Effect of amide protoporphyrin derivatives on immune response in Apis mellifera

Abstract

The intracellular microsporidian parasite Nosema ceranae is known to compromise bee health by induction of energetic stress and downregulation of the immune system. Porphyrins are candidate therapeutic agents for controlling Nosema infection without adverse effects on honeybees. In the present work, the impact of two protoporphyrin IX derivatives, i.e. PP[Asp]₂ and PP[Lys]₂, on Apis mellifera humoral immune response has been investigated in laboratory conditions in non-infected and N. ceranae-infected honeybees. Fluorescence spectroscopy analysis of hemolymph showed for the first time that porphyrin molecules penetrate into the hemocoel of honeybees. Phenoloxidase (PO) activity and the expression of genes encoding antimicrobial peptides (AMPs: abaecin, defensin, and hymenoptaecin) were assessed. Porphyrins significantly increased the phenoloxidase activity in healthy honeybees but did not increase the expression of AMP genes. Compared with the control bees, the hemolymph of non-infected bees treated with porphyrins had an 11.3- and 6.1-fold higher level of PO activity after the 24- and 48-h porphyrin administration, respectively. Notably, there was a significant inverse correlation between the PO activity and the AMP gene expression level (r = - 0.61696, p = 0.0143). The PO activity profile in the infected bees was completely opposite to that in the healthy bees (r = - 0.5118, p = 0.000), which was related to the changing load of N. ceranae spores in the porphyrin treated-bees. On day 12 post-infection, the spore loads in the infected porphyrin-fed individuals significantly decreased by 74%, compared with the control bees. Our findings show involvement of the honeybee immune system in the porphyrin-based control of Nosema infection. This allows the infected bees to improve their lifespan considerably by choosing an optimal PO activity/AMP expression variant to cope with the varying level of N. ceranae infection.

Figure 1

Flowchart of the experiments analyzing the effect of protoporphyrin IX derivatives on Apis mellifera humoral immune response, number of Nosema ceranae spores in bees, and bee survival.

Figure 2

Effect of amide derivatives of protoporphyrin IX on the number of Nosema ceranae spores in infected Apis mellifera. Statistically significant differences between the infected control group and porphyrin-treated groups on a particular day are indicated with asterisks; block letters (A,B,C,D,E) indicate statistically significant differences between the days in the porphyrin groups. The dashed line indicates groups of infected bees prior to porphyrin administration. The error bars represent standard errors of data.

Figure 3

Kaplan–Meier survival curves for uninfected and N. ceranae-infected honeybees treated with protoporphyrin IX derivatives (a) PP(Asp)₂ and (b) PP(Lys)₂ administered with the sucrose syrup supplement.

Figure 4

Confocal microscopic images of the midgut segment with porphyrin accumulation in the area of epithelial cells: (a) with anchored Nosema ceranae spores in infected honeybees and (b) free of N. ceranae spores in non-infected honeybees (λ_exc = 405 nm). DIC—differential interference contrast; corresponding fluorescence spectra were collected from the points indicated by arrows.

Figure 5

Fluorescence spectra of the honeybee hemolymph showing the presence of protoporphyrin IX amide derivatives (a) PP(Asp)₂ and (b) PP(Lys)₂ inside the hemocoel.

Figure 6

Phenoloxidase activity in honeybees on days 3 (a) and 4 (b) post infection; n.i.—non-infected honeybees; i.—Nosema-infected honeybees; Controls—honeybees fed with pure sucrose syrup; PP(Asp)₂ and PP(Lys)₂—honeybees fed with sucrose syrup containing porphyrin.

Figure 7

Effect of porphyrins PP(Asp)₂ and PP(Lys)₂ on immune-related AMP gene expression in honeybees (a) on day 3 post N. ceranae-infection; (b) on day 4 post N. ceranae-infection. Aba—abaecin, Def—defensin, Hym—hymenoptaecin. Non-infected control and infected control bees fed with sucrose syrup only. Bars marked with asterisks are statistically significantly different from the non-infected control; error bars represent standard errors of data.