Proteomics of Nasonia vitripennis and the effects of native Wolbachia infection on N. vitripennis

Nasonia materials

Two Nasonia lines were used in our experiments, native Wolbachia-infected N. vitripennis and Wolbachia-free N. vitripennis; both lines had been grown in our laboratory since 2011. The Wolbachia-infected line was naturally infected with single Supergroup A Wolbachia strain, as confirmed by Wolbachia surface protein (WSP). The Wolbachia-free N. vitripennis line was treated with rifampin to remove native Wolbachia at the beginning, and repeated polymerase chain reaction (PCR) with primers targeting the WSP gene was used to confirm the effects of removal. Wolbachia-free organisms used in our study were reared after 30 generations to reduce the effects of antibiotics. Two lines were maintained using standard insectary conditions at 28 °C for light and 25 °C for dark (L:D = 16:8 h) with 60% ± 10% relative humidity (Werren & Loehlin, 2009c). In our experiment, N. vitripennis parasitised Wolbachia-free Sarcophaga marshalli pupa, where the eggs, larvae, and pupae of the wasp were developed (Werren & Loehlin, 2009c), and the adults were reared with 10% honey water.

Ecological experiment

At 10–13 days after N. vitripennis oviposited into Sarcophaga marshalli pupae, the pupae were dissected to obtain virgin adults. The male and female pupa were identified individually and collected separately once upon eclosion. Four crossing types based on different Wolbachia infection statuses were carried out (♀ W+ × ♂ W+, ♀ W+ × ♂ W−, ♀ W− × ♂ W−, ♀ W− × ♂ W+). A virgin female and a virgin male adult were placed in a 7 mL tube, together with one fresh Sarcophaga marshalli pupa for oviposition. The offspring of the crossings were collected and counted, and the percent females (the proportion of females of the total number of individuals) was calculated. The offspring number and percent females based on the number of fly pupae per day were calculated for nine consecutive generations. Moreover, the effects of Wolbachia on longevity of N. vitripennis were studied. Approximately 300 samples were used for life testing. Each experiment was repeated with six groups.

Protein preparation

Wolbachia-infected and Wolbachia-free line were prepared for iTRAQ separately, each line contains three replicates. The female adults were collected after eclosion two to three days, and all adults of a sample were from the same generation. Approximately 200 adult wasps per sample were homogenized, and total proteins were extracted using the phenol extraction procedure. The protein content in the supernatant was quantified using a BCA Protein Assay Kit (Thermo Scientific, Milford, MA, USA).

Proteome analysis by iTRAQ

The proteome was quantified using iTRAQ-LC-MS/MS. Protein abundances that changed more than 1.2-fold were regarded as significantly differentially expressed. The extracted proteins were digested, and peptides were quantified as described by Wisniewski et al. (2009). For peptide labeling, the peptide mixture from each group was labeled with iTRAQ Reagent-8plex multiplex kit (AB SCIEX, Foster City, CA, USA) according to the manufacturer’s instructions. Wolbachia-free and Wolbachia-infected samples were labeled with iTRAQ reagents 113, 115, 117 and 114, 116, 118, respectively. The labeled peptide mixtures were pooled for strong cation-exchange chromatography. Samples were separated by nanoLC-MS/MS (EASY-nLC1000). The mass spectrometry data were analyzed using a Q-Exactive mass spectrometer (Thermo Finnigan, Waltham, MA, USA) in the positive ion mode with a selected mass range of m/z 300–1,800.

Quantitative real-time reverse transcription PCR

Total RNA was isolated using TransZol UP (TRANSGEN BIOTECH, Beijing, China) from Wolbachia-infected and Wolbachia-free N. vitripennis adults (50 individuals each). The first-strand cDNA was reverse-transcribed from 2 μg total RNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANSGEN BIOTECH, Beijing, China) at 42 °C for 15 min according to the manufacturer’s instructions. Specific primers for tested genes were designed on the basis of cDNA sequences from NCBI database by Primer Premier 6 software. All primers used in quantitative PCR (qPCR) are shown in Table S1. qPCR was performed on a real-time PCR machine (Bio-Rad, Hercules, CA, USA) with TransStart Tip Green qPCR Supermix (TRANSGEN BIOTECH, Beijing, China). The amplification reaction conditions were as follows: 95 °C for 3 min; followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 95 °C for 10 s; and then a melting curve was constructed from 65 to 95 °C. The relative expression of each gene was normalized to the expression of RPL13a and UBC using the 2^−ΔΔCT method. Results are expressed as means ± standard deviations. Three biological replicates were used in the experiment.

