Induction and isolation of mealworm antimicrobial peptides and their antimicrobial activity

Authors:Liu Xuelan1,Dong Yilei1,Dalle Zotte2,Fu Chunyan1,Zhang Heng1,Shi Tianhong1,Li Xia1,Wei Xiangfa1,Liu Ruiting1,Yan Peipei1*

单位:1.山东省学院家禽研究所;2.University of Padua, Padua

Introduction: Liu Xuelan, female, from Chengwu, Shandong, researcher, engaged in the research and development of feed additives. *Corresponding author, assistant researcher, engaged in research and development of feed additives.

Funds: International Science and Technology Cooperation Project of Shandong Academy of Agricultural Sciences (CXGC2022F36), Poultry Innovation Team Building Project of Modern Agricultural Industry Technology System of Shandong Province (SDAIT-011-06); Jinan "20 New Universities" funded project (202228037).

Source: Anhui Agricultural Sciences, Issue 6, 2024

Citation:Xuelan Liu, Yilei Dong, Dalle Zotte, et al.Induction and isolation of mealworm antimicrobial peptides and their antibacterial activity[J].Anhui Agricultural Sciences,2024,52(6):86-90,273.

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Antimicrobial peptides, also known as antimicrobial peptides and peptide antibiotics, are a kind of defensive small peptides produced by biological organisms when resisting the infection of pathogenic microorganisms, usually cationic peptides, containing 10~50 amino acids, small molecular weight, good water solubility, strong thermal stability, and no immunogenicity. It acts on gram-negative bacteria, gram-positive bacteria, fungi, parasites, etc., and can quickly detect and kill targets. Compared with traditional antibiotics, the biggest advantage of antimicrobial peptides is that they are safe, non-toxic, and have no side effects, and pathogenic bacteria are not easy to develop drug resistance.

Insects are one of the most abundant groups of terrestrial animals today, and they have formed a unique and efficient innate defense system during the long evolutionary process. When it is damaged or infected by microorganisms, it quickly synthesizes antimicrobial peptides in a short period of time and releases them into the hemolymph to act on bacteria, viruses, fungi, etc. Mealworms are important members of the insect family, and their feeding habits, strong fecundity, simple rearing, and low cost make them a good antimicrobial peptide resource, which has attracted extensive attention from scientific researchers.

objective

To explore the effects of different methods on the activity of mealworm antimicrobial peptides, this paper lays a foundation for the modification, molecular cloning and exogenous expression of mealworm antimicrobial peptides.

way

The 7th, 8th and 9th instar mealworm larvae were selected, Pasteurella and Salmonella were used as inducing bacteria, and antimicrobial peptides were induced by microinjection and acupuncture to study the antimicrobial effects of antimicrobial peptides in different instar mealworm larvae induced by different bacterial liquid concentrations and different methods.

outcome

◆ Induction of mealworm antimicrobial peptides by microinjection of specific concentration of bacterial solution

Microinjection of specific concentrations of Pasteurella bacteria for the induction of mealworm antimicrobial peptides. The results of Table 1 showed that the antimicrobial peptide activity induced by injection of specific concentrations of Pasteurella larvae in the 7th, 8th and 9th instar mealworm larvae was significantly greater than that in the non-injected group (P<0.05), the activity of antimicrobial peptides in the 7th and 8th instar mealworms in the 0.8pL group increased significantly with the extension of the action time (P<0.05), the antimicrobial peptide activity of the 7th instar mealworm in the 1.2pL group was the highest at 48 h, which was significantly higher than that at 24 h (P<0.05), and the antimicrobial peptide activity of the 8th and 9th instar mealworms was the highest at 24 h, which was significantly higher than that at 48h (P<0.05);The antimicrobial peptide activity of 7th instar mealworms in the 1.6pL group was the highest at 72 h, which was significantly higher than that at 24 and 48 h (P<0.05), the antimicrobial peptide activity at 24 and 48 h in 8th and 9th instar mealworms was significantly higher than that at 72 h (P<0.05), and there was no significant difference in antimicrobial peptide activity between 24 and 48 h (P>0.05). As can be seen from Table 1, it is advisable to induce antimicrobial peptides by Pasteurella microinjection method with 0.8 pL injection for 72 hours.

Note: Different lowercase letters in the same column in the table indicate significant differences at the 0.05 level.

Induction of mealworm antimicrobial peptides by microinjection of specific concentrations of Salmonella. The results of Table 2 showed that the antimicrobial peptide activity induced by injection of specific concentrations of Salmonella in the 7th, 8th and 9th instar mealworms was significantly greater than that in the non-injected group (P<0.05), the antimicrobial peptide activity in the 7th, 8th and 9th instar mealworms in the 0.8pL group increased significantly with the extension of the action time (P<0.05), the antimicrobial peptide activity of the 7th instar mealworm in the 1.2pL group was the highest at 72 h, which was significantly higher than that at 24h (P<0.05), and the antimicrobial peptide activity of the 8th and 9th instar mealworms was the highest at 24 h, which was significantly higher than that at 48h and 72 h (P<0.05);The activity of antimicrobial peptides in the 7th instar mealworm in the 1.6pL group was the highest at 72 h, which was significantly higher than that at 24 h (P<0.05), and there was no significant difference in the antimicrobial peptide activity between the 8th and 9th instar mealworms in each time period (P>0.05). The results showed that it was appropriate to induce antimicrobial peptides by microinjection of Salmonella with 0.8pL for 72 hours.

Note: Different lowercase letters in the same column in the table indicate significant differences at the 0.05 level.

◆ Induction of antimicrobial peptides in mealworms by different times of acupuncture

Induction of antimicrobial peptides by needle Pasteurella times on mealworms at different instars. The results of Fig. 1 showed that the number of acupuncture had no significant effect on the antimicrobial peptide antibacterial activity of the 7th instar mealworm when acupunctured with the same concentration of Pasteurella (P>0.05), the number of acupuncture had a significant effect on the antimicrobial activity of the induced antimicrobial peptide, and the antibacterial activity of the second and third acupuncture times was significantly greater than that of the first acupuncture (P<0.05), and the number of acupuncture times also had a significant effect on the antimicrobial activity of the induced antimicrobial peptides, and the antibacterial activity of the 3rd acupuncture was significantly greater than that of the 1st and 2nd acupuncture times (P<0.05).