Extensive influence of microsporidian infection on sucrose solution consumption, antioxidant enzyme activity, cell structure, and lifespan of Asian honeybees

Abstract

Apis cerana is the original host of Vairimorpha (Nosema) ceranae, a widespread fungal parasite that causes bee nosemosis, which severely threatens the health of bee colonies and the sustainable development of the apiculture industry. To evaluate the impact of V. ceranae infection on A. c. cerana workers, V. ceranae spores were purified and used to inoculate newly emerged workers to evaluate the effects of V. ceranae infection. This was followed by an in-depth investigation of V. ceranae spore load and host sucrose solution consumption. Activities of four major antioxidant enzymes (SOD, PPO, CAT, and GST) were determined. Paraffin sections of the host midgut tissue were prepared and subjected to microscopic observation. The survival rates of V. ceranae-inoculated and uninoculated workers were analyzed. The results showed that spore load gradually increased and peaked at 12 dpi. The consumption of workers in the V. ceranae-inoculated group was extremely significant higher (P < 0.0001) than that of workers in the un-inoculated group. The results of antioxidant enzyme activity were suggestive of positive host defense via catalase (CAT) and glutathione-S-transferase (GST) in the middle stage of infection, as well as the negative fungal impact on superoxide dismutase (SOD) and polyphenol oxidase (PPO) at the whole stage of infection, reflecting the complex host-parasite interaction. Additionally, we observed a disruption in the structure of the host midgut epithelial cells. Moreover, the survival rate of workers in V. ceranae-inoculated groups was nearly always lower than that of workers in the uninoculated groups. These results demonstrate a consistent increase in spore load with the proliferation of V. ceranae, leading to persistent energetic stress and midgut epithelial cell structural damage to the host, ultimately resulting in a shortened lifespan for the host. Our findings enhance the current understanding of the interactions between A. cerana and V. ceranae as well as provide a solid basis for exploring the mechanisms underlying host response and V. ceranae infection.

Keywords: Apis cerana; Vairimorpha (Nosema) ceranae; antioxidant enzyme; honeybee; host-parasite interaction.

Figure 1

(A) Microscopic observation of V. ceranae spores derived from Percoll discontinuous density centrifugation (400× amplification). (B) Agarose gel electrophoresis for PCR amplification products from purified spores. Lane M: DNA marker; Lane VC: specific primers for V. ceranae; Lane VA: specific primers for V. apis; Lane N: sterile water (negative control).

Figure 2

Spore load of V. ceranae in the midguts of A. c. cerana workers after inoculation (n=3). Whiskers indicate the mean ± SD. Data were analyzed using the One-way ANOVA (Tukey’s post-hoc test). The same lowercase letters above the curve indicate no significant difference (P > 0.05), whereas different lowercase letters above the curve indicate statistically significant differences (P < 0.05).

Figure 3

Average sucrose solution consumption of V. ceranae-inoculated and un-inoculated workers (n=105). Data were showed as mean ± SD, that analyzed using the Two-way ANOVA (Bonferroni’s post-hoc test), multiple t tests; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

Figure 4

Detection of SOD (A) and PPO (B) activities in midguts of V. ceranae- and un-inoculated workers (n=3). Data were showed as mean ± SD, that analyzed using the Two-way ANOVA (Bonferroni’s post-hoc test), multiple t tests; ns indicates nonsignificance, * indicates P < 0.05, ** indicates P < 0.01.

Figure 5

Detection of CAT (A) and GST (B) activities in midguts of V. ceranae- and un-inoculated workers (n=3). Data were showed as mean ± SD, that analyzed using the Two-way ANOVA (Bonferroni’s post-hoc test), multiple t tests; ns indicates nonsignificance, * indicates P < 0.05, ** indicates P < 0.01.

Figure 6

Microscopic observation of paraffin midgut sections of un-inoculated and V. ceranae-inoculated A. c. cerana workers. (A, B) Midgut tissue of un-inoculated worker at 7 dpi without spores; (C, D) Midgut tissue of inoculated worker at 7 dpi with V. ceranae spores; (E, F) Midgut tissue of un-inoculated worker at 8 dpi without spores; (G, H) Midgut tissue of inoculated worker at 8 dpi with V. ceranae spores; (I, J) Midgut tissue of un-inoculated worker at 9 dpi without spores; (K, L) Midgut tissue of inoculated worker at 9 dpi with V. ceranae spores; (M, N) Midgut tissue of un-inoculated worker at 10 dpi without spores; (O, P) Midgut tissue of inoculated worker at 10 dpi with V. ceranae spores. (A, C, E, G, I, K, M, O) were microscopic fields under 200× amplification, whereas (B, D, F, H, J, N, P) were microscopic fields under 400× amplification. Black dashed boxes show the selected region for observation under 200× amplification.

Figure 7

Survival rate of A. c. cerana workers after un-inoculation and inoculation with V. ceranae spores (n=3). Data were analyzed using the Log-rank (Mantel-Cox) test; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

Figure 8

Hypothetical schematic diagram of the impact of V. ceranae infection on A. c. cerana worker.