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

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.

1. 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]. However, the American apiculture industry experienced catastrophic losses of unknown origin since 2006. 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 36% over the winter of 2007–2008 [6]. Additionally, it leads to reduction of honey production and ultimately to colony collapse [7]. 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).

As aforementioned, honey bee pathogens, such as viruses, microsporidian, mites, and parasites, undoubtedly affect the development of honey bee colonies and are involved in CCD. Among these pathogens, microsporidia is one of the most common prevalences in bee populations [5,8]. There are two species of honey bee microsporidia: Nosema apis and N. ceranae [9]. Both are obligate intracellular parasitic fungi [10]. Microsporidia are the causation pathogens of nosemosis and usually cause chronic impacts on honey bee populations [9,11]. N. apis was first discovered in A. mellifera in Australia, North America, and Europe, and N. ceranae was originally found in A. ceranae in the 1990s [12,13]. From 2004 to 2006, N. ceranae infections were detected in A. mellifera in Taiwan and European countries [14,15]. Hereafter, the prevalence of N. ceranae was confirmed in the population of A. mellifera, and has become a globally distributed bee pathogen in this past decade [16]. N. ceranae coincided with early reports of CCD, and was suggested to be a factor in honey bee declines [5]. It affects adult bees and was recently found in collapsing A. mellifera colonies in Spain [17]. Experimental results suggest that N. ceranae is more virulent than N. apis; therefore, N. ceranae is considered to be an important bee pathogen that causes bee colony loss [16].

N. ceranae is a gut-pathogen, in terms that the N. ceranae infection typically causes cytopathic effects (CPE) in the infected midgut tissue of honey bees [11]. It was reported that an N. ceranae infection led to the reduction of honey production, malnutrition, shorter life span, and higher mortality of adult honey bees [7,11,18,19,20]. Moreover, the N. ceranae infection would change the carbohydrate metabolism and suppress metabolism related gene expression [21,22]. However, it is not easy for beekeepers to note N. ceranae infection in honey bee colonies due to the lack of obvious symptoms from latent infection of N. ceranae. Ignorance of latent infection of N. ceranae might raise the risk of long-term colony infection of N. ceranae, and then cause colony disease outbreaks [16]. Therefore, early detection of nosemosis at the latent infection stage is needed for management of honey bee colonies.

In this study, we attempt to identify the differential expressed genes between N. ceranae infection and non-infected honey bees, and to clarify the gene expression profile of highly expressed genes during an N. ceranae infection. To this aim, a method of suppression subtractive hybridization (SSH), which is a powerful method for selectively amplifying differentially expressed genes [23], was used for identification of the differential expression N. ceranae genes, as well as the host response genes. The highly expressed N. ceranae specific genes were further validated and investigated. These data could provide insight for the detection of early nosemosis in the field.

2. Materials and Methods

2.1. Purification of Microsporidia from Infected Honey Bee Midguts

Spores for the inoculation were isolated from live heavily infected bees of Apis mellifera, collected from a naturally infected hive located in the experimental apiary at the National Taiwan University campus in Taipei, Taiwan, and the National Ilan University in Yilan, Taiwan. After dissecting the intestinal tracts, the midgut tissues were macerated in distilled water using a manual tissue grinder and examined under phase-contrast microscopy (IX71, Olympus, Tokyo, Japan). Infected tissues were homogenized and filtered through three layers of stainless net and centrifuged at 3000× g for 15 min. The pellet was then transferred into aseptic 90% Percoll and 10% 1.5 M NaCl, followed by centrifugation at 15,000× g for 40 min. The band centrifuged to the bottom of the tube contained mature spores [24,25]. The purified spores were stored in distilled water at 4 °C. The spore concentration was determined by counting with a hemocytometer chamber and the suspension was freshly prepared before using.

2.2. Experimental Infection

The experimental infection was modified from Higes et al. (2007), briefly described below; frames of sealed brood were obtained from a healthy colony of A. mellifera (Nosema-free, confirmed by PCR, weakly), and were kept in an incubator at 32 ± 2 °C and 95% relative humidity to provide newly emerged Nosema-free honey bees. Three days after eclosion, the bees were starved for 2 h and 100 bees per group were each fed with 5 μL of 50% sucrose solution containing 1.25 × 10⁵ spores of N. ceranae, by a droplet, with the spore solution at the tip of a micropipette, until it had consumed the entire droplet [26]. Control honey bees were fed with 5 μL of 50% sucrose solution. The honey bees were reared in an incubator at 32 ± 2 °C and 95% relative humidity, and the survival individuals were recorded daily for 21 days. The Kaplan–Meier survival curve was generated by SPSS software. For the sample collections, 10 bees from each cage were collected at 7, 14, and 21 days post infection (dpi) and their ventriculi processed for microscopic examination. Spores were counted and spore production was recorded every week until 3 weeks pi. Control bees were also sacrificed at days 7, 14, and 21 dpi, and analyzed to confirm the absence of spores in ventriculus.

2.3. Detection of Infection

DNA was extracted from the midgut tissue of control bees and infected honey bees by using the following procedure modified from Tsai et al., 2002 [27]. In brief, midgut tissues from 10 control and infected honey bees were homogenized in TE buffer (0.1 M Tris, 0.01 M EDTA, pH 9.0) with a pestle. The macerated solution was centrifuged and the supernatant was discarded. The pellets were incubated with RNase A at 37 °C for 1 h and proteinase K at 56 °C overnight, followed by DNA extraction with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), and DNA precipitation with 1/10 volume of 3 M sodium acetate, and 2 times the volume of pure ethanol. The DNA pellet was air dried and then dissolved in water. The small subunit ribosomal DNA (SSU rDNA) fragment of the microsporidium was amplified using primer set 18f /1537r (Supplementary Table S1). For the internal control, 18S primer set 143F/145R was used (Supplementary Table S1). Each 50-μL PCR mix contained 5 μL 10× reaction buffer (Bioman), 4 μL 2.5 mM dNTPs, 0.5 μL 100 mM of each primer, 1 μL 1.25 U HiFi Taq polymerase (RBC), and 1 μL template DNA. PCR amplifications were performed as follows on an Primus 96 Plus Thermal Cycler (MWG-Biotech, Ebersberg, Germany): thermal cycler was preheated at 95 °C for 5 min, 35 cycles at 94 °C for 30 s, 50 °C for 1 min, and 72 °C for 2 min, followed by a 10 min final extension at 72 °C, and storage at 20 °C. The PCR product was cloned into T&A cloning vector (RBC Bioscience) and commercially sequenced (Genomics Biosci. & Tech. Company, New Taipei, Taiwan).

