| Feeding silkworms with mulberry leaves which were submerged and disinfected by bleaching powder liquid from hatching to maturing had become an effective method to prevent and control pebrine,was widely used in sericulture.But this method made silkworm’ s body weakened and breeding performance declined at the same time.To deal with these problems,researchers started exploring for improving the silkworm breeding performance,but further research on the physiological aspects of silkworm and changes in intestinal microflora were rarely reported.In this study,silkworms were fed with mulberry leaves which were submerged and disinfected by bleaching powder liquid of different effective chlorine concentration(0.3%,0.4%,0.5%,0.6%)from 4th instar to maturing,simultaneously the study set the control group that the bleaching powder liquid were replaced by water and another treatment group that silkworms were fed with mulberry leaves which were submerged and disinfected by bleaching powder liquid of 0.3% effective chlorine concentration from hatching to maturing.The growth and development index of silkworm larva and related physiological indicators of cocoon period were invested in this study;The enzyme activity of protease,amylase,lipase in the intestinal fluid and the enzyme activity of SOD,CAT and POD in the blood of silkworm were measured at day1,3,5 and 7 of the 5th instar larva,then the difference between different treatment groups and the control group were compared and analysed;The intestinal bacteria of the silkworm were isolated and purified with NA medium,and the differences of the number of intestinal colonies in the different treatment groups and the control group were analyzed;The difference of the colonies’ number,enzyme’s variety and relative enzyme production capacity of intestinal bacteria which can produce digestive enzymes(protease,amylase,lipase and cellulase)in different treatment groups were studied with selected medium.The enzyme-producing bacteria were identified with 16 S rDNA sequence alignment technique,and the differences of treatment groups and the control group were compared.According to these studies,we mainly got the following results:1.During treatment groups that silkworm larva were fed with mulberry leaves which were submerged and disinfected by bleaching powder liquid from the 4th instar,when the effective chlorine concentration was 0.3%,the weight gain of silkworm larva was promoted obviously;When the effective chlorine concentration was 0.5% and 0.6%,the weight gain of silkworm larva during 4th instar period was significantly inhibited,and the amount of ingested food and percentage of ingested food of 5th instar silkworm significantly decreased;The amount of ingested food and percentage of ingested food of 5th instar silkworm significantly decreased in the 0.3% effective chlorine concentration treatment group.The cocoon rate of each treatment group was significantly lower than that of the control group,and the death rate was significantly higher than that of the control.The whole larval life 0.3% effective chlorine concentration group had the greatest effect on the cocoon rate.The above results shows that the current method of feeding silkworms with mulberry leaves which were submerged and disinfected by 0.3% effective chlorine concentration bleaching powder liquid during the whole larval life will affect the digestion and absorption of the silkworm larva,and thus affect the health of the silkworm and have some influence on the physiological function of the silkworm.2.Effects of ingesting mulberry leaves which were submerged and disinfected by different effective chlorine concentration bleaching powder liquid on the digestive enzyme in the intestinal fluid and protective enzyme activities in the blood of 5th instar larva silkworm showed,the activity of protease and amylase was inhibited when the effective chlorine concentration was 0.3%,especially the activity of amylase was inhibited significantly.The activity of SOD,CAT and POD in the blood increased significantly;The activity of POD,CAT in blood significantly increased,and the activity of protease and amylase in the intestinal fluid of the silkworm significantly decreased and the activity of lipase significantly increased at 0.4%,0.5% and 0.6% effective chlorine concentration,the increase of lipase activity were speculated to be a resistance to active chlorine stress.The activity of protease and amylase in the intestinal fluid of the silkworm was inhibited in the treatment group that were fed with mulberry leaves which were submerged and disinfected by 0.3% effective chlorine concentration bleaching powder liquid during the whole larval life,especially the activity of the amylase was inhibited and the activities of CAT and POD significantly increased.These results indicated that feeding silkworm with mulberry leaves which were submerged and disinfected by 0.3% effective chlorine concentration bleaching powder liquid the whole larval life infected silkworm’s digestion and absorption of protein and carbohydrate in the mulberry leaf,especially that starch digestion was significantly inhibited.In addition,the strong oxidative properties of bleaching powder constitute a kind of stress on the silkworm body,so that the activity of blood protective enzyme system of silkworm was enhanced and coordinated to protect the silkworm from free radical damage.3.The amount of colonies in the intestine of the silkworm was inhibited in each effective chlorine concentration treatment group,and the inhibition enhanced with the effective chlorine concentration increase.The amount of the colonies decreased in the whole larval life 0.3% effective chlorine treatment group.The amount of colonies that can produce protease and amylase in the intestine of silkworm significantly decreased when feeding silkworms with mulberry leaves which were submerged and disinfected by bleaching powder liquid,and the average enzyme-producing capacity decreased at the same time.The lipase-producing bacteria was found only in the 0.6% group and the whole larval life 0.3% effective chlorine concentration group,and the ability of producing enzyme of strains in the 0.6% group was significantly higher than that of the whole larval life 0.3% effective chlorine concentration group,which may be related to the resistance of the lipase to the effective chlorine stress.In addition,the structure of intestinal ingestion enzyme-producing bacterial community in the intestine of silkworm changed after ingesting mulberry leaves which were submerged and disinfected by bleaching powder liquid.There were relatively rich enzyme-producing strain genus in the control group and the whole larval life 0.3% effective chlorine concentration group,they were Staphylococcus sp.,Lelliottia sp.and Buttiauxella sp..The enzyme-producing strain genus of the other treatment groups were relatively single,that was Staphylococcus sp.The above description shows that the effective chlorine stress inhibited the growth of the strains that can produce protease and amylase,and the enzyme-producing bacteria groups become single,which may be related to the change of digestive enzyme activity in the intestine and the nutriention digestion and absorption of the silkworm,the whole larval life 0.3% effective chlorine concentration group might have formed a response to effective chlorine stress due to long-term ingesting mulberry leaves that were submerged and disinfected by bleaching powder liquid.4.The activity of protease and amylase in the intestinal fluid of the silkworm decreased by ingesting mulberry leaves the whole larval life which were submerged and disinfected by 0.3% effective chlorine bleaching powder liquid.At the same time,the amount of strains which can produce protease and amylase and the ability of producing enzyme were inhibited.Thus,the ingestion and absorption of nutrients by silkworm were inhibited.In addition,the activity of POD and CAT in the blood of the silkworm increased upon the effective chlorine stress.In general,the constitution of the silkworm declined,the cocoon rate decreased and the death rate of pupa increased,which affected the growth and development,and the normal physiological activity of silkworm.Therefore,the way how to use bleaching powder should be improved in sericulture,or select more effective silkworm drugs to ensure the safety of silkworm production. |
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