Antiparasitic effect of copper alloy mesh on tomont stage of Cryptocaryon irritans in aquaculture.

Copper alloy sheets have been shown to prevent cryptocaryoniasis. Therefore, we studied the potential efficiency of copper alloy mesh (CAM) in aquaculture tanks to prevent cryptocaryoniasis outbreaks. The effectivenesses of CAM against the tomont stage of Cryptocaryon irritans and in protecting fish from cryptocaryoniasis were tested both in vitro and in vivo. The mortality rate of C. irritans tomonts increased as the contact time with CAM rose and peaked at 70 min (100% of mortality). Morphological changes were observed such as the shrinking of the protoplasm of the treated tomonts, resulting in a larger gap between the cytoplasm and the cyst wall. Mitochondrial dysfunction due to shrinkage in the inner portion, outer and inner mitochondrial membrane damage and cytoplasmic vacuolation was revealed by ultrastructural analysis. The use of CAM effectively preventing reinfection was also provided. In comparison with group B (infected fish without CAM), both groups A (uninfected fish as a control group) and C (infected fish treated with CAM) had a 100% survival rate until the end of the trial. CAM has the same anticryptocaryoniasis effect as copper alloy sheets but is more advantageous due to its lightweight, reduced labor cost and lower purchase cost. It is noticeable that CAM exposure also prevents the excessive accumulation of copper ions in aquaculture sea water.

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.

Quantum Battery Based on Hybrid Field Charging.

A quantum battery consisting of an ensemble two-level atom is investigated. The battery is charged simultaneously by a harmonic field and an electrostatic field. The results show that the hybrid charging is superior to the previous case of only harmonic field charging in terms of battery capacity and charging power, regardless of whether the interaction between atoms is considered or not. In addition, the repulsive interaction between atoms will increase the battery capacity and charging power, while the attractive interaction between atoms will reduce the battery capacity and discharge power.

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.

Transcriptomic analysis of Nibea albiflora skin in response to infection by Cryptocaryon irritans.

Massive infection caused by Cryptocaryon irritans is detrimental to the development of marine aquaculture. Recently, our lab found that Nibea albiflora has low sensitivity and low mortality to C. irritans infection. The present study was designed to investigate the mechanisms of the N. albiflora response to C. irritans infection by analyzing transcriptome changes in the skin. Skin samples of control and experimental groups with C. irritans infection were collected at 24 and 72 h (24 h control, 24 h post-infection, 72 h control, and 72 h post-infection). Three parallels were set for each group and sample time, and a total of 12 skin samples were collected for sequencing. Overall, 297,489,843 valid paired-end reads and 48,817 unigenes were obtained with an overall length of 59,010,494 nt. In pairwise comparisons, changes in expression occurred in 1621 (764 upregulated and 857 downregulated), 285 (180 upregulated and 105 downregulated), 993 (489 upregulated and 504 downregulated), and 37 (8 upregulated and 29 downregulated) genes at 24 h control vs 24 h post-infection, 72 h control vs 72 h post-infection, 24 h post-infection vs 72 h post-infection, and 24 h control vs 72 h control, respectively. Gene Ontology (GO) analysis of differentially expressed genes (DEGs) indicated that the number of genes enriched in GO sub-categories were ordered 24 h control vs 24 h post-infection > 24 h post-infection vs 72 h post-infection >72 h control vs 72 h post-infection > 24 h control vs 72 h control. Further analysis showed that immune-related GO terms (including immune system process, complement activation, and humoral immunity) were significantly enriched at both 72 h control vs 72 h post-infection and 24 h post-infection vs 72 h post-infection, but no immune-related GO terms were significantly enriched in the 24 h control vs 72 h control and at 24 h control vs 24 h post-infection, indicating that C. irritans infection mainly affected the physiological metabolism of N. albiflora at an early stage (24 h), and immune-related genes play an important role at a later stage (72 h) of infection. In KEGG pathway analysis, the complement and coagulation cascade pathway are involved in early infection. Hematopoietic cell lineage, natural killer (NK) cell-mediated cytotoxicity, and the intestinal immune network for IgA production are involved in later infection. Further analysis showed that the alternative pathway of complement and coagulation cascades plays an important role in the resistance of N. albiflora to early C. irritans infection. During late infection, CD34, IgM, and IgD were significantly upregulated in the hematopoietic cell lineage pathway. CCR9 was significantly downregulated, and IGH and PIGR were significantly upregulated in the intestinal immune network for IgA production. GZMB and IGH were significantly downregulated in NK cell-mediated cytotoxicity. These findings indicate that acquired immunity at the mRNA level was initiated during later infection. In addition, the IL-17 signaling pathway was enriched by downregulated DEGs at 24 h post-infection vs 72 h post-infection, suggesting the inflammatory response at 24 h was stronger than at 72 h and the invasion of the parasite has a greater impact on the host.