2.4. Construction of Subtractive Complementary DNA (cDNA) Library

Total RNA was extracted using TRIzol^® Reagent (ambion), according to the manufacturer’s instructions, from 10 of the control bee and infected bee tissues, at 7, 14 and 21 dpi, and then equal quantities of three RNA samples, per group, were pooled together to maximize detection of differential expression of genes that may show variation between individuals in the exact timing of expression. Single-stranded and double-stranded cDNA were synthesized from the infected and control RNA pools with the SMART^TM PCR cDNA Synthesis Kit (Takara, Kyoto, Japan), according to the manufacturer’s protocol.

Two subtractive cDNA libraries (forward and reverse subtractive cDNA libraries) from the control and infected honey bees, were constructed by using the PCR-Select cDNA Subtraction kit (Clontech), according to the manufacturer’s specifications. Briefly described below, cDNA from the infected honey bees was used as the tester (forward subtractive cDNA library, fscl)/driver (reverse subtractive cDNA library, rscl), and cDNA from the control honey bees as the driver (fscl)/tester (rscl). Both tester and driver cDNA were digested with RsaI to produce shorter, blunt-ended fragments. After digestion with RsaI, the tester cDNA was divided into two portions, each of which was ligated with a different adapter (Adaptor-1 and Adaptor-2R) at 16 °C for overnight. After ligation, each tester cDNA was separately hybridized at 68 °C for 8 h with an excess of driver cDNA after denaturation at 98 °C for 90 s. Then, the two hybridized samples were mixed together and hybridized at 68 °C overnight, with excess of denatured driver cDNA. The resulting mixture was added with 200 mL dilution buffer and amplified by two rounds of suppression PCR. Before the primary PCR, the reaction mixture was incubated at 75 °C for 5 min to extend the adaptors. Primary PCR was performed at 94 °C for 30 s, 66 °C for 30 s, and 72 °C for 90 s for 27 cycles, in a reaction volume of 25 μL. The PCR product was then diluted 10-fold, and 1 μL-diluted product was used as template in the next subsequently nested PCR. The subtracted secondary PCR products were purified with DNA Clean/Extraction Kit (GeneMark) and then ligated into T&A cloning vector (RBC) followed by transformation into Escherichia coli, DH5α competent cells (RBC), to generate subtracted cDNA libraries. Each step of SSH was tested following the manufacturer’s instructions. A total of 192 white colonies from forward (96 colonies) and reverse libraries (96 colonies) were checked by colony PCR. The PCR products (insert DNA > 100 bp) were further subject to the dot-blotting method, which were described from the manufacturer’s protocol, and the potential colonies were identified and commercially sequenced (Genomics Bioscience & Technology, New Taipei, Taiwan). DNA sequencing from the 3′ end and 5′ end of the cDNA was conducted with M13 forward or reverse primers, respectively, on a high throughput automated sequencer (MJ Research BaseStation and ABI3730) (Thermo Fisher Scientific Inc., Waltham, MA, USA), using standard protocols.

2.5. DNA Sequences Analysis

cDNA sequences from each SSH library was sequenced. CodonCode Aligner was used for base-calling (Q > 13), trimming vector sequences, and sequence assemblage. All of the sequences were filtered for size (less than 100 bp). Putative functions of the unique sequences were discovered by using BLASTn and BLASTp to translate each nucleotide query sequence into all reading frames and then search for matches in the National Center for Biotechnology Information (NCBI) non-redundant (nr) database. Significant hits (with E value < 10⁻¹⁰) in the NCBI nr database were further submitted to the Gene Ontology (GO) enrichment analysis (http://geneontology.org/page/go-enrichment-analysis) for protein functions. The Gene GO annotations classify proteins by molecular function, biological process, and cellular component. Each unique sequence was tentatively assigned GO classification based on annotation of the E-value for the “best hit” match. These data were then used to classify the corresponding genes, according to their GO functions.

2.6. RT-qPCR Validations of Microsporidia Specific Genes

To further validate the differential expressed microsporidia specific genes in N. ceranae infected honey bees, microsporidia genes have 2 ≥ expressed sequence tags (ESTs) present in the forward library, and dot-blotting difference ≥ 3-fold were selected and subject to quantitative RT-qPCR analysis. Gene specific primer sets for RT-qPCR were designed by Primer Express v3.0 (Supplementary Table S1). Total RNA was extracted using TRIzol^® Reagent (ambion), according to the manufacturer’s instructions, from 5 of the control and infected bee midguts at 7, 14, and 21 dpi. The extracted RNA (2 μg) was treated with DNaseI (Roche Molecular Biochemicals, Basel, Switzerland), recovered by a phenol/chloroform/isoamyl alcohol extraction, and precipitated with ethanol. The RNA was then treated with DNase I (Invitrogen Life Technologies, Waltham, MA, USA), following the manufacturer’s instructions, to reduce genomic DNA contamination before RT-qPCR. The DNase I-treated total RNA samples were conducted to the reverse-transcription by GScript RTase kit (GeneDireX, New Taipei, Taiwan), following the manufacturer’s instructions. The reaction mixtures were incubated at 42 °C for 1.5 h and then the reaction was stopped at 70 °C for 15 min. Real-time qPCR was performed by using Thermo Scientific Verso SYBR Green 1-step qRT-PCR ROX Mix kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) in a 96-well Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The qRT-PCR was performed as follows: 95 °C for 3 min, 40 cycle at 95 °C for 10 s, 59 °C for 30 s, 95 °C for 10 s. All samples were performed in triplicate. The relative gene expression levels were calculated by 2^−△△Ct method [28].

