Morphological alterations observed in light microscopy and transmission electron microscopy (TEM) demonstrated that the drug significantly changed cell symmetry by causing the formation of various autophagic vacuoles/vesicles. The transcriptome sequencing of rapamycin-treated protomont revealed that 2249 (1837 up-regulated and 977 down-regulated) differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 226 DEGs were successfully annotated in 21 pathways (p˂0.05), including most enriched pathways apoptosis and phagosome with 25 and 24 DEGs, respectively. Most unigenes were assigned to autophagy-related pathways; 24 DEGs were classified into phagosomes, and 15 DEGs were assigned to lysosome pathways. Cytoskeleton and cell progression-associated genes were down-regulated. Besides, cell death-inducing proteins were up-regulated. The metabolomic analysis revealed exposure to rapamycin treatment enhanced protomont metabolites, including L-Cysteine, which is related to autophagy. Rapamycin had influenced the gene and metabolites of protomont; activating autophagy with inhibition of mechanistic target of rapamycin, (mTOR). The process negatively influences protomont morphology, encystation, and survival. Further autophagy-related gene silencing can be investigated via genome sequencing of C. irritans to study encystation.

Effects of autophagy inhibition by 3-methyladenine on encystation, morphology, and metabolites of Cryptocaryon irritans.

Encystment is crucial for defense and reproduction in Cryptocaryon irritans. Therefore, understanding the encystment-related events in the protomont stage can help prevent and control C. irritans. Autophagy promotes protozoan parasite encystation. However, 3MA can inhibit autophagy. In this study, the effects of autophagy inhibition on encystation, survival rate, ultrastructural features, and metabolomic profiles of C. irritans, were evaluated using protomonts treated with 3MA (20 mM). The treatment with 3MA for about 4 h significantly lowered survival and encystation rates of protomonts to about 86.44% and 76.08%, respectively. Microstructural observations showed that the 3MA-treated protomonts showed deformed cell membranes and the cytoplasmic content spill. Furthermore, observation of the ultrastructure of 3MA-treated protomonts showed the destruction of organelles (Golgi bodies and mucocyst) and a lack of autophagosomes. However, no abnormality was observed in the control experiments. Furthermore, the metabolic analysis revealed suppression of metabolites, such as lipids, amino acids, and carbohydrates. These results demonstrate that 3MA can inhibit autophagy in C. irritans, thus hindering encystation, suppressing the metabolism of metabolites, and altering morphological ultrastructure in these parasites.

Label-free quantitative proteomics analysis of skin of yellow drum (Nibea albiflora) reveals immune mechanism against Cryptocaryon irritans.

To explore the resistance mechanism of locally infected skin of yellow drum (Nibea albiflora) against Cryptocaryon irritans infection, N. albiflora were infected with C. irritans at a median lethal concentration of 2050 theronts/g fish. Then, the skin of the infected group (24 hT and 72 hT) and the control group (24 hC and 72 hC) were sampled at 24 h and 72 h for quantitative proteomics analysis. A total of 643 proteins were identified, of which 61 proteins were significantly affected by interaction between time and infection, 83 and 119 proteins were significantly affected by the infection and time, respectively. In addition, 17, 61, 81 and 45 differentially expressed proteins (DEPs) were obtained from pairwise comparison (24 hT vs 24 hC, 72 hT vs 72 hC, 72 hT vs 24 hT and 72 hC vs 24 hC), respectively. DEPs in 24 hT vs 24 hC and 72 hT vs 72 hC were mainly enriched in Gene Ontology terms (transferase activity, protein folding and isomerase activity) and Kyoto Encyclopedia of Genes and Genomes pathways (biosynthesis of antibiotics, carbon metabolism and Citrate cycle). Among them, enriched DEPs were malate dehydrogenase 2 (MDH2), malate dehydrogenase 1 ab (MDH 1 ab), citrate synthase, etc. Immune-related DEPs such as complement component C3 and Cell division cycle 42 were involved in response to stimulus and signal transduction, etc. Also, DEPs such as collagen, heat shock protein 75 and MDH2 play a role in helping fish skin wounds to heal and provide energy. Furthermore, protein-protein interaction analysis indicated that 18 proteins such as MDH2, MDH 1 ab, complement C3 and collagen were interrelated. In conclusion, this study found that many proteins in N. albiflora contribute to resist against C. irritans and promote fish recovery.

Skin metabolome reveals immune responses in yellow drum Nibea albiflora to Cryptocaryon irritans infection.

The yellow drum Nibea albiflora is less susceptible to Cryptocaryon irritans infection than is the case with other marine fishes such as Larimichthys crocea, Lateolabrax japonicus, and Pagrus major. To investigate further their resistance mechanism, we infected the N. albiflora with the C. irritans at a median lethal concentration of 2050 theronts/g fish. The skins of the infected and the uninfected fishes were sampled at 24 h and 72 h followed by an extensive analysis of metabolism. The study results revealed that there were 2694 potential metabolites. At 24 h post-infection, 12 metabolites were up-regulated and 17 were down-regulated whereas at 72 h post-infection, 22 metabolites were up-regulated and 26 were down-regulated. Pathway enrichment analysis shows that the differential enriched pathways were higher at 24 h with 22 categories and 58 subcategories (49 up, 9 down) than at 72 h whereby the differential enriched pathways were 6 categories and 8 subcategories (4 up, 4 down). In addition, the principal component analysis (PCA) plot shows that at 24 h the metabolites composition of infected group were separately clustered to uninfected group while at 72 h the metabolites composition in infected group were much closer to uninfected group. This indicated that C. irritans caused strong metabolic stress on the N. albiflora at 24 h and restoration of the dysregulated metabolic state took place at 72 h of infection. Also, at 72 h post infection a total of 17 compounds were identified as potential biomarkers. Furthermore, out of 2694 primary metabolites detected, 23 metabolites could be clearly identified and semi quantified with a known identification number and assigned into 66 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Most of the enriched KEGG pathways were mainly from metabolic pathway classes, including the metabolic pathway, biosynthesis of secondary metabolites, taurine and hypotaurine metabolism, purine metabolism, linoleic acid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis. Others were glyoxylate and dicarboxylate metabolism, glutathione metabolism, and alanine, aspartate, and glutamate metabolism. Moreover, out of the identified metabolites, only 6 metabolites were statistically differentially expressed, namely, L -glutamate (up-regulated) at 24 h was important for energy and precursor for other glutathiones and instruments of preventing oxidative injury; 15-hydroxy- eicosatetraenoic acid (15-HETE), (S)-(-)-2-Hydroxyisocaproic acid, and adenine (up-regulated) at 72 h were important for anti-inflammatory and immune responses during infection; others were delta-valerolactam and betaine which were down-regulated compared to uninfected group at 72 h, might be related to immure responses including stimulation of immune system such as production of antibodies. Our results therefore further advance our understanding on the immunological regulation of N. albiflora during immune response against infections as they indicated a strong relationship between skin metabolome and C. irritans infection.