Anti-parasitic effects and toxicity of formalin on the parasite Mesanophrys sp. of the swimming crab Portunus trituberculatus.

In aquaculture of the swimming crab Portunus trituberculatus, massive deaths have been recorded in the winter months due to infection with a novel emerging parasite, Mesanophrys sp. However, no information was available regarding the prevention and control of this particular parasite. Therefore, the present study was conducted to evaluate the anti-parasitic efficacy and toxicity of formalin against the Mesanophrys sp. In vitro results showed that the anti-parasitic efficacy of formalin improved with concentration increasing from 0.0 to 20.0 ppm within 24 h. In particular, when treated with formalin at 16.0, 15.0, 11.0, 10.0, 9.0, and 6.0 ppm for 0.5, 1, 2, 4, 6, 12, and 24 h respectively, the Mesanophrys sp. mortality rate reached 100%. To gain insights into the effects the formalin treatment had on the parasite, cell micro- and ultra-structure were investigated. It was determined that the cells contracted gradually and became rounded, intracellular vacuoles were observed at early time points (Ф≤4.83 ± 1.26 µm) and then disappeared. Cilia were shed and macronuclear chromatin became condensed and agglutinated. Small holes and bubbles appeared on surface of the parasites. In an in vivo trial, formalin was applied prior to Mesanophrys sp. artificial infection as prophylaxis to P. trituberculatus. The results showed that formalin prophylactic treatment effectively prevented P. trituberculatus from Mesanophrys sp. infection, thus remarkably reducing the mortality of crabs compared with the non-formalin-exposed and infected crabs. Furthermore, the normal behavior and survival of P. trituberculatus were not impacted by the prophylactic treatment.

Isolation, characterization and virulence of Mesanophrys sp. (Ciliophora: Orchitophryidae) in farmed swimming crab (Portunus trituberculatus) in eastern China.

A disease outbreak occurred in swimming crab (Portunus trituberculatus) farmed in eastern China, with a mortality rate of more than 80%. To further investigate the characteristics and pathogenesis, we reported isolation, characterization and virulence of the causative agent of this disease from 10 sick crabs. Histopathological observation found that multiple tissues, especially haemolymph, contained lots of ciliates. The ciliate was isolated and cultured in vitro, and molecular and morphological studies were done. The results showed that SSU rDNA and LSU rDNA sequences of the ciliate were similar to Mesanophrys ciliates (>96.81%), while ITS1-5.8s-ITS2 sequence was similar to Mesanophrys pugettensis (95.37%) and identical to Orchitophrya stellarum (100%). Furthermore, the results of the morphological study confirmed that the ciliate was similar to Mesanophrys ciliates and O. stellarum cultured in supportive media, but different from O. stellarum cultured in living sperm cells of starfish (Leptasterias spp.). Also, the growth of the ciliate did not interfere with light, which was different from O. stellarum. Accordingly, the ciliate was classified as genus Mesanophrys and temporarily named as Mesanophrys sp. In addition, experimental infection confirmed that Mesanophrys sp. was the pathogen that infected farmed crabs. In summary, Mesanophrys sp. was first isolated and characterized in P. trituberculatus.

Antiparasitic Effect of Copper Alloy Surface on Cryptocaryon irritans in Aquaculture of Larimichthys crocea.

