pH Regulates the Formation and Hatching of Cryptocaryon irritans Tomonts, Which Affects Cryptocaryoniasis Occurrence in Larimichthys crocea Aquaculture.

Cryptocaryon irritans are the main pathogens of white spot disease in marine teleost. However, the occurrence of cryptocaryoniasis is influenced by several abiotic factors including the pH. To explore the effect of pH on the life cycle of C. irritans (encystment, cleavage, and hatchability), protomonts and tomonts of C. irritans were incubated in seawater of 10 different pH levels (2-11). pH 8 was used as the control. The change in morphology and infectivity of theronts that hatched from tomonts against Larimichthys crocea were then recorded. We found that pH 6-9 had no significant effect on the encystment, cleavage, and hatching of the parasites. However, pH beyond this limit decreased the cleavage and hatching of the tomonts. Furthermore, extreme pH decreased the number of theronts hatched by each tomont and the pathogenicity of the theronts, but increased the aspect ratio of the theronts. Infectivity experiments further revealed that extreme pH significantly decreased the infectivity of C. irritans against L. crocea. In conclusion, the C. irritans can survive in pH of 5 to 10, but pH 6-9 is the optimal range for the reproduction and infectivity of C. irritans. However, extreme pH negatively affects these aspects. IMPORTANCECryptocaryon irritans is a ciliate parasite that causes "white spot disease" in marine teleosts. The disease outbreak is influenced by hosts and a range of abiotic factors, such as temperature, salinity, and pH. Studies have shown that change in pH of seawater affects the structure (diversity and abundance of marine organisms) of marine ecosystem. However, how pH affects the life cycle and survival of C. irritans, and how future ocean acidification will affect the occurrence of cryptocaryoniasis, are not well understood. In this study, we explored the effect of pH on the formation and hatching of C. irritans tomonts. The findings of this study provide the foundation of the environmental adaptation of C. irritans, the occurrence of cryptocaryoniasis, and better management of marine fish culture.

Integration of metabolomic and transcriptomic analyses to characterize the influence of the gill metabolism of Nibea albiflora on the response to Cryptocaryon irritans infection.

The parasite Cryptocaryon irritans causes massive losses in the marine fish culture industry and is one of the most threatening pathogens affecting teleost species. The acute death of infected fish is primarily caused by the destruction of gill cells, resulting in osmotic imbalance and respiratory stress. C. irritans has wide host specificity; however, the yellow drum Nibea albiflora is highly resistant to this parasite. Metabolomic approaches in combination with transcriptomic analysis were used to characterize the host immune reaction and metabolic changes in yellow drum in response to C. irritans infection and to identify the key genes and compounds in the gills that have the strongest contribution to disease resistance. The yellow drum was challenged with theronts at a median death rate (2050 theronts per gram fish). The samples were collected from the gills 24 h and 72 h after the infection (hpi). The results of metabolomic analysis indicated that metabolites involved in energy metabolism were predominantly downregulated. In contrast, a compensatory increase in the expression of the genes involved in the citric acid cycle and glycolysis was detected 24 hpi. The suppression of metabolites was alleviated after feed intake recovery 72 hpi. The levels of amino acids were decreased, and the expression of aminoacyl-tRNA was increased. Additionally, elevated levels of arachidonic acid derivatives, primarily prostaglandins, were responsible for anti-inflammatory, osmotic, and hypoxia regulations. Purine metabolism was also involved in the immune response via generation of reactive oxygen species catalyzed by xanthine oxidase. A significant increase in the generation of retinoic acid, which could enhance mucosal adaptive immunity by stimulating the synthesis of antibodies and accelerating the restoration of epithelial integrity, was observed at 72 hpi. This result was consistent with high expression of the genes related to secreted immunoglobulin T 72 hpi. In conclusion, the present study comprehensively described the key compounds and genes related to C. irritans infection in yellow drum gills. Biomarkers that were significantly changed during the infection may represent future targets for nutritional intervention to enhance host immunity against C. irritans infection and to accelerate disease recovery.

The gill transcriptome reveals unique antimicrobial features that protect Nibea albiflora from Cryptocaryon irritans infection.

