Screening of Differentially Expressed Microsporidia Genes from Nosema ceranae Infected Honey Bees by Suppression Subtractive Hybridization.

Zih-Ting Chang,Chong-Yu Ko,Yu-Shin Nai,Yue-Wen Chen,Ming-Ren Yen

doi:10.3390/insects11030199

Abstract

The microsporidium Nosema ceranae is a high prevalent parasite of the European honey bee (Apis mellifera). This parasite is spreading across the world into its novel host. The developmental process, and some mechanisms of N. ceranae-infected honey bees, has been studied thoroughly; however, few studies have been carried out in the mechanism of gene expression in N. ceranae during the infection process. We therefore performed the suppressive subtractive hybridization (SSH) approach to investigate the candidate genes of N. ceranae during its infection process. All 96 clones of infected (forward) and non-infected (reverse) library were dipped onto the membrane for hybridization. A total of 112 differentially expressed sequence tags (ESTs) had been sequenced. For the host responses, 20% of ESTs (13 ESTs, 10 genes, and 1 non-coding RNA) from the forward library and 93.6% of ESTs (44 ESTs, 28 genes) from the reverse library were identified as differentially expressed genes (DEGs) of the hosts. A high percentage of DEGs involved in catalytic activity and metabolic processes revealed that the host gene expression change after N. ceranae infection might lead to an unbalance of physiological mechanism. Among the ESTs from the forward library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes. Out of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation. The results indicated that these genes were highly expressed during N. ceranae infection. Among nine N. ceranae genes, one N. ceranae gene (AAJ76_1600052943) showed the highest expression level after infection. These identified differentially expressed genes from this SSH could provide information about the pathological effects of N. ceranae. Validation of nine up-regulated N. ceranae genes reveal high potential for the detection of early nosemosis in the field and provide insight for further applications.

Highlights

The honey bee (Apis mellifera) is an essential pollinator of many crop plants in natural ecosystems and agricultural crops; it contributes more than $14 billion to agriculture annually in North America [1,2,3,4].the American apiculture industry experienced catastrophic losses of unknown origin since2006
The results indicated that these genes showed high expression during N. ceranae infection
28 honey bee genes and 24 genes in N. ceranae were identified as differentially expressed genes (DEGs) after honey bees were infected with N. ceranae via the suppression subtractive hybridization (SSH) approach

Summary

Introduction

The honey bee (Apis mellifera) is an essential pollinator of many crop plants in natural ecosystems and agricultural crops; it contributes more than $14 billion to agriculture annually in North America [1,2,3,4].the American apiculture industry experienced catastrophic losses of unknown origin since2006. The decline and disappearance of the bee species in the natural environment and the collapse of honey bee colonies were defined as colony collapse disorder (CCD) [1]. The consequences of this syndrome are evident as an unexplained disappearance of adult bees, a lack of attention to the brood, reduced colony vigor, and heavy winter mortality without any apparent pathological infection. Reductions of honey bee populations were estimated at 23% over the winter of 2006–2007 [5] and at. As a matter of fact, CCD is becoming a worldwide issue and honey bees are still suffering threats from either the natural environment (i.e., pesticide residues) or diseases caused by microorganisms (i.e., viruses, bacteria, and fungi)

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Discussion

Conclusion