3. Results and Discussion

3.1. Microsporidian Infection

The midgut tissues of the control and infected honey bees were homogenized and mature spores in the macerated solution were counted under microscopy. The formation of mature spores was observed after 14 to 21 dpi; moreover, the spore number increased from 14 to 21 dpi (Figure 1A,B). The DNA of honey bee midgets was extracted for confirming the infection of N. ceranae by PCR with 18f and 1537r primer sets (microsporidia degenerated primer set). The results of PCR revealed that only the infected honey bees were detected (Figure 1D). The infection of N. ceranae was detected at 7 dpi, though the mature spores were only observed at 14 dpi (Figure 1). This result was similar to the previous report that different parasite stages of N. ceranae should present in the midgut epithelial cells after the experimental infection of N. ceranae with honey bees at 6–7 dpi [11], indicated the honey bees were under N. ceranae infection. It has been described that the pathological effects of N. ceranae infected A. mellifera resulted in the reduction of the lifespan [11,21]. From our survival rate analysis, adult honey bees infected with N. ceranae showed lower survival possibility and survival time than those uninfected honey bees (Figure 1C). These data proved a consistent negative effect of the N. ceranae to adult bees.

3.2. Screening of the Subtracted cDNA libraries

Two subtracted cDNA libraries were established, and a total of 192 white colonies (96 colonies each) were first screened by colony PCR (Figure 2). The results of colony PCR showed that the positive rate was 97.9% for fscl and 95.8% for rscl. The size of inserted cDNA fragments varied from 180 bp to 1 kb (Figure 2). The positive PCR products were then subjected to dot blotting for screening the differential ESTs. A total of 112 differentially expressed sequence tags (ESTs) were identified in the subtracted libraries and sequenced. Moreover, 65 and 47 ESTs were identified from infected (forward) and non-infected (reverse) subtracted libraries, respectively (

Table 1. Information for the expression sequence tags (ESTs).

Library Type	Value	Mean EST Sequence Length (bp)
Forward library	65	426
Reverse library	47	395
Total ESTs a	112	411

^a ESTs = Expression sequence tags.

and Figure 2B,C,E,F). These clones revealed the genes with high differential expression levels and were further sequenced.

3.3. Analysis of EST Sequences

A total of 112 ESTs were sequenced and further analyzed. The average sequence length of ESTs in each library was 426 bp for the infected (forward) library and 395 bp for the non-infected (reverse) library (Table 1). According to the NCBI BLASTn analysis, 75.4% ESTs (49 ESTs) were identified as microsporidia genes and 20% ESTs (13 ESTs) belonged to Apis spp. in the fscl (

Table 2. Analysis of 65 differential expressed sequence tags (ESTs) identified from forward cDNA library. NCBI = National Center for Biotechnology Information.

