Nosema Tolerant Honeybees (Apis mellifera) Escape Parasitic Manipulation of Apoptosis

Abstract

Apoptosis is not only pivotal for development, but also for pathogen defence in multicellular organisms. Although numerous intracellular pathogens are known to interfere with the host's apoptotic machinery to overcome this defence, its importance for host-parasite coevolution has been neglected. We conducted three inoculation experiments to investigate in the apoptotic respond during infection with the intracellular gut pathogen Nosema ceranae, which is considered as potential global threat to the honeybee (Apis mellifera) and other bee pollinators, in sensitive and tolerant honeybees. To explore apoptotic processes in the gut epithelium, we visualised apoptotic cells using TUNEL assays and measured the relative expression levels of subset of candidate genes involved in the apoptotic machinery using qPCR. Our results suggest that N. ceranae reduces apoptosis in sensitive honeybees by enhancing inhibitor of apoptosis protein-(iap)-2 gene transcription. Interestingly, this seems not be the case in Nosema tolerant honeybees. We propose that these tolerant honeybees are able to escape the manipulation of apoptosis by N. ceranae, which may have evolved a mechanism to regulate an anti-apoptotic gene as key adaptation for improved host invasion.

Fig 1. Quantification of apoptosis in the midgut epithelium of honeybees infected with N. ceranae.

(A) The frequency of apoptotic cells was calculated as the numbers of TUNEL+ve relation to all (DAPI+ve) nuclei. For this, DAPI and TUNEL stained images (top) were merged (bottom left); nuclei were binarised and automatically counted using ImageJ (bottom right; red = TUNEL+ve, white = TUNEL–ve). Scale bars = 25 μm. (B) Comparison of apoptotic TUNEL+ve cells detected in the posterior end of the midgut in Nosema infected sensitive and tolerant honeybees on day 6 p.i. Scale bars = 50 μm. (C) Apoptosis ratio (mean ± s.e.) during Nosema ceranae infection in Nosema sensitive (SN, solid circles) and tolerant (TN, solid squares) honeybees, and their uninfected controls (SC, open circles and TC, open squares) at 1 day (green) and 6 days (blue) after inoculation. Sample sizes are given in S2 Table. Significance between treatment groups **, P < 0.01.

Fig 2. Relative expression (mean ± s.e.) of candidate genes important for apoptosis in Nosema infected honeybees.

Nosema sensitive (SN, solid circles) and tolerant (TN, solid squares) honeybees infected with 10⁵ N. ceranae spores, and their controls uninfected (SC, open circles and TC, open squares), were sampled at 1 day (green) and 6 days (blue) after inoculation. The genes JNK/bsk (Jun N–terminal kinase/ basket), p53 (tumor protein p53-like), iap–2 (inhibitor of apoptosis protein 2; predicted homologous gene to Diap–1 in D. melanogaster), casp–2 (caspase–2–like; homologous gene to Dcp–1), casp–10 (caspase–10–like; homologous gene to Dredd) were predicted from Drosophila melanogaster. Sample sizes are ranging between six and ten pools of three individual honeybee midguts (see also S3 Table). Significance between treatment groups ***, P < 0.001.