The Analysis Of The Homolymph Metalloprotein Difference Of The Fifth-instar Larvae Under Pesticide And Effects Of Metallic Elements On The Phoxim-induced Toxicity Symptom Of Fifthinstar Larvae And

Bombyx mori, which belongs to Bombycidae of Lepidoptera, is a kind of insect with economically importance, and has a long history of domestication in China. Phoxim is the most commonly used pesticide in pest control, and has seriously restricted the development of silk industry in China. Metal elements play an important role in biological systems and carry out vital biological functions in a variety of metalloenzymes and metalloproteins.Metallome is an emerging discipline focused on the study of the distribution, chemical species, content, structure, feature, and function of metal elements in biological system. Metalloproteins are important competent of metallome. Approximately 30% of proteins present in cells are metalloproteins or metal – containing protein complexes, and around 40% of those metals in the proteins confer biological functionality. The methods for separating metalloproteins are mainly liquid chromatography and gel electrophoresis. The approaches of metal detection in metalloproteins are mainly High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry(HPLC-ICP-MS) and Laser Ablation Inductively Coupled Plasma Mass Spectrometry(LA-ICP-MS). In our study, The(size exclusion method) SEC and non-denatured gel electrophresis(GE) were used to separate the proteins in the homolymph of fifth-instar larvae under phoxim, chorfenapyr and cypermethrin, then the detection of elements was carried out by ICP-MS and LA-ICPMS. FeCl2, CuCl2, and RbCl were used to feed the fifth-instar larvae, which could relieve the phoxim – induced toxicity symptom; The main results were summarized as follows:1. The proteins in homolymph of fifth-instar larvae under phoxim, chorfenapyr and cypermethrin were separated by using SEC(30mmol/L Tris- CH3 COOH, pH7.4 running buffer at the flow rate of 0.7 mL/min, 50μL sample volume), then ICP-MS was utilized for the detection of the metallic elements. The proteins of silkworms were well separated under this chromatographic conditions. The results revealed that there were some differences of the protein peaks between the pesticide–induced group and the control group. Then the difference of he metallic elements(Fe, Cu, Zn and Mn) of metalloproteins in the homolymph of silkworms was maesured by HPLC-ICPMS. The results showed that there were abundant Fe, Cu, Zn and Mn elements in the metalloproteins in homolymph. Compared with the control group, the content of metalloproteins(with Fe, Cu, Zn and Mn elements) in the homolymph of fifth-instar larvae treated by phoxim, chorfenapyr and cypermethrin was remarkably decreased, and the peak of the metalloprotein with Cu element was even undetectable.2. In our study, non-denatured polyacrylamide gel electrophoresis(PAGE) was used to separat the proteins in the silkworms homolymph under the condition of low current and temperature, then the gel was stained with Coomassie Biosafe. The results indicated that there was a significant difference of the protein bands between the groups under pesticide and the blank group, among which 5 protein(most with small molecular weight) bands were especially obvious. Then LA-ICP-MS was used for the detection of the metal elements of detect Fe, Cu, Zn and Mn in the different protein bands. It demonstrated that there were abundant metallic elements could be detected in the bands, indicating the existence of metalloproteins in the 5 different protein bands.3.The mulberry leaves were respectively sprayed withFeCl2(50 mg/L), CuCl2(150 mg/L), and RbCl(0.5 mg/L) to feed the silkworms. After phoxim exposure 48 h, the death rate of the phoxim- exposed silkworms was 47%, whereas the ones of the FeCl2 + phoxim-treated group, CuCl2 + phoxim-treated group, and RbCl + phoximtreated group were 27%, 32%, 22%, respectively. The reason may be that FeCl2, CuCl2, and RbCl can alleviate the poisoning symptoms of the silkworms under phoxim and increase the survial rate of the larvas. It could be seen that phoxim infection significantly inhibited the activities of SOD, CAT, and CarE compared with control group after phoxim exposure at 48 h. However, the added FeCl2, CuCl2, and RbCl could remarkably promoted the activities of SOD, CAT and CarE. CuCl2 improved the activity of SOD obviously; FeCl2 could improve the activity of CAT; FeCl2 and CuCl2 had better effect on the improvement CarE activity.