No	EST Number	Insert Sequence Length (bp)	NCBI_BLAST_N			NCBI_BLAST_X
			Species	Gene Name	E-Value	Species	Gene Name	E-Value
1	SSH-Forward-lib-1	302	Nosema ceranae	Hypothetical protein (AAJ76_1600052943)	3.00E-127	Nosema ceranae	Hypothetical protein (AAJ76_1600052943)	9.00E-55
2	SSH-Forward-lib-2	396	Nosema ceranae	Dynein light chain 1 (AAJ76_5000122115)	4.00E-77	Nosema ceranae	Dynein light chain 1	2.00E-24
3	SSH-Forward-lib-3	302	Nosema ceranae	Hypothetical protein (AAJ76_1600052943)	2.00E-124	Nosema ceranae	Hypothetical protein (AAJ76_1600052943)	5.00E-55
4	SSH-Forward-lib-4	421	Nosema ceranae	Hypothetical protein (AAJ76_700047245)	1.00E-177	Nosema ceranae	Hypothetical protein NCER_101230 (AAJ76_700047245)	2.00E-64
5	SSH-Forward-lib-5	958	Nosema ceranae	60s ribosomal protein L10a (AAJ76_1200039265)	0.00E+00	Nosema ceranae	60s ribosomal protein L10a	2.00E-145
6	SSH-Forward-lib-7	413	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	0.00E+00	Nosema ceranae	Actin	8.00E-86
7	SSH-Forward-lib-8	532	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	0.00E+00	Nosema ceranae	Actin	1.00E-82
8	SSH-Forward-lib-9	346	Nosema ceranae	14-3-3 protein 1-like protein (AAJ76_300017093)	8.00E-158	Nosema ceranae	14-3-3 protein 1-like protein	4.00E-66
9	SSH-Forward-lib-10	354	Nosema ceranae	Heat shock protein 90 (AAJ76_1170002375)	2.00E-153	Nosema ceranae	Heat shock protein 90	7.00E-61
10	SSH-Forward-lib-11	262	Apis mellifera	PREDICTED: Apis mellifera protein G12-like (LOC102654405)	1.00E-100	Apis mellifera	PREDICTED: protein G12-like	1.00E-18
11	SSH-Forward-lib-12	181	Apis mellifera	Apis mellifera ribosomal protein LP1 (RpLP1) (NM_001185144)	8.00E-63	Apis mellifera	Unknown	6.00E-17
12	SSH-Forward-lib-13	326	Nosema ceranae	Ubiquitin (AAJ76_1300017036)	1.00E-115	Nosema ceranae	Ubiquitin	1.00E-47
13	SSH-Forward-lib-14	231	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	1.00E-88	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	8.00E-34
14	SSH-Forward-lib-15	212	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	3.00E-93	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	2.00E-33
15	SSH-Forward-lib-19	959	Apis mellifera	PREDICTED: Apis mellifera insulin receptor B (IR-B), transcript variant 1	0.00E+00	Bacteroides dorei	Hypothetical protein HMPREF1063_05178	1.00E-18
16	SSH-Forward-lib-20	163	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	1.00E-47	Nosema ceranae	Actin	3.00E-16
17	SSH-Forward-lib-21	814	Schistosoma rodhaini	Schistosoma rodhaini genome assembly S_rodhaini_Burundi (LL960759 )	3.00E+00	-	-	-
18	SSH-Forward-lib-23	231	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	2.00E-93	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	6.00E-35
19	SSH-Forward-lib-25	319	Nosema ceranae	Mitochondrial sulfhydryl oxidase (AAJ76_600077605)	3.00E-136	Nosema ceranae	Mitochondrial sulfhydryl oxidase	9.00E-58
20	SSH-Forward-lib-26	133	Alexandrium tamarense	Alexandrium tamarense mRNA (AB233356)	4.00E-14	Durinskia baltica	Cytochrome oxidase subunit 3, partial	2.00E-32
21	SSH-Forward-lib-27	537	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	0.00E+00	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	2.00E-116
22	SSH-Forward-lib-29	186	Apis florea	PREDICTED: Apis florea V-type proton ATPase 16 kDa proteolipid subunit (LOC100867892)	5.00E-66	Melipona quadrifasciata	V-type proton ATPase 16 kDa proteolipid subunit, partial	2.00E-32
23	SSH-Forward-lib-30	244	Nosema ceranae	Hypothetical protein (AAJ76_6800017741)	4.00E-106	Nosema ceranae	Hypothetical protein (AAJ76_6800017741)	9.00E-40
24	SSH-Forward-lib-32	323	Nosema ceranae	Hypothetical protein (AAJ76_1600052943)	3.00E-135	Nosema ceranae	Hypothetical protein (AAJ76_1600052943)	1.00E-56
25	SSH-Forward-lib-34	885	Apis mellifera	PREDICTED: Apis mellifera early nodulin-75 (LOC724199)	0.00E+00	Apis dorsata	PREDICTED: extensin-like	2.00E-27
26	SSH-Forward-lib-36	320	Nosema ceranae	Polar tube protein 1 (PTP1) gene	2.00E-88	Vibrio parahaemolyticus	V-type ATPase subunit C	7.00E-14
27	SSH-Forward-lib-37	232	Nosema ceranae	Polar tube protein 2 (AAJ76_1900025375)	3.00E-85	Nosema ceranae	Polar tube protein 2	2.00E-34
28	SSH-Forward-lib-41	594	Nosema ceranae	Heat shock protein 90 (AAJ76_1170002375)	0.00E+00	Nosema ceranae	Heat shock protein 90	6.00E-122
29	SSH-Forward-lib-44	682	Apis mellifera	PREDICTED: Apis mellifera uncharacterized LOC100578731 (LOC100578731)	0.00E+00	Apis mellifera	PREDICTED: uncharacterized protein LOC100578731 isoform X1	6.00E-123
30	SSH-Forward-lib-46	411	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	0.00E+00	Nosema ceranae	Actin	1.00E-87
31	SSH-Forward-lib-48	294	Nosema ceranae	Hypothetical protein (AAJ76_1900028347)	2.00E-129	Nosema ceranae	Hypothetical protein AAJ76_1900028347	5.00E-51
32	SSH-Forward-lib-49	85	Apis dorsata	PREDICTED: Apis dorsata V-type proton ATPase 16 kDa proteolipid subunit-like (LOC102679198)	3.00E-27	Cyprinus carpio	Hypothetical protein cypCar_00031997	6.00E-06
