| Chinese mitten crab(Eriocheir sinensis) is one of the most important economic species of freshwater aquaculture in China, and plays a special role in the aquaculture industry. Many pathogens, such as bacteria, virus, fungi and parasites can cause different diseases in Eriocheir sinensis. In the past, the traditional method of using antibiotics to prevent and cure disease leading very serious side effects, including severe resistance, food insecurity and environmental pollution. In order to apply the healthy concept in aquiculture in the future, we test the immunity and disease resistance effects of vegetable oil and astaxanthin in fattening diets for Chinese mitten crab and find the optimal amount of additives. The effects of different vegetable oil replacement ratio(0, 25%, 50%, 75% and 100%) on fattening crab to the resistance to Aeromonas hydrophila challenge, immune performance and microbial diversity of adult Enriocheir sinensis were explored and measured. Moreover, different content(0, 26.60, 41.62, 75.35, and 81.37 mg/kg) of astaxanthin which is a expensive extractive with high inoxidizability was used as a functional additive in the diets of male adult Enriocheir sinensis, the same biomarkers were determined to evaluate to the resistance and health level. This study provides a theoretical basis for Eriocheir sinensis diet exploitation and healthy aquiculture.1. Fish oil is a key content of fattening diets of crab which can promote the development and improve the resistence. Although the benefits are dramatic, the high cost and the toxic product of residual diets oxidation after long-term use of fish oil could obviously reduce its value. In recent year, the vegetable oil, which is cheap and can partially replace fish oil, became a research hotspot, and many issues to consider are their effects on the fish growth, animal welfare and the final product quality, but the important effect of crab health, which is resistant to pathogens is little known when evaluating vegetable oil. In our study, significant difference of survival rate was found in all 5 groups of fattening period crabs, high content of fish oil in diets improved the resistance of male crabs to Aeromonas hydrophila challenge, the no replacement group has the highest survival rate of 37.5%, followed by 12.5% of 25% and 75% vegetable oil replacing group. Dramatically, the survival rate of 100% replacing group was 0%.The female crabs of 75% and 100% vegetable oil replacing group have a higher level of resistance to Aeromonas hydrophila challenge compared with other groups, and the survival rate were 20% and 40%, respectively. In order to find the reason of different survival rate in each group, the pathological section, immune related genes and intestinal flora were performed.Pathological sections results shown that, compared with controls, pathological damage is not obvious in hepatopancreas and gills for 25% vegetable oil replacing group in the lethal male crabs infected by Aeromonas hydrophila, hepatopancreas cells shed and gills lamellae dissolution inordinately in other groups. The most serious symptoms were found in 100% vegetable oil replacing group with a large area of hepatopancreas cells cavity structure, necrosis and turbulence, followed by the epithelium cytolysis of gill, the structures of some gill lamellae even disappeared only left the chitin cavitation. A large mass of impurities were found among the gill lamellae. For the female crabs, pathological damage was not obvious in hepatopancreas and gills in 75% and 100% vegetable oil replacing groups. But a large numbers of hepatopancreas cells cavity structure shed, and gill epithelium cells proliferation or atrophy, corrugated, and irregular were found in the groups with 0 and 50% vegetable oil replacements. Gill lobe cavity expansion and impurities accumulation were also found among the gill lamellae.The results of immune related genes showed that different replacement ratio in diets can significantly affect the alkaline phosphatase(ALP) activity in serum and gonad of male crabs in all groups(P<0.05). The ALP activities of serum and gonad in 25% replacing group decreased significantly. In addition, the activities of acid phosphatase(ACP) and superoxide dismutase(SOD) did not change obviously. In contrast, the activities of serous SOD and gonadal ACP of female crabs were significantly reduced in the no replacement group(P<0.05), but increased in 75% and 100% replacing group(P<0.05). Moreover, the different diets also significantly impacted the relative expression level. The