Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum

  • Kayvan Etebari1,

    Affiliated with

    • Robin W Palfreyman2,

      Affiliated with

      • David Schlipalius3,

        Affiliated with

        • Lars K Nielsen2,

          Affiliated with

          • Richard V Glatz4 and

            Affiliated with

            • Sassan Asgari1Email author

              Affiliated with

              BMC Genomics201112:446

              DOI: 10.1186/1471-2164-12-446

              Received: 9 March 2011

              Accepted: 9 September 2011

              Published: 9 September 2011

              Abstract

              Background

              Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported.

              Results

              De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes.

              Conclusion

              This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, DsIV genes expressed in the host and also improves our current understanding of this host-parasitoid interaction.

              Background

              Endoparasitoids of the insect order Hymenoptera inject their eggs inside a host insect where they hatch and subsequently feed on the host until its death. For successful parasitism, endoparasitoids bring about a change in their hosts' conditions in favour of the developing parasitoid larvae. For this purpose, female wasps introduce secretions such as venom or ovary fluids, which may contain symbiotic viruses (polydnavirus (PDV) and/or virus-like particles), and other maternal factors, into the host [1]. In addition to suppression of the host immune system to protect the developing parasitoid, several studies have shown that parasitoids and their introduced maternal factors have significant effects on host metabolism and development such as plasma protein composition, food consumption, endocrine system activity and even on regulatory microRNA levels [28].

              Diadegma semiclausum Hellén is an ichneumonid endoparasitoid that carries a PDV with a circular, double-stranded and segmented DNA genome encoding proteins that suppress the host immune response and cause developmental arrest/delay [9]. PDVs are the most highly characterized of the known mutualistic viruses [10], replicating only within the calyx cells of the reproductive tract of female wasps [11]. No virus replication occurs in parasitized larvae; however expression of encapsidated PDV genes induces different physiological modifications such as interruption of the larval endocrine system and suppression of the host immune system [1215].

              PDVs are classified into two genera based on their host wasp families; Ichnovirus containing ichnoviruses (IVs) and Bracovirus containing bracoviruses (BVs) [11, 16]. These genera are morphologically distinct and their gene functions vary. Analysis of virion structural components from IVs indicates that the set of structural genes is conserved among wasps associated with IVs and might originate from an ancestral virus [17]. Recently, Bigot et al. suggested that IVs originated from ascoviruses by lateral transfer of ascoviral genes into wasp genomes [18]. Interestingly, the DNA that is encapsidated within PDVs appears more similar to that of eukaryotes than that of other viral genomes [19]. It has been demonstrated that less than 2% of encapsidated PDV genes have homologs in other viruses in contrast to the proviral or structural genes [20]. Most of the PDV DNA appear to be non-coding, except for some groups of genes that are involved in host immune suppression pathways [21]. IV genomes generally encompass more than 20 circular DNA segments ranging in size from 2-28 Kb with estimated total genome sizes ranging from 75 Kb to greater than 250 Kb [20].

              Research so far has concentrated mainly on individual genes or small defined groups of host or PDV genes, to explore their function or differential expression following parasitization. Deep sequencing data can provide extensive information about host-parasitoid interactions at the transcriptome level. The large amount of sequencing data that can readily be produced by next-generation sequencing platforms, such as the Illumina GAII, reduces the need for prior sequence knowledge for gene expression profiling and are now making direct sequencing approaches the method of choice for whole transcriptome analysis in many species [2226]. The large numbers of short reads produced by next-generation sequencers provide opportunities for development of new applications where sequencing only a portion of a molecule is sufficient. However, the analysis of transcriptome data produced by these technologies for organisms with limited genomic information still presents challenges because the sequenced fragments must be aligned against existing good quality reference genomes [27].

