| Macrobrachium nipponense is an important freshwater species for aquaculture in China. With therapid production and development of the prawn industry, prawn diseases have become severe in recentyears. The exploration of prawn immune response is therefore of great signifcance. Our previous study hasdemonstrated that the treatment of imidazole derivative KK-42can increase the survival rate of juvenileLitopenaeus vannamei. In order to research whether the similar effect of KK-42on M. nipponense existed,we firstly investigated the survival rate of M. nipponense challenged Aeromonas hydrophila after KK-42treatment; secondly we observed the histological changes of hepatopancreas of M.nipponense challengedA. hydrophila and analysed the expressions of humoral immunity factors such as α2M and chitinase. Theresults were as follows.About600prawns with body length of3.5~5.0cm randomly were divided into three groups: KK-42treatment, control, blank. The prawns of KK-42treatment or control group were soaked for1min inKK-42solution at a concentration of1.95×10-4mol/L or0mol/L, respectively. After12h, the prawns ofthese groups or blank group were injected with20μL of A. hydrophila suspension or sterile saline solutioninto the abdomen muscle. The survival rate for48h after A. hydrophila-challenge test was calculated; thehepatopancreas and hemolymph were collected, and the histological changes of hepatopancreas ofM.nipponense was observed by paraffin section; and the expressions of α2M and chitinase genes wereassayed using Real-time PCR; and the activities of α2M and chitinase were monitored byspectrophotometer method.The survival rate of blank group keeped upon90%although few prawns died because of molt. AfterA.hydrophila injection, the survival rate of KK-42treatment group respectively increased by25%,141%and133%at6,12,24h compared with control group which rapidly descended and retained lower level at24h.The histology result revealed that the hepatopancreas tubules of control group had complete structureand clearly distinguished F-cell, the lumen with floccules extended, basement membranes fractured andmany big vacuoles observed, the structure restored and few vacuoles observed at0,6,12,24h, respectively.The hepatopancreas tubules' lumen was relatively smaller at6h in KK-42treatment group, few hepatopancreas tubules were damaged at12h and the structure was similar to those of0h at24h.A partial fragment of α2M gene was cloned from M. nipponense by PCR, Sequence comparisonshowed that the fragment shares90%identity with that of Macrobrachium rosenbergii. The quantitativereal-time PCR analysis showed that α2M was mainly expressed in haemocytes; a2M mRNA level was thehighest in stages D, and the lower in stages A, B and C stages. After KK-42treatment, the hepatopancreasα2M mRNAlevel significantly increased compared with the corresponding controls at6~48h(P<0.01),but the haemocyte α2M mRNA level couldn't showed significant differentiation. Atfer A. hydrophilainjection, the α2M mRNA level of hepatopancreas or haemocyte significantly increased at3,6,12,48h or3h, respectively(P<0.05).However, the haemocyte or hepatopancreas α2M mRNA level significantlyincreased at6,12,48h or12h, respectively, compared with the corresponding control in KK-42treatmentgroup(P<0.05). The haemolymph α2M activity pattern was similar to α2M mRNA level.A partial fragment of chitinase gene(Mnchi-3)was obtained, sequence comparison showed that thededuced amino acid of Mnchi-3cDNA fragment involved chitinase catalyticactive site'-LDGLDMDWE-'and the fully conserve glutamate residue '-G-', and which revealed homology around90%compared to those in other species. The real-time PCR analysis analysis showed that Mnchi-3wasexpressed mainly in hepatopancreas, and Mnchi-3mRNA level was the highest in stages D2/D3. AfterKK-42treatment, the hepatopancreas Mnchi-3mRNA contents significantly increased by150%comparedwith the corresponding controls at12h(P<0.05). Atfer A. hydrophila injection, the Mnchi-3mRNA levelsignificantly increased at3h; the Mnchi-3mRNA contents increased by127%,304%,120%, respectively,compared with the corresponding controls at12,24,48h in KK-42treatment group(P<0.05). Thehepatopancreas chitinase activity significantly increased at24h in KK-42treatment group(P<0.05).Conclusion:Administration of imidazole derivative KK-42can increase the survival rate of M.nipponense, reduce the degree of damage of hepatopancreas of M. nipponense challenged A. hydrophila,and induce the α2M, Mnchi-3gene expressions and enzyme activities. |
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