significant up-regulated expression of immune related genes, such as myeloid differentiation factors 88(My D88), fatty binding protein 3(FABP3), SOD and lysozyme(LZM) were found in hepatopancreas of the male crabs from 25%, 50% and 75% replacing groups, but the expression of hemocyanin(Hc) only increased in hepatopancreas of male crabs from 50% replacing group(P<0.05). In addition, the expression of serous myd88 in female crabs from 75% and 100% replacing group was significantly up-regulated(P<0.05).In order to find the relationship between intestinal flora and the immunity and disease resistance and the effects of bacteria from gills and water on the intestinal flora, the PCR amplification of 16 S r RNA V4 variable region of fore intestine, hind intestine, gills and aquculture water of Eriocheir Sinensis were sequenced by pyrosequencing technique and performed using an Illumina Mi Seq instrument. The results demonstrated that it is obvious similarity in the intestinal bacterial composition of male crabs and female crabs, the dominant phylum are Tenericutes, Firmicutes, TM7, Bacteroidetes and Proteobacteria in the fore intestine, in contrast to Tenericutes, Firmicutes, TM7, Bacteroidetes, and Proteobacteria in the hind intestine, as well as Acidobacteria, Actinobacteria, TM7, Bacteroidetes, and Proteobacteria in gills, and Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Chloroflexi, Cyanobacteria, Thermi, Verrucomicrobia, TM7, Acidobacteria in aquculture water. These results suggested that dominant phylum component of Eriocheir sinensis are affected by the vegetable oil replacement ratio in diets, however, the changes in these 5 groups on microflora relative abundance are different.2. There was a significant difference in 5 groups with various contents of astaxanthin in fattening period for male Chinese mitten crabs. The result showed that both 41.62 mg/kg and 75.35 mg/kg astaxanthin in the diets can improve the resistance to Aeromonas hydrophila challenge of crabs, and had the highest survival rate of 42% for the 41.62 mg/kg group. Different additive amount of astaxanthin in diet significantly affect the relative expression level of immune related genes. The expression of My D88, Caspase and SOD in hemocyte and hepatopancreas were both down-regulated significantly in the 26.60 mg/kg, 41.62 mg/kg and 75.35 mg/kg group(P<0.05).The dominant bacteria number and composition were measured by culture-based enumeration techniques, and then the isolates were sequenced by 16 S r RNA gene analysis and obtained the accession number in NCBI for all 5 groups crabs. 106 bacterial strains(accession number from KU570293~KU570368, KU570370, KU570372~KU570383, KU601302~KU601316 to KU720553) were isolated from the intestinal tract, gills and aquaculture water, 92 bacterial strains were dominant bacteria, among them, 29 bacterial strains were in the intestinal tract, 32 bacterial strains were in the gills and 31 bacterial strains were in the aquaculture water. The dominant bacteria were Citrobacter, Pseudomonas and Aeromonas in the intestinal tract, total number of culture-based dominant bacteria of group B in intestinal tract was the highest(1.06×108 cfu/g). The number of potential pathogenic bacteria of group E(75.35 mg/kg) was significantly decreased(P<0.05). The dominant bacteria were Citrobacter, Bacillus, Pseudomonas and Aeromonas in the gill, total number of dominant bacteria of group 5(75.35 mg/kg) was significanthigher than the other four groups. The dominant bacteria were Bacillus, Aeromonas, Citrobacter, Pseudomonas and Chryseobacterium in the aquaculture water, but the numbers of dominant bacteria have no significant difference in the 5 groups. The number of potential pathogenic bacteria in intestinal tract and gills was account for high proportion in 41.26 mg/kg dietary astaxanthin group. To conclude, dietary astaxanthin with various concentrations could significantly affect the survival ratio of crabs challenged with the Aeromonas hydrophila and culture-based dominant bacteria composition of intestinal tract and gill in adult male crabs.In summary, we test the immunity and disease resistance effects and check the intestinal flora of vegetable oil replacement of fish oil and astaxanthin in fattening diets for Chinese mitten crab in the present study. The results help to find the optimal amount of additives. It will provide the theoretical basis for Eriocheir sinensis diet exploitation and useful probiotics information for healthy aquiculture in future. |
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