| Copper is the essential trace element of bee,which plays important roles in improving tissue antioxidant capacity,promoting animal growth and development,and improving the immune function of animals.In this study,the Italian bee(Apis mellifera L)was used as the research object to feed artificial diets supplemented with different levels of copper in bee larvae during artificial rearing and adult bee maintained in vitro in laboratory cages,and to explore the effects of copper on honeybee growth and physiological function.Accounting to the impact of growth development,physiology function,lifespin and other indicators,and to the appropriate level of copper was obtained which added to the development of bee larvae and adult.The following experiments were carried out:The first one: study on the suitable level of copper in feeding of bee larvae.In the experiment,1440 larvae of the Italian honeybee were selected,randomly divided into 6groups,and each group had 10 repetitions,each of which had 24 larvae,and was raised in24-hole cell culture plate.The control groupwas fed with a basical diet prepared with D-glucose,D-fructose,yeast extract,fresh royal jelly,deionized water mixture of fodder,and the copper level was 2.69 μg/g,the remaining 5 treatments were fedwith glucose acid copper is added in the basis of fodder fodder,copper levels were 1.00 μg/g 、 2.00 μg/g 、3.00μg/g 、4.00 μg/g and 5.00 μg/g.In the laboratory,artificial feeding was carried out,and the larvae were replaced daily until the pupae were removed.Determination of different day age larval weight,body length,pupa rate,eclosion rate,haemolymph biochemical index,activity of antioxidant enzymes,lipase activity and antioxidant enzyme gene expression related measure,high affinity copper transporters gene expression,defense element quantity indicators such as gene expression,and statistical analysis.The experimental results showed that:(1)Larvae of artificial fodder to add copper make bee pupa rate and feather out of the room rate is significantly reduced(P < 0.05),copper level affect the bee larva body length significantly(P < 0.05),copper affects the bee larvae weight was not significant.(2)Copper has a significant influence on the biochemical indicators of blood larvae of honeybee larvae.Cu levels in diets ranged from 1.00 to 5.00 μg/g.With the increase of copper levels,the total protein and total cholesterol in the haemolymph of bee larvae were significantly reduced(P<0.05).(3)copper has significant influence on antioxidant enzyme activity and lipase activity of bee larvae(P<0.05).With the increase of copper content in the diet,the LPS activity of bee larvae increased significantly(P<0.05).(4)the effects of copper on the expression of antioxidant enzymes related genes and the expression of defensin genes in the larvae of Italian honeybee at different ages were significant(P<0.05).The relative expression levels of Cu/Zn-SOD,CAT and Defensin1 genes increased first and then decreased with the increase of copper content in diet(P<0.05).The second one: The effect of copper on the physiological function of the audlt bees.3000 new emerged audlt bees were collected and randomly divided into 6 groups with 5 repeat,each repeat take 100 bees rearing in the feeding box;The control group(Group A)was fed with a mixture of sugar and deionized water and copper level of 0.00 mg/kg of fodder,five treatment groups(Group B,C,D,E and F)were fed by the sucrose,deionized water and glucose acid mixture of the copper with the copper level of acid copper were 2.00 μg/g,4.00μg/g,6.00 μg/g,8.00 μg/g and 10.00 μg/g sugar water.The feeding box was placed in a constant temperature and humidity incubator and maintained at 32° C and 50% ± 5% R.H.rearing conditions.Determined the indicators of lifespan,body tissue copper content,hemolymph biochemical indicators,antioxidant enzyme activity,ceruloplasmin activity,lipase activity and expression of antioxidant enzyme-related genes,high affinity copper transporter gene expression,defensin gene expression and so on.Midgut slices were prepared.The results showed that:(1)from the point of bees survival curve,the survival rates of the treatment group B and C were significantly higher than those of treatment group D,E,F(P <0.05);When the copper level in the diet was 2.00 μg/g,the structure of the midgut’s midgut membrane achieve maximum preformance.(2)Copper has a significant effect on the body tissue copper content and hemolymph biochemical index content.When the copper level in the diet is continuously increased within the range of 0.00 to 10.00 μg/g,the copper content of the honeybee body tissue is significantly increased(P <0.05),the total protein and total cholesterol content of bees in the hemolymph was significantly reduced.(3)copper had a great effect on activity of antioxidant enzymes,lipase activity to bees.D treatment group of bees Cu/zn-SOD activity was significantly higher than other treatment group,E treatment group had a significantly higher LPS activated than other group(P<0.05).(4)copper has asignificant influence on the expression of antioxidant enzyme related genes,high affinity copper transporter gene expression and defense gene expression in Italian honeybees of different ages.The relative expression of Cu/Zn-SOD gene in group E was significantly higher thanothers(P<0.05).The relative expression of CAT gene increased significantly(P<0.05),and the relative expression of ctr1 was significantly increased(P<0.05)when the copper level in the diet increased continuously in the range of 0.00~10.00 μg/g.The effect of copper on the relative expression of defensin1 gene was significant(P<0.05),and the relative expression level of the C treatment group was significantly higher than that of the treatment group D,E and F(P<0.05).According to the above studies,when the larvae of Italian honeybee were bred indoors,the appropriate level of copper in the diet was 1.14~2.41 μg/g.The effect of copper level on the physiological function of honeybee was significant,especially for the longevity of honeybee,copper content in body tissues,antioxidant capacity and related gene expression. |
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