              In this study, we used deep sequencing to explore the impact of D. semiclausum parasitization on its host, Diamondback moth Plutella xylostella L. (Lepidoptera, Plutellidae), a notorious pest of cruciferous plants. P. xylostella has developed resistance to many groups of chemical insecticides and also Bacillus thuringiensis endotoxin [28, 29]. This has made P. xylostella one of the world's most destructive insect pests and the estimated global cost of controlling this insect is around US$1 billion annually [28]. This emphasizes the necessity for the continued development of innovative alternative control measures and resistance management strategies. Parasitoids or parasitoid-produced regulatory molecules can be used to improve conventional pest control strategies in sustainable agriculture. We have used a deep sequencing approach to give a comprehensive view of immune- and metabolic-related genes that are differentially expressed in parasitized versus non-parasitized P. xylostella larvae, revealing a significant number of D. semiclausum ichnovirus genes (DsIV) which have not been reported previously. This type of study may facilitate new controls for pest larvae by identifying molecules that are crucial for larval immune defence, development, pesticide resistance and other important metabolic regulatory functions.

              Results and Discussion

              P. xylostella transcriptome profile

              A transcriptome is the complete set of expressed RNA transcripts in one or more cells. Transcriptome profiling of organisms under stress or parasitization challenge helps us to obtain a better understanding of subsequent related cellular activities in organisms including growth, development, and immune defence. Recently, the newly developed deep sequencing approaches have significantly changed how the functional complexity of the transcriptome can be investigated [22, 25, 30].

              To analyse the transcriptome of P. xylostella host larvae following parasitization by D. semiclausum, RNA samples isolated from the whole host larvae at various time points after parasitization were pooled together prior to sequencing. This may lead to bias in the results (e.g. a gene may first be up-regulated and down-regulated subsequently); however, we aimed at obtaining an overview of what occurs during parasitism and generating a transcriptome of P. xylostella which has not been previously available and to isolate as many DsIV genes as possible. Illumina GAII RNAseq deep sequencing analysis produced approximately 26.6 and 27.1 million single-end reads from RNA extracted from the whole body of non-parasitized (control) and parasitized larvae of P. xylostella, respectively. De novo assembly using the CLC Genomic Workbench produced 172,660 contigs with a minimum contig size of 100 bp; of these, 66% were between 100-199 bp and only 59,255 (34%) of the contigs were above 200 bp (Table 1). Only contigs above 200 bp were selected for further analysis and 6% of these had nucleotide lengths above 1000 bp (Figure 1). We found 4992 contigs that shared their greatest homology with Tribolium castaneum (Coleoptera) genes with a minimum E-value of 1e-06. Bombyx mori (silkworm) was another species with which a range of P. xylostella genes showed high levels of homology. One reason for the higher number of hits against the beetle genome is that three times more T. castaneum genes than B. mori genes are reported in databases.
              Table 1

              Summary of contig statistics resulting from Illumina deep sequencing of parasitized and non-parasitized P. xylostella larvae

              Parameters

              Number

              Total de novo assembled contigs with CLC software

              172,660

              Contigs used for BLAST (cut-off: above 200 bp length)

              59,255

              Best BLAST matches (cut-off: E-value > E-6)

              26119

              No Hits (contigs without any BLAST match)

              33136

              Not mapped with any Gene Ontology (GO) database

              6087

              Annotated sequences (cut off: GO weight 5E value > E-6)

              20704

              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig1_HTML.jpg
              Figure 1

              Distribution of contig size in assembledP. xylostellatranscriptome. De novo assembly of RNAseq data by CLC genomic workbench generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Only contigs above 200 bp were selected for further analysis.