Data analysis

The raw MS/MS spectra data were searched and identified using Mascot 2.2 and Proteome Discoverer 1.4 (Thermo Fisher Scientific, Waltham, MA, USA). The database used in this study was NCBI_Nasonia_vitripennis_25492_20141104.fasta, loaded by Uniprot in November 2014. Assembled protein identifications were qualitatively analyzed using Proteome Discoverer 1.4 software. All data were reported based on 99% confidence for protein identification, as determined by a false discovery rate (FDR) of less than or equal to 1%. Statistical analysis was conducted using one-way analysis of variance (ANOVA). Results with P values of less than or equal to 0.05 by Tukey’s test were considered significant. Among the statistically significant proteins detected by ANOVA (P < 0.05), proteins showing more than 1.2-fold changes in abundance were regarded as significantly differentially expressed.

Bioinformatics

All proteins that were found in abundance among Wolbachia-infected and Wolbachia-free group were further analyzed for functional and biological relevance. These proteins were classified by their gene functions and biological pathways using the freely available gene ontology (GO) database provided by the GO consortium. The retrieved sequences were locally searched against NCBI nr using the NCBI BLAST+ client software (ncbi-blast-2.2.28+−win32.ext) to find homologous proteins, from which functional annotations were transferred to the target proteins. The top 10 BLAST hits with an E-value of less than 1e⁻³ for each query protein were retrieved and loaded into Blast2GO (Version 2.7.2) for GO mapping and annotation. In addition, differentially expressed proteins, proteins participating in the immune system, and proteins involved in the reproductive process were evaluated using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; http://string.embl.de/) to build a functional protein association network.

Percent females of crosses and wasp longevity with different infection statuses

Among the four crosses (♀ W+ × ♂ W+, ♀ W+ × ♂ W−, ♀ W− × ♂ W−, and ♀ W− × ♂ W+), the percent females of uninfected females and infected males (♀ W− × ♂ W+, 0.11) was significantly lower than that of the other three hybridization groups (P < 0.001; Fig. 1A), confirming that the reproductive regulation of Wolbachia on N. vitripennis was CI. The average offspring numbers of Wolbachia-infected wasps with one (W+*1), three (W+*3), and five pupa (W+*5) per day were 215.17 ± 19.30, 391.83 ± 12.95, and 444.17 ± 20.32 respectively, whereas the offspring numbers of Wolbachia-free wasps with one (W−*1), three (W−*3), and five pupa (W−*5) per day were 176.17 ± 13.53, 304.83 ± 24.86, and 407.17 ± 11.18, respectively (Fig. 1B). With the increase in pupae number, the progeny size of Wolbachia-infected N. vitripennis increased faster, implying that Wolbachia significantly enhanced host fecundity (one pupae: df = 1, F = 1.572, P = 0.002; three pupa: df = 1, F = 1.208, P < 0.001; five pupa: df = 1, F = 2.965, P = 0.003).

The average longevities of Wolbachia-infected and Wolbachia-free females were 18 and 16 days (F_1,288 = 0.19, P = 0.07), respectively, whereas those of Wolbachia-infected and Wolbachia-free males were nine and eight days (F_1,347 = 0.36, P = 0.12), respectively (Fig. 2). The existence of Wolbachia did not significantly alter the life span of N. vitripennis.

Identification, GO analysis, and KEGG analysis of Nasonia proteomics by iTRAQ

A total of 3,109 proteins were identified from 22,946 unique peptides based on the N. vitripennis database with a peptide FDR of less than or equal to 0.01. From these, 3,096 proteins were quantified in three Wolbachia-infected and three Wolbachia-free N. vitripennis libraries (Table S2). Statistical analysis showed that 40.11% of the identified proteins had coverage greater than 20, whereas only 4.54% of proteins had coverage below 2. In addition, 2,520 proteins (81.03%) were identified with at least two peptides. Proteins with masses of more than 10 kDa had broader coverage in protein mass distribution (Fig. S1).

All 3,096 proteins were annotated using Blast2GO (Table S3) and analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (Table S4). In total, 2,578 proteins were annotated by 9,297 GO terms, covering a wide range of BPs (33.47%), MFs (41.47%), and CCs (25.26%; Fig. 3). Notably, 13 proteins participated in the immune system process, and 15 proteins were involved in both the reproductive process and reproduction (Table 1). In particular, these immune proteins were involved in regulation of the immune system process, immune response, innate immune response, and immune response-regulating cell surface receptor signaling pathways. Reproduction proteins participated in the regulation of reproductive processes, sexual reproduction, developmental processes involved in reproduction, reproductive system development, and other pathways.

