Variation In Activities Of Two Metabolic Enzymes In Silkworm Inoclulated With BmNPV And Preliminary Separation And Detection Of Metalloprotein In Silkworm Heamolymph

Bombyx mori, also known as silkworm, is a kind of insect with important economic value and also a typical model organism for the study of Lepidoptera. BmNPV (Bombyx mori nuclear polyhedrosis virus) is the main disease virus in silkworm rearing which causes a variety of Bombyx mori larvae disease and leads great economic loss in sericulture. Virus invasion affect silkworm normal physiological metabolism, especially cause the change of enzyme activity. The Study found that carboxylesterase and acetylcholinesterase activities may be associated with the resistance to the virus, but its variation trend is unclear. Metallic elements are important components of silkworms and are ubiquitous in their bodies, and they are also involved in every stage of growth and development of silkworms. In general, the metallic elements in the organism can be divided into two classes (macro elements and trace elements) according to their contents, most of the metallic elements participate in various life activities through the combination with proteins. Therefore, the separation and detection of metalloproteins were essential for the study of the structures and functions of them in silkworms. In this study, silkworms with and without Bombyx mori nuclear polyhedrosis virus (BmNPV) resistance were chosen as experiment materials, their fifth-instar larvae were inoculated with BmNPV, inductively coupled plasma mass spectrometry (ICP-MS) was applied for the detection of 11 kinds of metallic elements in the heamolymph of silkworms, then size exclusion-high performance liquid chromatography (SEC-HPLC) was coupled with ICP-MS for the separation and determination of the metalloproteins in silkworms. The results were as follows:1. Silkworms with BmNPV resistance and non-resistance were selected to be raised, and then their fifth-instar larvae were inoculated with BmNPV, finally their heamolymph and midgut tissues were studied in the next 5 days for the evaluation of the activity of carboxylesterase (CarE) and acetylcholinesterase (AchE). It revealed that the activity of CarE in midgut tissues in resistant silkworms inoculated with BmNPV improves much compared with the one in silkworms not inoculated with BmNPV, suggested that the silkworms with BmNPV resistance had made effective response and defense to the virus they were infected with; but the activity of the AchE in midgut and heamolymph tissues in resistant silkworms had no obvious change, compared with non-resistance, so it was supposed that the silkworms with BmNPV resistance still kept relatively stable metabolic processes after infected with BmNPV.2. The method for the detection of 11 different metallic elements (Na、Mg、K、Ca、 Mn、Co、Ni、Cu、Fe、Zn、Rb) in silkworm heamolymph of four groups (silkworm with BmNPV resistance, silkworm without BmNPV resistance, silkworm with BmNPV resistance inoculated with BmNPV, silkworm without BmNPV resistance inoculated with BmNPV) by using ICP-MS was established. The results demonstrated that in the silkworm heamolymph of the four groups, the contents of Na, Mg, K and Ca were significantly higher than the other elements and could reach μg/mL level, the contents of the left elements such as Mn、Co、Ni、Cu、Fe、Zn and Rb were relatively lower and were at the level of μg/L, among which the content of Co was below μg/L. In the silkworms with and without BmNPV resistance, the variation trend of the same element was basically coincide in the 5 days, but the variation degree of Mn, Fe, K, Ca and Mg in the first 4 days were slightly larger than that in the former. After the silkworms (with and without BmNPV resistance) were inoculated with BmNPV, the metallic element contents between the two were different. The variation law of most of the metallic elements was coincident either in the silkworms with or without BmNPV resistance, which might be attributed to the BmNPV infection; but there were still some differences of the variation of Cu, Ni and Rb between the two kinds of silkworms, which might have some relationships with the BmNPV resistance.3. In this assay, SEC-HPLC (30mmol/L Tris-CH3COOH, pH 7.4, running buffer at the flow rate of 0.7 mL/min,50μL sample volume) was utilized for the separation of the metalloproteins in silkworm heamolymph of four groups (silkworm with BmNPV resistance, silkworm without BmNPV resistance, silkworm with BmNPV resistance inoculated with BmNPV, silkworm without BmNPV resistance inoculated with BmNPV); then ICP-MS was used for the determination of the metallic elements in the metalloproteins. The results showed that the retention times of the silkworm heamolymph metalloproteins ranged from 8 to 22 min, Cu and Fe elements could both combine with the proteins with high or small molecular weights, most of the Zn and Co elements could combine with the proteins whose retention times range from 12 to 20 min, Rb and most of Mg, Ca and Mn elements could combine with the proteins with small molecular weights whose retention times were more than 20 min. Compared the peaks of the four groups, it was found that 5 days after the silkworms were inoculated with BmNPV, an obvious peak with Cu elements (retention time 8 min-10 min) appeared in the heamolymph of silkworms without any BmNPV resistance, i.e. lots of metalloproteins with Cu elements and high molecular weights were found. In addition, it demonstrated that 3 or 4 days after the silkworms were inoculated with BmNPV, the contents of metalloparotein with Cu elements between the four kinds of silkworms were much different from each other.