              The short-read data generated by deep sequencing may be problematic when annotating alternative splice variants [24]. However, many gene expression profiling studies that use high-throughput sequencing can also provide valuable annotation information, such as existence of novel genes, exons or splice events which can be used to annotate putative gene sequences for other species. For instance, 39% of the reads from the brain transcriptome of the wasp, Polistes metricus, were matched to the honeybee (Apis mellifera) genome sequence and EST resources, for annotation [31]. Using a series of filtering and critical cut-off values for BLAST E-value and gene ontology (GO) weighting, 20,704 sequences were annotated by B2GO software http://​www.​blast2go.​org through UniProt KB/TrEMBL and other available databases. GO-annotated consensus sequences were assigned to biological process clusters such as cellular component and molecular function, and distributed among various sub-categories such as metabolism, growth, development, apoptosis, immune defense, molecular processing, signal transduction, transcription regulator activity, catalytic activity etc. (Figure 2).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig2_HTML.jpg
              Figure 2

              GO annotation of consensus sequences (Level 2). 20704 sequences were annotated by B2GO software. This program categorized 14075 contigs in biological process, 18127 contigs in molecular function and 10048 contigs in cellular components. The data from InterPro terms, EC codes and KEGG were merged with GO terms for a wide functional range cover in B2GO annotation.

              Comparison of the transcriptome pattern of P. xylostella for eight different GO terms (molecular function - Level 2) with those of silkworm http://​www.​silkdb.​org showed high similarity in the distribution of genes across GO categories indicating that the transcriptome analysed was not biased towards particular categories (Figure 3).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig3_HTML.jpg
              Figure 3

              Comparison ofP. xylostella(DBM; diamondback moth) transcriptome pattern with that ofB. mori(silkworm), based on 8 different molecular function Gene Ontology (GO) terms (Level 2). Silkworm data was obtained from http://​www.​silkdb.​org.

              Overall, from the assembled contigs of over 200 bp in length, 44% showed similarity with genes or proteins in the NCBI database. The rest may represent unknown genes, non-coding RNA or misassembled contigs that are expected due to the presence of large repetitive or duplicated regions. David et al. (2010) suggested that a significant proportion of transcript signatures detected outside predicted genes represent regulatory non-coding RNAs, because these large numbers of non-coding RNA can be antisense, intergenic or overlapping with protein-coding genes [22].

              Effects of parasitism on the transcription of host immune-related genes

              Our sequencing data analyses indicate that parasitism has a significant impact on the transcriptome profile of P. xylostella larvae. The 'Oases' package was used to assemble data for differential expression analysis. Initially, reads from the parasitized and non-parasitized larvae were cleaned and combined, before de novo transcriptome assembly was carried out using Oases 0.1.18. [32]. The individual sets of reads were then mapped back to the previously assembled contigs and counted as a proxy for gene expression. After filtering our dataset using criteria such as number of reads, contig length, E-value (for nearest homolog identity) and greater than 2-fold change, 928 contigs were short-listed. Figure 4 shows that most of the differentially expressed transcripts for the selected GO terms (Level 2), molecular function and biological process, were up-regulated. Among the contigs, only those related to immunity and development, which were differentially transcribed after D. semiclausum parasitization, are displayed in Tables 2 and 3, respectively, since these are the genes most relevant to parasitism. In instances where differential expression of P. xylostella genes following D. semiclausum parasitization may not be consistent with proteomic observations in other host-parasitoid systems previously reported (e.g. [2]), it is possible that post-transcriptional inhibitory effects of parasitoids' maternal factors (e.g. PDVs or venom) contribute to these differences. It has been reported that PDV genes are able to interrupt translation of host genes with their host translation inhibitory factors (HTIF) which was initially characterized from Campoletis sonorensis IV (CsIV) [33]. Accordingly, in Heliothis virescens it was shown that lysozyme activity declined after parasitization by C. sonorensis or injection of CsIV; however, the transcript levels of the gene increased after parasitization. This suggested that CsIV may regulate host cell gene expression at the translation level. We also found that lysozyme transcript levels increased following parasitization (Table 2). Another study also showed that lysozyme concentration and activity in P. xylostella larvae parasitized by Cotesia plutellae was decreased [2]; however, transcript levels were not measured. Recently, Barandoc and Kim [34] showed that the translation of storage protein in P. xylostella was inhibited by two BV genes (CpBV15α and CpBV15β). In another study, it was shown that expression of antimicrobial peptides (diptericin, cecropin A and drosomycin) were either unchanged or minimally induced in parasitized Drosophila melanogaster larvae by Leptopilina boulardi [35]. The authors concluded that antimicrobial genes are regulated differently independent of those mediating cellular encapsulation.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig4_HTML.jpg
              Figure 4