KEGG analysis showed that 1,975 proteins were assigned to 1,962 KEGG orthologies and were involved in 337 maps. The top 20 maps were analyzed (Fig. S2), and the ribosome contained the most proteins.

Significantly differentially expressed proteins

Based on the iTRAQ-LC-MS/MS proteomic analysis, proteins with more than 1.2-fold differences and P values of less than 0.05 were regarded as significantly differentially expressed. There were only 23 proteins that were significantly differentially expressed between Wolbachia-infected and uninfected N. vitripennis based on the proteomics analysis, including 15 upregulated and eight downregulated proteins (Table 2). Fifteen proteins among the 23 proteins were involved in catalytic activity and binding in the MF category and in metabolic processes in the BP category. These differentially expressed proteins belonged to the biosynthesis, metabolism, and degradation processes, including Ras signaling, peroxisomes, vascular smooth muscle contraction, inflammatory mediator regulation of TRP channels, and chemical carcinogenesis. Interestingly, neither immune-related proteins nor reproductive proteins were significantly differentially expressed. Some immune genes, particularly those involved in the Toll or IMD pathways, as well as some antimicrobial peptides, were not found in either Wolbachia-infected or Wolbachia-free organisms. Protein LOC 100121395 (NV15328), which was uncharacterized in the research database, was found as putative odorant binding protein 69. Uncharacterized protein LOC1003315494 and LOC100678792 may be venom proteins by BLAST. NV11606, which was described as hydroxyacid oxidase 1, may participate not only in the carbon metabolism, but also in redox signaling and lipid homeostasis. NV16308, alcohol dehydrogenase class-3, also participated in multiple metabolic pathways, including glycolysis/gluconeogenesis, carbon metabolism, metabolism of xenobiotics by cytochrome P450, retinol metabolism, tyrosine metabolism, and fatty acid degradation.

Quantitative real-time reverse transcription PCR

Seven genes encoding significantly differentially expressed proteins (NV11606, NV11923, NV11878, SP6, NV18383, NV16308, and NV21224-PA; Table 2) were selected for quantitative real-time reverse transcription PCR (RT-qPCR) validation; these genes encoded calcium-independent phospholipase, serine protease, alcohol dehydrogenase, hydroxyacid oxidase, trypsin, and two uncharacterized proteins. Moreover, seven genes encoding putative immune-related proteins or reproductive proteins (NV16743, NV10430, NV15633, NV13603, NV12583, NV18120, and NV10830; Table 1) were chosen for RT-qPCR. With the exception of NV11606 and SP6, all RT-qPCR results showed similar changes compared with the proteomic analyses (Fig. 4), confirming the reliability of our proteomic analysis.

Interactions between putative immune-related proteins, reproductive proteins, and significantly differentially expressed proteins

The online database STRING 10.0 (Search Tool for Retrieval of Interacting Genes/Proteins database) is a system that searches for interactions between known and predicted proteins based on both direct physical and functional correlations among proteins. To explore whether the significantly differentially expressed proteins influenced the immune or reproductive processes in N. vitripennis indirectly, the relationships among 23 significantly differentially expressed proteins, 13 proteins with putative roles in the immune system process, and 15 proteins involved in the reproductive process were studied.

The results showed that all significantly differentially expressed proteins located on the edge of the network map were not associated with any immune-related proteins or reproductive proteins, and there were no direct interactions between significantly differentially expressed proteins (Fig. 5). Some immune-related proteins and reproductive proteins formed a network with complex interactions. For example, four Ras family proteins formed a complex interaction network, with functions in the cell cycle, cell adhesion force, cell phagocytic activity, and other processes.

Lipid metabolic pathway

Next, we analyzed the pathways involved for all proteins from Drosophila melanogaster, Aedes aegypti, Solenopsis invicta, Apis mellifera, and N. vitripennis using the KEGG database and filtered out fatty acid metabolism pathways (Table 3). Fatty acid metabolism involves a total of 16 pathways. The proteins involved in the glycerophospholipid metabolism pathway, glycerolipid metabolism, and fatty acid degradation were relatively highly expressed. In N. vitripennis, proteins involved in primary bile acid biosynthesis were more than D. melanogaster, Aedes aegypti, Solenopsis invicta and Apis mellifera, approximately 6–13-fold.