Transcriptional responses in honey bee larvae infected with chalkbrood fungus

Abstract

Background: Diseases and other stress factors working synergistically weaken honey bee health and may play a major role in the losses of bee populations in recent years. Among a large number of bee diseases, chalkbrood has been on the rise. We present here the experimental identification of honey bee genes that are differentially expressed in response to infection of honey bee larvae with the chalkbrood fungus, Ascosphaera apis.

Results: We used cDNA-AFLP Technology to profile transcripts in infected and uninfected bee larvae. From 64 primer combinations, over 7,400 transcriptionally-derived fragments were obtained A total of 98 reproducible polymorphic cDNA-AFLP fragments were excised and sequenced, followed by quantitative real-time RT-PCR (qRT-PCR) analysis of these and additional samples.We have identified a number of differentially-regulated transcripts that are implicated in general mechanisms of stress adaptation, including energy metabolism and protein transport. One of the most interesting differentially-regulated transcripts is for a chitinase-like enzyme that may be linked to anti-fungal activities in the honey bee larvae, similarly to gut and fat-body specific chitinases found in mosquitoes and the red flour beetle. Surprisingly, we did not find many components of the well-characterized NF-kappaB intracellular signaling pathways to be differentially-regulated using the cDNA-AFLP approach. Therefore, utilizing qRT-PCR, we probed some of the immune related genes to determine whether the lack of up-regulation of their transcripts in our analysis can be attributed to lack of immune activation or to limitations of the cDNA-AFLP approach.

Conclusions: Using a combination of cDNA-AFLP and qRT-PCR analyses, we were able to determine several key transcriptional events that constitute the overall effort in the honey bee larvae to fight natural fungal infection. Honey bee transcripts identified in this study are involved in critical functions related to transcriptional regulation, apoptotic degradation of ubiquitinated proteins, nutritional regulation, and RNA processing. We found that immune regulation of the anti-fungal responses in honey bee involves highly coordinated activation of both NF-kappaB signaling pathways, leading to production of anti-microbial peptides. Significantly, activation of immune responses in the infected bee larvae was associated with down-regulation of major storage proteins, leading to depletion of nutritional resources.

Figure 1

Polyacrylamide gel electrophoresis. cDNA-AFLP PCR products generated using different selective (2⁺/2⁺) primer combinations for the 24 h time point. PCR fragments that are in the range of 100-400 bp have been visualized using two different types of the polyacrylamide gels (A) PCR fragments were separated using 6.5% polyacrylamide gel and NEN Model 4300 DNA Analyzer (B) fragments separated on 8% polyacrylamide that ran on a V16-2 gel system apparatus and stained with 10,000× dilution of SybrGold for 20 minutes. Lanes: (M) DNA marker; (C) control group; (1-8) denotes treated group plus the primer combination used to generate the PCR product. Each pair of a control sample and an experimental sample (e.g., C1, C2, C3) was generated by PCR amplification using different primer sets (1-8). Some of the differentially expressed PCR fragments are indicated by arrows.

Figure 2

qRT-PCR analysis of selected cDNA-AFLP transcripts (A-C) and transcripts not derived from cDNA-AFLP (D-H):. (A) Glycosyl Hydrolase, (B) Serine Protease, (C-D) storage proteins Hex 70b and Vg, and (E-H) components on the honey bee immune response pathways including anti-microbial peptides. Control group of samples are represented by blue bars, the experimental samples are represented by green bars. Samples were done in triplicate and normalized by dividing the amount (pg or fg as described in Materials and Methods) of the gene of interest by the amount for the housekeeping gene, actin. Results are reported as an average of the triplicates plus the standard deviation. Significant by ANOVA at P < 0.05(*), P < 0.01(**), and P < 0.001(***).