33	SSH-Forward-lib-51	231	Nosema ceranae	Hypothetical protein (AAJ76_2600030604)	7.00E-38	Nosema ceranae	Hypothetical protein AAJ76_2600030604	2.00E-05
34	SSH-Forward-lib-53	668	Nosema ceranae	Forkhead hnf3 transcription factor (AAJ76_500091146)	0.00E+00	Nosema ceranae	Forkhead hnf3 transcription factor	7.00E-126
35	SSH-Forward-lib-54	305	Nosema ceranae	Histone H4 (AAJ76_150002436)	8.00E-127	Nosema ceranae	Histone H4	1.00E-39
36	SSH-Forward-lib-55	731	Apis mellifera	PREDICTED: Apis mellifera facilitated trehalose transporter Tret1-like (LOC724874)	0.00E+00	Apis mellifera	PREDICTED: facilitated trehalose transporter Tret1-like isoform X4	2.00E-07
37	SSH-Forward-lib-56	279	Apis mellifera	Apis mellifera heat shock protein cognate 4 (Hsc70-4) (NM_001160050)	2.00E-117	Bombus terrestris	Heat shock protein cognate 70	6.00E-50
38	SSH-Forward-lib-57	420	Apis mellifera	Apis mellifera heat shock protein cognate 4 (Hsc70-4) (NM_001160050)	7.00E-164	Melipona quadrifasciata	Heat shock 70 kDa protein cognate 4	2.00E-69
39	SSH-Forward-lib-59	231	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	4.00E-93	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	6.00E-35
40	SSH-Forward-lib-60	668	Nosema ceranae	Forkhead hnf3 transcription factor (AAJ76_500091146)	0.00E+00	Nosema ceranae	Forkhead hnf3 transcription factor	1.00E-124
41	SSH-Forward-lib-61	811	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	0.00E+00	Nosema ceranae	Actin	1.00E-84
42	SSH-Forward-lib-62	163	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	1.00E-47	Nosema ceranae	Actin	6.00E-16
43	SSH-Forward-lib-63	651	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	0.00E+00	Nosema ceranae	Actin	4.00E-141
44	SSH-Forward-lib-65	534	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	0.00E+00	Nosema ceranae	Hypothetical protein (AAJ76_2000141845)	3.00E-87
45	SSH-Forward-lib-66	615	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	0.00E+00	Nosema ceranae	Actin	2.00E-133
46	SSH-Forward-lib-68	671	Apis mellifera	PREDICTED: Apis mellifera ankyrin repeat domain-containing protein 49-like (LOC408773)	0.00E+00	Apis dorsata	PREDICTED: ankyrin repeat domain-containing protein 49-like	3.00E-90
47	SSH-Forward-lib-70	163	Nosema ceranae	Hypothetical protein (AAJ76_500092411)	3.00E-49	Nosema ceranae	Actin	2.00E-16
48	SSH-Forward-lib-71	550	Nosema ceranae	Histone H3 (AAJ76_800037613)	0.00E+00	Nosema ceranae	Histone H3	4.00E-89
49	SSH-Forward-lib-73	248	Apis mellifera	PREDICTED: Apis mellifera ribosomal protein S7 (RpS7) (XM_624940) (LOC552564)	1.00E-101	Bombus impatiens	PREDICTED: 40S ribosomal protein S7	8.00E-22
50	SSH-Forward-lib-74	892	Nosema ceranae	14-3-3 protein 1-like protein (AAJ76_300017093)	0.00E+00	Nosema ceranae	14-3-3 protein 1-like protein	0.00E+00
51	SSH-Forward-lib-75	522	Nosema ceranae	Nucleolar transformer 2-like protein (AAJ76_1900029507)	0.00E+00	Nosema ceranae	Nucleolar transformer 2-like protein	2.00E-92
52	SSH-Forward-lib-76	521	Apis mellifera	PREDICTED: Apis mellifera uncharacterized (ncRNA) (LOC107964104)	0	-	-	-
53	SSH-Forward-lib-78	616	Nosema ceranae	Hypothetical protein (AAJ76_1900028347)	0.00E+00	Nosema ceranae	Hypothetical protein AAJ76_1900028347	7.00E-79
54	SSH-Forward-lib-79	293	Nosema ceranae	Hypothetical protein (AAJ76_1900028347)	5.00E-125	Nosema ceranae	Hypothetical protein AAJ76_1900028347	6.00E-51
55	SSH-Forward-lib-80	294	Nosema ceranae	Hypothetical protein (AAJ76_1900028347)	2.00E-124	Nosema ceranae	Hypothetical protein AAJ76_1900028347	6.00E-48
56	SSH-Forward-lib-82	226	Nosema ceranae	Adp-ribosylation factor (AAJ76_1600043340)	3.00E-100	Nosema ceranae	Adp-ribosylation factor	2.00E-40
57	SSH-Forward-lib-83	447	Nosema ceranae	DNA-directed RNA polymerase ii 16kda polypeptide (AAJ76_3400016364)	0.00E+00	Nosema ceranae	DNA-directed RNA polymerase ii 16kda polypeptide	6.00E-78
58	SSH-Forward-lib-84	227	Nosema ceranae	Histone H3 (AAJ76_800037613)	2.00E-90	Nosema ceranae	Histone H3	2.00E-38
59	SSH-Forward-lib-86	346	Nosema ceranae	Sec61beta (AAJ76_5600014344)	7.00E-129	Nosema ceranae	Sec61beta	2.00E-37
60	SSH-Forward-lib-87	354	Nosema ceranae	Heat shock protein 90 (AAJ76_1170002375)	2.00E-151	Nosema ceranae	Heat shock protein 90	2.00E-60
61	SSH-Forward-lib-88	886	Nosema ceranae	Elongation factor 1 (AAJ76_1200007387)	0.00E+00	Nosema ceranae	Elongation factor 1	0.00E+00
62	SSH-Forward-lib-90	252	Nosema ceranae	Ubiquitin (AAJ76_1300017036)	3.00E-90	Nosema ceranae	Ubiquitin	3.00E-38
63	SSH-Forward-lib-94	529	Nosema ceranae	60s ribosomal protein L23 (AAJ76_200068052)	0.00E+00	Nosema ceranae	60S ribosomal protein L23	1.00E-101
64	SSH-Forward-lib-95	320	Hottentotta judaicus	Hottentotta judaicus clone Hj0016 gamma(m)-buthitoxin-Hj1c pseudogene mRNA (HQ288097)	1.00E-14	Hottentotta judaicus	Gamma-buthitoxin-Hj1a	3.00E-18
65	SSH-Forward-lib-96	401	Nosema ceranae	Histone H4 (AAJ76_150002436)	1.00E-151	Nosema ceranae	Histone h4	5.00E-52