Cryptocaryonosis is the greatest threat to most teleost species among all parasitic diseases, causing mass loss to the marine aquaculture industry. Epidemiological investigation of teleost susceptibility to Cryptocaryon irritans infection revealed that yellow drum (Nibea albiflora) is highly resistant. In order to further understand the activation of the immune system in the gill, which is one of the main mucosal-associated lymphoid tissues and a target of parasites, transcriptome analysis of the yellow drum gill was performed. Gill samples were collected from fish challenged after 24 hr and 72 hr with theronts at a median death rate (2050 theronts per gram fish). Gene expression profiles showed that TLR5 was the only receptor that activated the downstream immune response. The infection activated complement cascade through alternative pathway and increased the expression of C5a anaphylatoxin chemotactic receptor 1. In addition, possible antimicrobial molecules, including lipoprotein and haptoglobin, which are responsible for trypanolysis in humans, were among the top significantly upregulated genes at 24 hr. After 72 hr, the expression of secreted immunoglobulin T-related genes was induced. These results suggested a rapid innate and adaptive immune response at the mucosal level. In conclusion, the results provide new perspectives on mucosal immune resistance in yellow drum against cryptocaryonosis and provide the possibility of mining resistance genes for future therapy.

Updating specific PCR primer for detection of Cryptocaryon irritans from reared Larimichthys polyactis.

Artificial breeding of small yellow croaker (Larimichthys polyactis) was recently achieved, providing a bright future for its commercial farming. In May 2019, a disease outbreak occurred among small yellow croakers in an aquaculture farm near Xiangshan Bay, charactering by white spots spotted on the surface of fish skin, gills and fins. The parasite was preliminarily identified as Cryptocaryon irritans based on morphological feature of the parasite and the symptoms on fish. However, the previously published specific primer pairs failed to confirm the existence of C. iriitans. Six nucleotides mismatches were discovered after mapping specific forward primer back to targeted gene. Therefore, an updated PCR specific primer was developed within the 9th highly variable region of 18S rRNA gene and conserved in all C. irritans sequences available in GenBank database. The specificity was verified in silico by Primer-BLAST against GenBank nucleotide. Laboratory cultured ciliates (Mesanophrys, Pseudokeronopsis and Uronema) as well as natural microbial community samples collected from sea water and river water was used as negative control to verify the specificity of the primer in situ. Besides, tank transfer method was used to evaluate the treatment of the parasite infection. By tank transfer method, 2.00 ± 0.61 out of 10 fish that already sever infected were successfully survived after 8 days treatment, meanwhile the control group died out at d 6. More loss to the treatment group during first five days was observed and may attribute to the combined effect from infection and stress the recent domesticated fish suffered during rotation. Therefore, tank transfer method was also effective to prevent small yellow croaker from further infection, however the loss of the small yellow croaker suffered from stress during rotation also needs to be carefully concerned. In conclusion, this study reported the first diagnose of C. irritans infection on small yellow croaker, provided updated specific primer to detect C. irritans infection on fish body and reported the effect of tank transfer on small yellow croaker treatment.

Combination effects of ellagic acid with erlotinib in a Ba/F3 cell line expressing EGFR H773_V774 insH mutation.

BACKGROUND: Epidermal growth factor receptor H773_V774 insH (EGFR-insH) is an EGFR exon 20 insertion mutation in non-small cell lung cancer (NSCLC), which is naturally resistant to available EGFR tyrosine kinase inhibitors (TKIs) and lacks a patient-derived cell line. METHODS: A Ba/F3 cell line expressing EGFR-insH mutation (Ba/F3-insH cell line) was generated using an IL3-deprivation method. A cell proliferation assay was performed to screen natural compounds that exhibit a synergistic effect with erlotinib. Trypan blue staining was used to assess cell growth and crystal violate staining was recruited to evaluate clonogenic potential. Flow cytometry was used to detect EGFR expression and cell apoptosis. A xenograft model was created to evaluate the effect of ellagic acid (EA) with erlotinib on tumor growth. RESULTS: EA was identified to synergistically inhibit the proliferation of Ba/F3-insH cells with erlotinib. The growth and clonogenic potential of Ba/F3-insH cells were definitely constrained by EA with erlotinib, whereas, the apoptosis of Ba/F3-insH cells was dramatically promoted by the combination. In a xenograft model of the Ba/F3-insH cell line, the combination treatment also exhibited a synergistic reduction in tumor growth. CONCLUSIONS: In this study, we generated a Ba/F3 cell line expressing EGFR H773_V774 insH mutation and identified a synergistic treatment (EA with erlotinib) that markedly inhibited the viability of Ba/F3-insH cells in vitro and in vivo. KEY POINTS: Our results indicated that the combination of ellagic acid with erlotinib has synergistic effects against EGFR H773_V774 insH mutation.

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.