| Phenol,as a priority pollutant of many industrial point sources,such as oil processing industry,pesticide factory,and chemical trades,is often found in aquatic environment.In this study,according to the LC50 of phenol on Moina macrocopa at 48 h(2.45 mg/L),we set up five phenol concentration groups(0.25,0.75,1.25,1.75,2.25 mg/L)and one control group for 24 h and 48 h.Firstly,we observed the histological changes on the epidermis of M.macrocopa exposed to different concentrations of phenol using light microscopy and electron microscopy.Then,we measured the molting frequency,chitinase,β-NAGase,ecdysone,NO and NOS.Finally,we looked for the stable reference genes,and analyzed the m RNA expression of Ec R,HR3,βFTZ-F1 and CP of M.macrocopa exposed to phenol.The experimental results are as follows:(1)The thickness of epidermis was first increased and then decreased with the increase of phenol concentration at 24 h.The thickness of epidermis was reached the peak at 0.25 mg/L phenol(P<0.01),and significantly decreased at 2.25 mg/L phenol compared with the control group(P<0.05).The thickness of treatment groups were significantly thinner than that of control group at 48 h(P<0.05,P<0.01).The ultrastructural changes of epidermis cell were observed by transmission electron microscopy(TEM).The shape of nucleus changed from regular to irregular when exposed to phenol.At the same time,the other phenomenons were observed,such as nucleolus ruptured,chromosome condensation marginalized,nuclear membrane swelled.In addition,the swelling of mitochondria,the vacuolation of cristae,and the fracture of rough endoplasmic reticulum were observed.The results indicated that the epidermal structure of M.macrocopa was obviously damaged.(2)With the increase of phenol concentrations and the extension of treatment periods,the molting frequency of M.macrocopa was a clear decrease when exposed to phenol,especially in the highest concentration group,and was significantly less than the control group(P<0.01).The chitinase activity and ecdysone content were decreased with the increase of phenol concentrations,which were significantly decreased among 0.75~2.25 mg/L phenol(P<0.05,P<0.01).At 1.25 mg/L for 24 h,compared to that of the control group,the activity of β-NAGase was increased.With the concentration increase,the activities dropped sharply,and there was no difference between the other treatment groups and the control group.The NO content was similar to that of NOS activity.(3)Based on the four methods to look for the stable reference gene from three candidate genes,16 S r RNA was identified as the best reference gene for q RT-PCR of M.macrocopa.With the increase of phenol concentration,the m RNA expression of Ec R first increased and then decreased at 24 h and 48 h.In the lowest concentration group of phenol,the m RNA expression of Ec R was significantly higher than the control group(P<0.01).The HR3 was significantly reduced(P<0.01,P<0.05).The expression of βFTZ-F1 was similar to the Ec R,and the 0.75 mg/L phenol was significantly higher than the control group(P<0.01,P<0.05).The m RNA expression of CP gene was decreased at 24 h,especially in the highest concentration group,and were significantly less than the control group(P<0.05),and no significant differences were observed between other treatment groups and the control group.The treatment groups of CP gene were significantly lower than the control group at 48 h(P<0.01,P<0.05).The following conclusions are drawn from the comprehensive experimental results:(1)The thickness and shape of epidermis were observed a clear change.These were indicated that phenol had caused high toxicity to aquatic organisms.(2)The molt of M.macrocopa was inhibited by phenol,and the chitinase,β-NAGase,ecdysone,NO and NOS were also affected.The differences between the treatment groups and control group were markedly different with the increase of phenol concentration and the extension of stress time.These suggested that phenol had toxic effect on the molt of M.macrocopa.(3)The m RNA expressions of Ec R,HR3,βFTZ-F1 and CP of M.macrocopa were abnormal after phenol treatment,which suggested that phenol could affect the expression of molting-related genes on the transcriptional levels of M.macrocopa. |