and Figure 2B,C). From the rscl, 93.6% ESTs (44 ESTs) showed significant homology to the insect host, particularly Hymenoptera insects (A. mellifera and A. florea) (

Table 3. Analysis of 53 differential expressed sequence tags (ESTs) identified from reverse cDNA library.

No	EST Number	Insert Sequence Length (bp)	NCBI_BLAST_N			NCBI_BLAST_X
			Species	Gene Name	E-Value	Species	Gene Name	E-Value
1	SSH-Reverse-lib-1	387	Colletes stepheni	Colletes stepheni isolate CSTM4 internal transcribed spacer 1, partial sequence (GU132310)	3.00E-03	Elephantulus edwardii	Histone deacetylase complex subunit SAP130-like	1.5
2	SSH-Reverse-lib-2	216	Apis mellifera	PREDICTED: Apis mellifera facilitated trehalose transporter Tret1-like (LOC724874)	3.00E-50	Apis mellifera	Facilitated trehalose transporter Tret1 isoform X1	10.00E-15
3	SSH-Reverse-lib-3	156	Apis mellifera	PREDICTED: Apis mellifera chitin synthase chs-2 (LOC412215)	6.00E-51	-	-	-
4	SSH-Reverse-lib-4	467	Apis mellifera	PREDICTED: Apis mellifera histone H2A-like (LOC552322)	0.00E+00	Nasonia vitripennis	PREDICTED: histone H2A	4.00E-57
5	SSH-Reverse-lib-5	829	Apis mellifera	PREDICTED: Apis mellifera early nodulin-75 (LOC724199)	0.00E+00	Apis dorsata	PREDICTED: extensin-like	2.00E-28
6	SSH-Reverse-lib-6	331	Bombus impatiens	PREDICTED: Bombus impatiens zinc finger protein 665-like (LOC100740332)	1.00E-39	-	-	-
7	SSH-Reverse-lib-7	488	Apis mellifera	PREDICTED: Apis mellifera protein SMG9 (LOC411774)	0.00E+00	Apis mellifera	PREDICTED: protein SMG9-like	3.00E-95
8	SSH-Reverse-lib-10	382	Apis mellifera syriaca	Apis mellifera syriaca mitochondrion ( KP163643)	5.00E-38	-	-	-
9	SSH-Reverse-lib-12	233	Megachile rotundata	PREDICTED: Megachile rotundata probable maleylacetoacetate isomerase 2 (LOC100882531)	2.00E-14	-	-	-
10	SSH-Reverse-lib-14	192	Apis mellifera	PREDICTED: Apis mellifera caspase-like (LOC411381)	3.00E-69	Apis mellifera	PREDICTED: caspase-like	7.00E-29
11	SSH-Reverse-lib-15	546	Apis dorsata	PREDICTED: Apis dorsata nuclease-sensitive element-binding protein 1-like (LOC102681001)	0.00E+00	-	-	-
12	SSH-Reverse-lib-16	218	Ceratitis capitata	Ceratitis capitata clone 17a mRNA ( DQ406807)	0.019	-	-	-
13	SSH-Reverse-lib-17	629	Apis florea	PREDICTED: Apis florea histone H3.3 (LOC100869036)	4.00E-30	-	-	-
14	SSH-Reverse-lib-18	166	Hottentotta judaicus	Hottentotta judaicus clone Hj0016 gamma(m)-buthitoxin-Hj1c pseudogene mRNA (HQ288097)	2.00E-12	-	-	-
15	SSH-Reverse-lib-19	246	Apis mellifera	PREDICTED: Apis mellifera probable alpha-ketoglutarate-dependent dioxygenase ABH4-like (LOC551104)	6.00E-98	Apis mellifera	PREDICTED: alpha-Ketoglutarate-dependent dioxygenase alkB homolog 4-like	1.00E-25
16	SSH-Reverse-lib-20	658	Apis mellifera	PREDICTED: Apis mellifera facilitated trehalose transporter Tret1-like (LOC724874)	0.00E+00	Apis mellifera	PREDICTED: facilitated Trehalose transporter Tret1-like isoform X1	7.00E-81
17	SSH-Reverse-lib-21	976	Boechera divaricarpa	Boechera divaricarpa GSS (HF949778)	0.00E+00	Apis mellifera	PREDICTED: transcription Initiation factor IIA subunit 1 isoform 1	8.00E-48
18	SSH-Reverse-lib-22	469	Apis mellifera	PREDICTED: Apis mellifera ribosomal protein L3 (RpL3) (LOC552445)	0.00E+00	Apis mellifera	PREDICTED: 60S ribosomal protein L3	1.00E-80
19	SSH-Reverse-lib-25	482	Apis mellifera	PREDICTED: Apis mellifera UNC93-like protein MFSD11-like (LOC552407)	0.00E+00	Apis mellifera	PREDICTED: UNC93-like protein MFSD11-like	8.00E-77
20	SSH-Reverse-lib-26	273	Apis mellifera	PREDICTED: Apis mellifera vegetative cell wall protein gp1 (LOC726855)	1.00E-108	Apis mellifera	PREDICTED: vegetative cell wall protein gp1-like isoform X1	8.00E-30
21	SSH-Reverse-lib-28	440	Apis mellifera	PREDICTED: Apis mellifera uncharacterized (LOC102653963)	0	-	-	-
22	SSH-Reverse-lib-32	556	Apis mellifera	PREDICTED: Apis mellifera NPC intracellular cholesterol transporter 2 (LOC724386)	0.00E+00	Apis mellifera	PREDICTED: protein NPC2 homolog	1.00E-78
23	SSH-Reverse-lib-35	269	Apis mellifera	PREDICTED: Apis mellifera vegetative cell wall protein gp1 (LOC726855)	1.00E-100	Apis mellifera	PREDICTED: vegetative cell wall protein gp1-like isoform X1	3.00E-25
24	SSH-Reverse-lib-38	318	Apis mellifera	PREDICTED: Apis mellifera RNA-dependent helicase p72 (LOC411250)	6.00E-131	-	-	-
25	SSH-Reverse-lib-39	233	Megachile rotundata	PREDICTED: Megachile rotundata probable maleylacetoacetate isomerase 2 (LOC100882531)	2.00E-14	-	-	-
26	SSH-Reverse-lib-41	454	Apis mellifera	PREDICTED: Apis mellifera chitotriosidase-1-like (LOC100577156)	3.00E-30	Apis mellifera	PREDICTED: chitotriosidase-1-like isoform X3	2.00E-10
27	SSH-Reverse-lib-45	377	Colletes stepheni	Colletes stepheni isolate CSTM4 internal transcribed spacer 1 (GU132310 )	2.00E-04	Elephantulus edwardii	PREDICTED: histone Deacetylase complex subunit SAP130-like	0.99
28	SSH-Reverse-lib-49	386	Colletes stepheni	Colletes stepheni isolate CSTM4 internal transcribed spacer 1 (GU132310 )	2.00E-04	Elephantulus edwardii	PREDICTED: WD repeat-containing protein 27	4.4
29	SSH-Reverse-lib-52	247	Apis mellifera	PREDICTED: Apis mellifera RNA-dependent helicase p72 (LOC411250)	1.00E-101	Apis dorsata	DEAD-box ATP-dependent RNA helicase 20-like	0
30	SSH-Reverse-lib-58	213	Apis mellifera	PREDICTED: Apis mellifera phosphotidylinositol 3 kinase 21B ortholog (LOC408577)	4.00E-80	Curtobacterium sp.	Hypothetical protein	4.8
31	SSH-Reverse-lib-59	327	Apis mellifera	PREDICTED: Apis mellifera ribosomal protein S7 (RpS7) (LOC552564)	2.00E-138	Apis mellifera	PREDICTED: 40S ribosomal protein S7	2.00E-58
32	SSH-Reverse-lib-60	199	Apis mellifera	PREDICTED: Apis mellifera solute carrier organic anion transporter family member 5A1-like (LOC409650)	1.00E-60	-	-	-
33	SSH-Reverse-lib-61	237	Apis mellifera	PREDICTED: Apis mellifera Jun-related antigen (Jra) (LOC726289)	7.00E-91	Salmonella enterica	Hypothetical protein, partial	2.4
34	SSH-Reverse-lib-63	274	Apis mellifera	PREDICTED: Apis mellifera vegetative cell wall protein gp1 (LOC726855)	1.00E-107	Apis mellifera	PREDICTED: vegetative cell wall protein gp1-like isoform X1	3.00E-18
35	SSH-Reverse-lib-67	377	Colletes stepheni	Colletes stepheni isolate CSTM4 internal transcribed spacer 1 (GU132310 )	0.003	Danio rerio	glutamate receptor 3 precursor	5.3
36	SSH-Reverse-lib-68	386	Colletes stepheni	Colletes stepheni isolate CSTM4 internal transcribed spacer 1 (GU132310 )	2.00E-04	-	-	-
37	SSH-Reverse-lib-69	376	Colletes stepheni	Colletes stepheni isolate CSTM4 internal transcribed spacer 1 (GU132310 )	0.003	Nothobranchius furzeri	PREDICTED: histone deacetylase complex subunit SAP130	4.7
38	SSH-Reverse-lib-70	275	Apis mellifera	PREDICTED: Apis mellifera upstream activation factor subunit spp27-like (LOC408520)	3.00E-109	-	-	-
39	SSH-Reverse-lib-71	377	Colletes stepheni	Colletes stepheni isolate CSTM4 internal transcribed spacer 1 (GU132310 )	2.00E-04	Elephantulus edwardii	PREDICTED: WD repeat-containing protein 27	4.9
40	SSH-Reverse-lib-72	329	Apis mellifera	PREDICTED: Apis mellifera transcription factor mblk-1-like (Mblk-1)	1.00E-139	-	-	-
41	SSH-Reverse-lib-74	563	Apis dorsata	PREDICTED: Apis dorsata uncharacterized (LOC102671476)	0.00E+00	Apis dorsata	PREDICTED: uncharacterized protein LOC102671476	6.00E-95
42	SSH-Reverse-lib-75	281	Apis mellifera	Phosphatidylinositol 4-phosphate 5-kinase type-1 alpha (LOC724991)	4.00E-115	-	-	-
43	SSH-Reverse-lib-79	946	Apis mellifera	PREDICTED: Apis mellifera protein FAM76A-like (LOC408294)	0.00E+00	Apis mellifera	PREDICTED: ATPase family AAA domain-containing protein 2-like	1.00E-63
44	SSH-Reverse-lib-84	383	Apis mellifera	PREDICTED: Apis mellifera ribosomal protein L21 (RpL21) (LOC726056)	3.00E-134	Apis mellifera	PREDICTED: 60S ribosomal protein L21	3.00E-47
45	SSH-Reverse-lib-87	404	Apis mellifera	PREDICTED: Apis mellifera protein split ends (LOC412243)	3.00E-167	-	-	-
46	SSH-Reverse-lib-92	532	Hottentotta judaicus	Gamma(m)-buthitoxin-Hj1c pseudogene mRNA (HQ288097)	2.00E-14	Apis cerana	Hypothetical protein APICC_07509	5.6
47	SSH-Reverse-lib-94	487	Colletes stepheni	Colletes stepheni isolate CSTM4 internal transcribed spacer 1 (GU132310 )	3.00E-04	Nothobranchius furzeri	PREDICTED: histone deacetylase complex subunit SAP130	4.2

