| American foulbrood disease,AFB,is a bacterial and malignant infectious disease that infects honeybee larvae.The Gram-positive bacterium,Paenibacillus larvae(P.larvae),is the etiologic pathogen of AFB.P.larvae can produce spores with strong resistance,which renders them especial difficulty of complete elimination from hoenybee colonies.Honeybee larvae are infected through ingesting food contaminated with P.larvae spores.In the midgut of honeybee larvae,P.larvae spores can germinate and propagate into vegetative cells,which will grow and pass through the intestinal epithelium into the hemolymph.Finally,honeybee larvae die of bacteremia.P.larvae continues to reproduce by utilizing the dead larvae as the nutrition source,and forms millions of spores when the nutrition is exhausted,forming a new contamination source of AFB.The outbreak of AFB often leads to the collapse of the whole colony,which causes great economic losses to the honeybee industry.At present,the prevention and treatment of AFB mainly depend on antibiotics.However,with the emergence of antibiotics-related problems,such as drug-resistant strains,antibiotic residues in honeybee products,the disorder of honeybee intestinal flora,and the shortening of bee life,it is urgent to explore natural active products that can replace or partially replace antibiotics for the prevention and treatment of AFB in beekeeping production.Based on the abovementioned facts,an isolated P.larvae strain,named P.l.YZU,was obtained from a local honeybee farm,then leaves of Trapa bispinosa Roxb and Euryale ferox Salisb,which are two plants rich in local resources but usually left unattended as agricultural wastes,were selected as the initial experimental materials.The inhibitory effects of ethanolic extracts of plants and the corresponding extracts of different solvents on the growth P.l.YZU vegetative cells were evaluated by agar diffusion experiments.Usingi the minimal inhibitory concentration(MIC)as the screening index of effective antibacterial substances,the antibacterial components were analyzed by LC-MS both qualitatively and quantitatively.We determined that gallic acid(GA)had inhibitory effects on the growth of P.l.YZU vegetative body,and then the inhibitory effect of GA on the spore germination of P.l.YZU and its toxicity to the primary intestinal cells of honeybee larvae were determined by in vitro assays.By artificially culturing honeybee larvae in the laboratory,the toxicity of GA to honeybee larvae,the protective effect of GA on honeybee larvae challenged by P.l.YZU spores,and its effects on the composition of volatile substances on the body surface of the larvae,the expression of antimicrobial peptide genes,and the bacterial flora structure of the honeybee larvae were evaluated.There are four groups of larvae in this study:control group(HL group),P.l.YZU spore challenged group(SL group),GA-treated group(GL),and GA and P.l.YZU spore co-treatment group(GSL group).The main conclusions of this study are as listed as follows:(1)GA significantly inhibited the spore germination and vegetative growth of P.l.YZU.When GA and chloramphenicol were used in combination,they exhibited synergistic bacteriostatic effects,which could significantly reduce the concentration of antibiotics and enhance its bacteriostatic effect.GA exhibted bacteriostatic effects against the growth of P.l.YZU vegetative cells through destroying bacterial plasma membrane,and its inhibitory effects on spore germination of P.l.YZU increased significantly with the increase of GA concentration(P<0.05).(2)At the final concentration 125 μg/mL(equivalent to 1× MIC for P.l.YZU vegetative cells),GA had no significant effects on the viability of primary intestinal cells of honeybee larvae,neither were there any significant effects on the survival rate and weight of honeybee larvae.GA could significantly improve the survival rate and weight of honeybee larvae chanllenged with P.l.YZU spores(P<0.05).(3)Compared with larvae in group SL,the release of necrotic elements(oleic acid and linoleic acid)in larvae of group GL was significantly reduced,while the release of protective hydrocarbons(pentadecane and heptadecane)was significantly increased(P<0.05).At the same time,the release of necrophin from larvae in GSL group was significantly higher than that from group HL(P<0.05),indicating that the addition of GA did not interfere with the identification and removal of suspected diseased larvae by workers in the honeybee colony,and would not affect the social group immunity of the honeybee colony.(4)Results from the frozen sections of the midgut tissue of honeybee larvae showed that the midgut tissue of the larvae in GL group remained intact and the thickness was normal.However,in larvae from SL group,the morphology of the midgut tissue was severely changed,the peritrophic membrane was degraded and a large number of bacteria was observed in the intestinal cavity.In GSL group,although the intestinal wall became thinner and the morphology of peritrophic membrane changed,the growth of P.larvae in the intestinal cavity was inhibited to some extent due to the co-treatment of GA.These results indicated that GA did not affect the development of the midgut tissue of honeybee larvae,and GA could alleviate the damage of peritrophic membrane caused by the proliferation of P.l.YZU in intestinal cavity.(5)GA could significantly reduce the bacterial loads of P.l.YZU in honeybee larvae(P<0.05).In terms of the transcriptional expression of antibacterial peptide genes,GA treatment could significantly increase the expression of antibacterial peptide genes in honeybee larvae(P<0.05).Compared with larvae in group GL,the priming effects of the expression of antimicrobial peptide genes in larvae of group SL happened earlier and lasted longer,indicating that adding GA would not cause significant immune burden to larvae.(6)In terms of the bacterial flora structure of honeybee larvae,results from the 16S rRNA amplicon sequencing analysis showed that infection with P.l.YZU would increase the relative abundance of pathogenic bacteria(such as Paenibacillus)and decrease the relative abundance of beneficial bacteria(such as Lactobacillus).In the meantime,GA could optimize the bacterial flora structure of honeybee larvae and significantly increase the relative abundance of beneficial bacteria in the intestinal tract.These results were further confirmed by the successful isolation of senven lactic acid bacteria from the intestine of honeybee larvae in GL group,which was consistent with the sequencing results obtained by 16S rRNA amplicon analysis.In this study,the inhibitory effects of GA on spore germination and vegetative growth of P.larvae were confirmed.The non-toxic effects of GA on honeybee larvae and its protective effects on P.larvae infected larvae were demonstrated through in vivo experiments.These results provide basises for the potential application of GA,a natural compound,in the prevention and treatment of AFB.They also provide insights into reducing the application of antibiotics in beekeeping production aiming to promote the sustainable and healthy development of beekeeping industry. |