Copper and alloys containing >60% copper by weight are antimicrobial. In aquaculture, copper alloys are used as part of corrosion-resistant cages or as part of copper coating. To test whether a copper alloy surface prevents the outbreak of parasitosis in the aquaculture of Larimichthys crocea, we covered the bottom of the aquaculture tank with sheets of copper alloy containing 74% to 78% copper, and we cultured L. crocea juveniles that had been artificially infected with the protozoan parasite Cryptocaryon irritans Our results showed that these copper alloy sheets effectively blocked the infectious cycle of C. irritans within a 1-week period and significantly reduced the number of C. irritans trophonts and tomonts, thereby decreasing the mortality rate of L. crocea In in vitro assays, the cytoplasmic membranes of protomonts disintegrated and the cytoplasm overflowed after just 5 minutes of contact with copper alloy surfaces. Although the same cytoplasmic membrane disintegration was not observed in tomonts, the tomonts completely lost their capacity for proliferation and eventually died following direct contact with copper alloy sheets for 1 h; this is likely because C. irritans tomonts took in >100 times more copper ions following contact with the copper alloy sheets than within the control aquaculture environment. Exposure to copper alloy sheets did not lead to excessive heavy metal levels in the aquacultured fish or in the culture seawater.IMPORTANCECryptocaryon irritans, a parasitic ciliate that penetrates the epithelium of the gills, skin, and fins of marine fish, causes acute suffocation and death in cultured fish within days of infection. Much of the existing research centers around the prevention of C. irritans infection, but no cure has been found. Studies demonstrate that copper has strong antimicrobial properties, and fish grown in copper-containing cages have lower rates of C. irritans infection, compared to those grown in other currently used aquaculture cages. In this study, we found that an alloy containing 74% to 78% copper by weight effectively killed C. irritans cells and prevented cryptocaryoniasis outbreaks within a 1-week period. These findings offer a new perspective on the prevention and control of cryptocaryoniasis.

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.

Sustainable development of factory aquaculture through automation of ultraviolet parasiticide for the prevention and control of cryptocaryoniasis.

BACKGROUND: Cryptocaryon irritans infestations on marine teleosts are a considerable burden on factory mariculture. Ultraviolet (UV) light can kill C. irritans under laboratory conditions. However, a rational method for using UV in factory aquaculture to control cryptocaryoniasis has not been developed. This study focused on evaluating the killing effect of UV on protomonts and tomonts of C. irritans and established an automatic UV parasiticide device for the prevention and control of cryptocaryoniasis in marine teleosts. RESULTS: The survival rate of protomonts and tomonts decreased with an increase in the UV irradiation dose. All the protomonts and tomonts died within 14 and 24 min, respectively. The lowest UV lethal doses of protomonts and tomonts of C. irritans were 2.0 × 106 and 3.5 × 106 µWs cm-2 , respectively. Exposure of protomonts and tomonts to lethal doses of UV radiation led to shrinkage and severe dissolution of the protoplasm, causing abnormal development of cells. The survival rate of artificially infected Larimichthys crocea (treatment group, group A) was 83.33% at the end of the test (day 14) after disinfection using the automatic UV parasiticide device, whereas that of the control group (group C) was 90.00% (p < 0.05). However, all artificially infected L. crocea without disinfection using the automatic UV parasiticide device (untreated group, group B) died on day 8. CONCLUSION: The automation of traditional physical methods conforms to the sustainable development of aquaculture and provides a theoretical reference for the prevention and control of cryptocaryoniasis in mariculture. © 2022 Society of Chemical Industry.

In vitro and in vivo corrosion and histocompatibility of pure Mg and a Mg-6Zn alloy as urinary implants in rat model.

Pure Mg and a Mg-6wt.% Zn alloy were investigated as potential candidates for biodegradable implants for the urinary system. The in vitro corrosion behavior was studied by potentiodynamic polarization and immersion tests in simulated body fluid (SBF) at 37°C. The in vivo degradation and histocompatibility were examined through implantation into the bladders of Wistar rats. The alloying element Zn elevated the passivation potential and increased the cathodic current density. Both in vitro and in vivo degradation tests showed a faster corrosion rate for the Mg-6Zn alloy. Tissues stained with hematoxylin and eosin (HE) suggested that both pure Mg and Mg-6Zn alloy exhibited good histocompatibility in the bladder indwelling implantation and no differences between pure Mg and Mg-6Zn groups were found in bladder, liver and kidney tissues during the 2weeks implantation. Overall, this work presented instructive information on the degradation properties and histocompatibility of pure Mg and the Mg-6Zn alloy in the urinary system.

The generation of continuous-variable entanglement frequency comb.

Continuous-variable (CV) entanglement frequency comb can be produced by enhanced Raman scattering in an above-threshold optical oscillator cavity in which a hexagonally-poled LiTaO3 crystal resides as a Raman gain medium. The Stokes and anti-Stokes Raman signals are enhanced by a coupled quasi-phase-matching optical parametric process and the entanglement natures among these Raman signals and pump are demonstrated by applying a sufficient inseparability criterion for CV entanglement. Such entanglement frequency comb source with different frequencies and continuously tunable frequency interval may be very significant for the applications in quantum communication and networks.