and Figure 2E,F). This result indicated that the microsporidia genes were highly expressed during the infection process. Only a few host genes showed dramatic responses of up-regulated during N. ceranae infection, while most of the up-regulated host genes were identified from the rscl, suggesting the suppression of host gene expression by N. ceranae infection. Based on this result, several immune-related genes (i.e., Jra, pi3k) of honey bees were identified from the rscl. As a matter of fact, that N. ceranae infection could change the metabolism of the host and lead to negative effects on the immune systems of honey bees [29]. It has also been reported that honey bees infected with N. ceranae showed a down-regulation of some immune-related genes, such as abaecin, apidecin, defensin, hymenoptaecin, glucose dehydrogenase (GLD), and vitellogenin (Vg), suggesting that N. ceranae infection suppresses immune defense mechanisms in honey bees [30,31,32].

The result of Gene Ontology (GO) analysis showed a total of 30 differentially expressed genes (DEGs) (78.9%) from the host were identified and further categorized into molecular function, biological process, and cellular component (Figure 3; Supplementary Table S2). Moreover, 16 GO terms belonged to three major categories (Figure 3; Supplementary Table S2). Based on the GO analysis, the genes involved in catalytic activity (GO:0003824) were highly up-regulated and were followed by cellular process (GO:0009987)/membrane (GO:0016020), and binding (GO:0005488)/transporter activity (GO:0005215)/cell (GO:0005623) in the fscl. For the rscl, host genes involved in the metabolic process (GO:0008152) and cell (GO:0005623) showed high changes during N. ceranae infection, followed by catalytic activity (GO:0003824)/binding (GO:0005488)/cellular process (GO:0009987)/membrane (GO:0016020), and transporter activity (GO:0005215)/organelle (GO:0043226) (Figure 3; Supplementary Table S2). In conclusion, according to our data, a high percentage of host DEGs were involved in catalytic activity (GO:0003824), cellular process (GO:0009987), membrane (GO:0016020)/cell (GO:0005623), and transporter activity (GO:0005215) (Figure 3; Supplementary Table S2). As a matter of fact, the up-regulation of the α-glucosidase gene, and genes that were involved in the trehalose transport, and the down-regulation of the trehalase and the glucose-methanol-choline oxidoreductase, three encoding genes during N. ceranae infection. The unbalance of gene expressions result in modifications of carbohydrate metabolism in infected honey bees; suggesting that these changes in gene expressions and carbohydrate metabolism were manipulative activity of the N. ceranae to get nutrients from the host [21,30]. Based on our data, the genes involved in the metabolic process (GO:0008152) from the rscl showed differential expression, revealed the host gene expression change after N. ceranae infection, and led to the unbalance of physiological mechanisms.

3.4. Identification and Validation of Highly Expressed N. ceranae Specific Genes

From the forward (infected) library, 75.4% ESTs (49 ESTs belonged to 24 genes) were identified as N. ceranae genes (Table 2). Of 24 N. ceranae genes, nine DEGs, which have more than three identified ESTs and >3-fold change by dot bolt analysis, were selected for further validation by qRT-PCR, including four hypothetical proteins (AAJ76_1600052943, AAJ76_500092411, AAJ76_2000141845, and AAJ76_1900028347), 14-3-3 protein 1-like protein (AAJ76_300017093), Heat shock protein 90 (AAJ76_1170002375), Ubiquitin (AAJ76_1300017036), Forkhead hnf3 transcription factor (AAJ76_500091146), and Histone H3 (AAJ76_800037613) (Figure 4). Similar to the expectations, the results indicated that these genes showed high expression during N. ceranae infection. Moreover, the results also showed that all microsporidia specific gene expressions had the highest gene expression level at 7 dpi, and decreased at 14 and 21 dpi (Figure 5). It should be noted that among these nine N. ceranae specific genes, a hypothetical protein (AAJ76_1600052943) (the previous library clone no. = sr22 and provisional named sr22 gene) has consistently the highest expression, which up-regulated to ~1.1 × 10⁴ fold at 7 dpi to the mock infection 2340, and 1148 fold at 14 and 21 dpi, respectively (Figure 5). For the other eight genes, three genes, histone H3, ubiquitin, and hypothetical protein (AAJ76_2000141845), showed higher expression levels, which ranged from ~2100 to 4400 fold to the mock infection at 7 dpi; the hypothetical protein (AAJ76_2000141845) also showed consistently high expression at 14 to 21 dpi (Figure 5).

