| Procambarus clarkii is an important aquatic economic animal in China.In 2019,the total production of Procambarus clarkii reached 2.0896 million tons,and the total area of culture reached 19.29 million mu,which increased by 27.52%and 14.80%respectively compared with 2018.With the explosive growth of culture industry,the harm of pathogenic bacteria to Procambarus clarkii culture industry also increased.In the culture environment of Procambarus clarkii,Aeromonas hydrophila is ubiquitous,which is also related to the harm of fish and crustaceans.It can be isolated from healthy Procambarus clarkii and is considered as a potential pathogen.In recent years,Procambarus clarkii aquaculture industry widely uses antibiotics to control bacterial diseases.A large number of antibiotics will make many bacteria resistant.Therefore,it is urgent to find a kind of natural plant additive which is good for animal health and environment.Plant extracts are widely used as a kind of natural and safe active substances.Among them,tea tree oil as a natural plant additive has been used in animal breeding.In view of this,this study aimed to determine the antibacterial mechanism of tea tree oil on Aeromonas hydrophila and its effect on the growth performance of Procambarus clarkii,so as to provide theoretical basis for the application of tea tree oil in aquaculture.The test mainly includes the following two parts1.Study on the antibacterial mechanism of tea tree oil against Aeromonas hydrophila in vitroThe effects of tea tree oil on the antibacterial activity,membrane permeability,membrane integrity and cell ultrastructure of Aeromonas hydrophila were studied(1)The inhibition zone of tea tree oil to Aeromonas hydrophila showed that Aeromonas hydrophila was highly sensitive to tea tree oil.(2)The results showed that the minimum inhibitory concentration(MIC)of tea tree oil against Aeromonas hydrophila was 0.039 mg/ml.When the concentration of tea tree oil was higher than 1 mic,the growth of bacteria was almost completely inhibited.The germicidal efficacy of high concentration tea tree oil increased with the extension of action time and the increase of concentration.1 mic tea tree oil could completely kill the bacteria within 2hours.During the treatment of 1/2 MIC tea tree oil to Aeromonas hydrophila,the total bacterial colony number of the whole group was lower than that of the control group.(3)The permeability of tea tree oil to Aeromonas hydrophila membrane showed that the K~+concentration and DNA exudation of tea tree oil treatment group were significantly higher than those of control group;The exudation of 1/2 MIC and 1 mic tea tree oil groups tended to be stable after 2 h;The permeability of cell membrane was as follows:1 mic tea tree oil group>1/2 mic tea tree oil group>control group.(4)Effect of tea tree oil on Aeromonas hydrophila biofilm:by measuring the absorbance at595 nm after diluted with 33%glacial acetic acid solution,it was found that there was a significant difference between the treatment group and the control group(P<0.01).The effect of 1 mic tea tree oil group on the removal of biofilm was about 55%higher than that of the control group.The clearance rate of tea tree oil to biofilm was concentration dependent.PI staining showed that with the increase of tea tree oil concentration,the number of living cells decreased significantly,indicating that tea tree oil could destroy the biofilm of Aeromonas hydrophila.(5)The effect of tea tree oil on the microstructure of Aeromonas hydrophila:in the control group,the cell edge was clear,neat and smooth,the cell membrane had no defect and fracture,and the cell contents were clear and evenly distributed in the cells;The cytoplasmic wall was tightly bound without loss.The surface of cell wall of 1/2 MIC treated cells was uneven with obvious deletion;Part of the cell contents overflowed.In mic group,there was no clear internal structure,cell wall was seriously damaged,and plasmolysis occurred.(6)Effects of tea tree oil on virulence genes of Aeromonas hydrophila results:compared with the control group,the expression levels of serine protease gene aha and outer membrane porin gene Omp A were significantly down regulated after 1/2 MIC tea tree oil treatment(P<0.01);The expression levels of virulence gene thermosensitive extracellular protease gene eprcai,cytotoxin gene rtx A and adhesin gene ahp A were significantly down regulated(P<0.05).The results showed that tea tree oil could reduce the virulence of Aeromonas hydrophila by regulating the virulence factors;At the same time,it increases the permeability of the cell membrane of Aeromonas hydrophila,destroys the integrity of the cell membrane,causes the leakage of cell contents,and finally leads to the death of the cell.2.Effects of tea tree oil on growth performance and serum biochemical enzyme activities of Procambarus clarkiiThe purpose of this experiment was to study the effects of different levels of tea tree oil on the growth performance of Procambarus clarkii.The healthy and lively Procambarus clarkii with uniform size,intact appendages were randomly divided into three groups,with 3replicates in each group,40 prawns in each replicate,a total of 360 prawns in total.They were raised in a cement pond and fed with the experimental diet for 60 consecutive days.The results are as follows:(1)Effects of dietary tea tree oil on growth performance of Procambarus clarkii results:compared with the control group,there were no significant differences in survival rate,specific growth rate,feed coefficient and fatness of Procambarus clarkii fed diets supplemented with 50 mg/kg and 100 mg/kg tea tree oil(P>0.05).The weight gain rate increased with the increase of tea tree oil dosage.(2)Effects of tea tree oil on serum biochemical enzyme activities of Procambarus clarkii were studied:There was no significant difference in antioxidant capacity(T-AOC)and malondialdehyde(MDA)of Procambarus clarkii fed with tea tree oil(P>0.05);The total protein(TP)and superoxide dismutase(SOD)in serum were significantly affected(P<0.05),which reached the maximum in 100 mg/kg tea tree oil group;The serum acid phosphatase(ACP)in 100 mg/kg tea tree oil group was significantly higher than that in 50mg/kg tea tree oil group and control group(P<0.01);The serum glutathione peroxidase(GSH-Px)increased with the increase of tea tree oil dosage.The results showed that tea tree oil could promote the growth of Procambarus clarkii and improve its antioxidant capacity. |
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