Accumulation Of Zn²⁺ In The Hemolymph Of Larvae Of Hermetia Illucens L. And Its Preliminary Effects On The Enzyme Activity And Protein Of Hemolymph

Objective: Different concentrations of Zn²⁺ were added into the artificial feed and the accumulation of Zn²⁺ in the hemolymph of larvae of Hermetia illucens L. for three consecutive generations under such stress and the CAT, T-AOC, PO, MT and 4KD, 8KD small molecular protein of effects.content was examined in the hemolymph of 5-day-old larva of 1st and 3rd hermetia illucens L. This thesis will provide the theoretical basis for the operation of Hermetia illucens L. excrement harmless treatment system, on new insight research and development of its by-products.Methods:Different concentrations of Zn²⁺（0, 75, 150, 300, 600, 1200mg/kg） were added into the artificial feed to breed Hermetia illucens L. for three consecutive generations; the enzyme activity and protein content was examined in the hermetia of 5-day-old larvae of 1st and 3rd Hermetia illucens L. The accumulation data of Zn²⁺ in the hemolymph was determined by the plasma atomic emission spectrometry; The activity of CAT and T-AOC and concentration of PO and MT was detected by ELISA assay. After ultrafiltration and centrifugation.the protein content of crude extracts from the hemolymph was measured by A-280. Tricine-SDS-PAGE was utilized to separate the protien crude extracts; the aminoacid sequence of the target peptide 4KD was analyzed by LC-MS-MS.Results:（1） The content of Zn²⁺ in the hemolymph of larvae of 1st, 3rd Hermetia illucens L. under the stress of Zn²⁺ increased with the treatment concentration of Zn²⁺, and a significant difference among all treatment groups was identified（p<0.05）; the content of Zn²⁺ in the hemolymph of larvae of 3rd Hermetia illucens L. was significantly higher than that of 1st（P<0.05）.（2） When the treatment concentration of Zn²⁺ falls in the range of 75-300mg/kg, the enzyme activity of CAT and T-AOC and levels of PO and MT in the hemolymph of larvae of 1st, 3 rd Hermetia illucens L. enhanced with the increase of the treatment concentration of Zn²⁺, and also with the increase of the number of generation while when the treatment concentration of Zn²⁺ fall in the range of 600-1200mg/kg, the activity and levels of correspording enzymes were decreased with the increase of Zn²⁺.（3） When the treatment concentration of Zn²⁺ falls in the range of 0-300mg/kg, the content of protein increased with the increase of treatment concentration of Zn²⁺, but decreased with the increase of treatment concentration of Zn²⁺ when it falls in the range of 600-1200mg/kg, and the content of antimicrobial peptides of 3rd was significantly lower than that of 1st.（4） TrisSDS-PAGE was performed to separate the protein crude extracts from the hemolymph of 5-day-old of 3rd Hermetia illucens L., The targe peptides bands tended to be more obviously with the increase of Zn²⁺ when the concentration ranges in 75-300mg/kg,while these bands became weaker with the increase of Zn²⁺ when the concentration ranges in 600-1200mg/kg.（5） The primary structure of 4KD peptide from 1200mg/kg treatment group and control group was analyzed by LC-MS-MS, 4 groups of peptide sequence was determined, while 3 groups showed different amio-acid sequerce.Conclusion:（1） Zn²⁺ may be accumulated in the hemolymph of larvae of Hermetia illucens L. and such accumulation rise as the number of generation increased.（2） The enzyme activity in larvae of Hermetia illucens L. may be enhanced under the stress of lowdose Zn²⁺ in the hemolymph of 5-day-old larvae of 1st, 3rd Hermetia illucens L. at the day of 5,while inhibited by the stress of high-dose Zn²⁺, and the enzyme activity of CAT,T-AOC and the content of PO,MT in the hemolymph enhanced as the generation number increased.（3） With a view to the content of protein in the hemolymph of 5-day-old larvae of 1st, 3rd Hermetia illucens L. the content of antimicrobial peptides in the hemolymph of larvae of Hermetia illucens L. may be increased under the stress of low-dose Zn²⁺, while the synthesis may be inhibited under the stress of high-dose Zn²⁺ and such accumulation declined as the generation increased.（4） High does exposure of Zn²⁺ in Hermetia illucens L. larvae can change the amino-acid sequence of its protein.