Ubiquitin is a small protein that is involved in the ubiquitin-proteasome system (UPS). The UPS is the crucial intracellular protein degradation system in eukaryotes. It has been reported that the infection of N. bombycis resulted in up-regulation of protein degradation-related enzymes in the host midgut, including the S-phase kinase-associated protein 1, and the ubiquitin-conjugating enzyme E2G. Both of which are involved in the ubiquitin-proteasome pathway [33]. From our data, the high expression of ubiquitin was observed in N. cerana, indicating the host defense mechanism to the parasite and, therefore, triggers the protein degradation-related enzyme in N. cerana.

Besides, the high expression of histone H3 was detected in the N. ceranae infection process; histone proteins are essential for DNA packing, chromosome stabilization, and gene expression in the nucleus of a eukaryotes cell. Each nucleosome consists of two copies of each core histone—H2A, H2B, H3, and H4—to be an octameric protein complex. Histones H3 and H4 are the most highly conserved histones, and play a regulatory role in chromatin formation, such as retaining the ability to impede transcription [34]. Therefore, it was assumed that high expression of N. ceranae histone H3 might enhance the regulation of the chromatin formation and facilitate the microsporidia DNA replication.

For the other up-regulated N. ceranae genes, they showed up-regulated expression profiles during N. ceranae infection (Figure 5). The functions of several genes revealed the response of N. ceranae to the honey bee host and also showed the replication activity of N. ceranae during infection. For the 14-3-3 protein 1-like protein (AAJ76_300017093), it is a serine/threonine binding protein. The 14-3-3 protein 1-like protein inhibits apoptosis through sequestration of pro-apoptotic client proteins [35]. Interestingly, the high expression levels of ubiquitin of N. ceranae was detected at 7 dpi and then decreased at 14 and 21 dpi, while the expression of 14-3-3 protein 1-like protein was higher than that of ubiquitin at 21 dpi; besides, the heat shock protein 90 (Hsp90) was also identified as a high expressed N. ceranae gene that facilitates metastable protein maturation, stabilization of aggregation-prone proteins, quality control of misfolded proteins, and assists in keeping proteins in activation-competent conformations [36]; it seems that during the infection process, N. ceranae not only needs to against the stress from the host, but also attempts to stabilize the physical pathway itself for self-propagation purposes. This hypothesis is worthy to be addressed in the future.

The gene forkhead hnf3 transcription factor (AAJ76_500091146) presented throughout the animal kingdom, as well as in fungi and yeast (Mazet et al. 2003). The function of forkhead hnf3 transcription factor is characterized by a type of DNA-binding domain known as the forkhead box (FOX) [37]. It was also described that the forkhead hnf3 transcription factor is involved in a wide range of biological functions, including development, growth, stress resistance, apoptosis, cell cycle, immunity, metabolism, reproduction, and ageing [38]. As a matter of fact, in terms of these genes, it might play an important role for the replication of microsporidia, but it still needs to be further evaluated. The genome of the honey bee (A. mellifera) had been sequenced, and it consisted of a little more than 10,000 genes, lower than other insects (D. melanogaster, 13,600 genes; A. gambiae, 14,000 genes; B. mori, 18,500 genes) [39]. In additional, the draft assembly (7.86 MB), and highly compact (~1 gene/kb) genome of the N. ceranae genome, has been fully sequenced in 2009 [40]. N. ceranae has a strongly AT-biased genome (74% A+T) and a diversity of repetitive elements, complicating the assembly. A total of 2614 predicted protein-coding sequences were predicted and 1366 of these genes have homologs in the microsporidian Encephalitozoon cuniculi, the most closely related published genome sequence. These databases could be a reference for studying and clarifying the virulence of N. ceranae and the interaction between their host, A. mellifera, in the near future.

In this study, the method of SSH was mainly used for the identification of the differential expression N. ceranae genes. From our data, several N. ceranae genes were identified as differentially expressed genes through the SSH, which could provide information about the pathological effects of N. ceranae. It should be also noted that the microRNA (miRNA) of N. ceranae might play important roles in the transcriptome of N. ceranae during infection, and reduction of the replication of N. ceranae via knockdown of transcripts was also demonstrated [41]. Thus, our data could provide the candidate genes for evaluation of nosemosis-controlling effects during infection. Since the condition (i.e., hybridization) of SSH might have some impacts on the result (i.e., the sequence through-put). Therefore, more modern methods, such as whole transcriptomics for deep sequencing, could be used for further investigations. In terms of the validation of nine up-regulated N. ceranae genes, the N. ceranae genes, which were identified by the SSH method, revealed high coordinates to the expectations. These genes showed high potential for the detection of early nosemosis in the field and provided insight for further applications.

4. Conclusions

In this study, 28 honey bee genes and 24 genes in N. ceranae were identified as DEGs after honey bees were infected with N. ceranae via the SSH approach. For the host DEGs, 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 the unbalance of physiological mechanisms. Of 24 N. ceranae genes, nine DEGs were subject to real-time quantitative reverse transcription PCR (real-time qRT-PCR) for validation, and all of them showed high expression levels during different time post infections. Regarding the validation results, these genes revealed highly potential for the detection of different stages of nosemosis in the field. It should also be noted that one N. ceranae gene (AAJ76_1600052943, sr22) showed the highest expression level after infection. This gene might be further developed as a biomarker for early nosemosis detection. The data from this study could provide information on the pathological effects of N. ceranae and new insight for further applications on honey bee pathogen detections.

5. Patent

Taiwan, I481618. Lo, C. F., Wang, C. H., and Nai, Y. S. Molecular Markers for nosema infection.