Comparative Analysis of Proteolytic Enzyme Activity in the Midgut of Galleria mellonella Larvae Selected for Antibiotic Resistance

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The accumulation of antibiotics in biocenoses carries risks of development of multidrug-resistant microorganisms through horizontal transfer of resistance genes, metabolic changes in various animals, including humans. It is hypothesized that these changes may have a significant impact on the structure of digestive enzymes in the gut. This may be achieved through the switching between different classes and isoforms of proteases, the expression of new isoforms, and the consequent effect on digestion and resistance to pathogens. The present study was completed on the larvae of the wax moth Galleria mellonella, which were cultivated on a diet that contained low doses of antibiotic for thirty generations (R-line). The R-line obtained demonstrated a considerable degree of resistance in the insects to exposure to the entomopathogenic bacterium Bacillus thuringiensis. The per os treatment of R-line larvae demonstrated a complete loss of susceptibility to the bacterium, and a decrease in total proteolytic enzyme activity from the first day after infection, primarily due to suppression of serine protease activity.

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T. Klementeva

Institute of Systematics and Ecology of Animals SB RAS

Email: ovp0408@yandex.ru
俄罗斯联邦, Novosibirsk

N. Kruykova

Institute of Systematics and Ecology of Animals SB RAS

Email: ovp0408@yandex.ru
俄罗斯联邦, Novosibirsk

D. Kornienko

Institute of Systematics and Ecology of Animals SB RAS; Novosibirsk State Medical University

Email: ovp0408@yandex.ru
俄罗斯联邦, Novosibirsk; Novosibirsk

A. Esaulko

Stavropol State Agrarian University

Email: ovp0408@yandex.ru
俄罗斯联邦, Stavropol

V. Glupov

Institute of Systematics and Ecology of Animals SB RAS

Email: ovp0408@yandex.ru
俄罗斯联邦, Novosibirsk

O. Polenogova

Institute of Systematics and Ecology of Animals SB RAS

编辑信件的主要联系方式.
Email: ovp0408@yandex.ru
俄罗斯联邦, Novosibirsk

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2. Fig. 1. Survival of Galleria mellonella larvae of the native strain (N) and the strain of insects reared on a diet with an antibiotic (R) after per os exposure to Bacillus thuringiensis bacteria. Letters a and b indicate intergroup differences calculated using the Log-rank test (p < 0.001).

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3. Fig. 2. Zymogram (SDS-PAGE) of homogenates of the midgut section of the wax moth Galleria mellonella: control samples of the native line (N-C) and the line of insects grown on a diet with an antibiotic (R-C), as well as 48 h after per os exposure to Bacillus thuringiensis (Bt) bacteria of the native line (N-Bt) and the line of larvae on a diet with an antibiotic (R-Bt). M – molecular weight standards (Servicebio, China).

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4. Fig. 3. Proteolytic enzyme activities in midgut homogenates of wax moth Galleria mellonella larvae of the native line (N) and the line of insects reared on an antibiotic diet (R) after per os exposure to Bacillus thuringiensis (Bt) bacteria. (a) – Total proteolytic enzymes; (b) – PMSF (serine protease inhibitor); (c) – E-64 (cysteine protease inhibitor); (d) – EDTA (metalloprotease inhibitor). Different letters (A–C) indicate significant intergroup differences within one time point, calculated using the Kruskal–Wallis test followed by Dunn’s test (p < 0.05).

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