Comparative Transcriptome Investigation of Nosema ceranae Infecting Eastern Honey Bee Workers

Abstract

Apis cerana is the original host for Nosema ceranae, a widespread fungal parasite resulting in honey bee nosemosis, which leads to severe losses to the apiculture industry throughout the world. However, knowledge of N. ceranae infecting eastern honey bees is extremely limited. Currently, the mechanism underlying N. ceranae infection is still largely unknown. Based on our previously gained high-quality transcriptome datasets derived from N. ceranae spores (NcCK group), N. ceranae infecting Apis cerana cerana workers at seven days post inoculation (dpi) and 10 dpi (NcT1 and NcT2 groups), comparative transcriptomic investigation was conducted in this work, with a focus on virulence factor-associated differentially expressed genes (DEGs). Microscopic observation showed that the midguts of A. c. cerana workers were effectively infected after inoculation with clean spores of N. ceranae. In total, 1411, 604, and 38 DEGs were identified from NcCK vs. NcT1, NcCK vs. NcT2, and NcT1 vs. NcT2 comparison groups. Venn analysis showed that 10 upregulated genes and nine downregulated ones were shared by the aforementioned comparison groups. The GO category indicated that these DEGs were involved in a series of functional terms relevant to biological process, cellular component, and molecular function such as metabolic process, cell part, and catalytic activity. Additionally, KEGG pathway analysis suggested that the DEGs were engaged in an array of pathways of great importance such as metabolic pathway, glycolysis, and the biosynthesis of secondary metabolites. Furthermore, expression clustering analysis demonstrated that the majority of genes encoding virulence factors such as ricin B lectins and polar tube proteins displayed apparent upregulation, whereas a few virulence factor-associated genes such as hexokinase gene and 6-phosphofructokinase gene presented downregulation during the fungal infection. Finally, the expression trend of 14 DEGs was confirmed by RT-qPCR, validating the reliability of our transcriptome datasets. These results together demonstrated that an overall alteration of the transcriptome of N. ceranae occurred during the infection of A. c. cerana workers, and most of the virulence factor-related genes were induced to activation to promote the fungal invasion. Our findings not only lay a foundation for clarifying the molecular mechanism underlying N. ceranae infection of eastern honey bee workers and microsporidian-host interaction.

Keywords: Apis cerana cerana; Nosema ceranae; differentially expressed gene; honey bee; infection mechanism; microsporidian; transcriptome.

Figure 1

Microscopic detection and PCR validation of N. ceranae spores. (A) Microscopic detection (400 times amplification). (B) AGE for PCR amplified fragments, Lane Nc: Specific primers for N. ceranae, Lane Na: Specific primers for N. apis, Lane N: Sterile water (Negative control), Lane M: DNA marker.

Figure 2

Microscopic observation of paraffin sections of N. ceranae-inoculated and un-inoculated A. c. cerana workers’ midguts. (A) Worker’s midgut at 11 dpi with N. ceranae under 200 times amplification. (B) Worker’s midgut at 11 dpi with N. ceranae under 400 times amplification. (C) Worker’s midgut at 11 dpi without N. ceranae under 200 times amplification. (D) Worker’s midgut at 11 dpi without N. ceranae under 400 times amplification. Black dashed box shows the region for observation under 400 times amplification.

Figure 3

Analysis of DEGs in N. ceranae. (A) Venn analysis of upregulated genes in NcCK vs. NcT1, NcCK vs. NcT2, and NcT1 vs. NcT2 comparison groups. (B) Venn analysis of downregulated genes in NcCK vs. NcT1, NcCK vs. NcT2, and NcT1 vs. NcT2 comparison groups. (C) Heatmap of common DEGs in the NcCK, NcT1, and NcT2 groups.

Figure 4

GO category of DEGs. (A) DEGs in NcCK vs. NcT1. (B) DEGs in NcCK vs. NcT2; (C) DEGs in NcT1 vs. NcT2.

Figure 5

Pathways annotated by DEGs. (A) DEGs in NcCK vs. NcT1. (B) DEGs in NcCK vs. NcT2. (C) DEGs in NcT1 vs. NcT2.

Figure 6

Heatmap of virulence factor-associated DEGs shared by the NcCK, NcT1, and NcT2 groups.

Figure 7

RT-qPCR verification of DEGs in N. ceranae infecting A. c. cerana workers. (A) DEGs in NcCK vs. NcT1. (B) DEGs in NcCK vs. NcT2. Error bars represent the variance of RT-qPCR results of each DEG. Bars with asterisk symbol indicate statistical differences (p < 0.05).