              The gene ontology score for some selected GO terms (level 2) for significantly up- and down-regulated transcripts. (A) Molecular Function (B) Biological Process. Binding and catalytic activity had the highest GO scores for Molecular Function among those genes that were differentially expressed after parasitization. In the Biological Process category, two GO terms of metabolic and cellular processes showed the highest GO scores. In each group, the abundance of up-regulated genes was higher than down-regulated genes.

              Table 2

              A list of P .xylostella immune-related genes that were differentially transcribed after parasitization by D. semiclausum

              Sequence ID

              TSA Accession No.

              Nt. Length

              Fold change

              Protein

              Species

              E value

              Nt. ID

              (%)

              Accession no.

              Locus 2992

              JL943792

              517

              11

              Moricin-like peptide C2

              Galleria mellonella

              5.00E-12

              75

              ABQ42576.1

              Locus 11864

              JL943746

              452

              7.06

              Serine proteinase inhibitor

              Procambarus clarkii

              5.00E-14

              41

              AAQ22771.1

              Locus 13987

              JL943753

              281

              6.97

              Gloverin

              Plutella xylostella

              3.00E-49

              100

              ACM69342.1

              Locus 42625

              JL943821

              487

              6.71

              Beta-1,3-glucan recognition protein 3

              Helicoverpa armigera

              5.00E-30

              45

              ACI32828.1

              Locus 652

              JL943855

              803

              6.25

              Proline-rich protein

              Galleria mellonella

              6.00E-18

              38

              ACQ99193.1

              Locus 10530

              JL943744

              1440

              6.09

              Transferrin

              Plutella xylostella

              0

              99

              BAF36818.1

              Locus 19881

              JL943772

              503

              4.96

              Odorant binding protein

              Heliothis virescens

              1.00E-49

              66

              ACX53743.1

              Locus 8118

              JL943865

              992

              4.51

              Serine proteinase

              Samia cynthia ricini

              6.00E-89

              55

              BAF43531.1

              Locus 4070

              JL943818

              449

              4.37

              Cecropin E

              Plutella xylostella

              8.00E-17

              100

              BAF36816.1

              Locus 6056

              JL943850

              650

              4.3

              Cecropin 1 (antibacterial peptide)

              Plutella xylostella

              1.00E-16

              98

              ADA13281.1

              Locus 2740

              JL943785

              349

              4.23

              Thrombin inhibitor infestin

              Triatoma infestans

              7.00E-19

              47

              AAK57342.1

              Locus 742

              JL943860

              1405

              3.99

              Lysozyme II

              Artogeia rapae

              4.00E-15

              76

              AAT94286.1

              Locus 8845

              JL943866

              517

              3.81

              Peptidoglycan recognition protein

              Plutella xylostella

              2.00E-86

              97

              BAF36823.1

              Locus 3377

              JL943803

              744

              3.78

              Peptidoglycan recognition protein S6

              Bombyx mori

              3.00E-58

              61

              NP_001036858.1

              Locus 5206

              JL943842

              712

              3.67

              Hemolin

              Plutella xylostella

              7.00E-131

              97

              ACN69054.1

              Locus 11896

              JL943747

              1453

              3.28

              Prophenoloxidase-activating proteinase 3

              Plutella xylostella

              0

              93

              BAF36824.1

              Locus 4541

              JL943828

              843

              3

              Trypsin-like serine proteinase 1

              Plutella xylostella

              2.00E-37

              42

              ADK66277.1

              Locus 3869

              JL943816

              1604

              2.91

              Bifunctional protein folD

              Culex quinquefasciatus

              2.00E-101

              63

              XP_001846734.1

              Locus 30146

              JL943795

              236

              2.91

              Nucleotide excision repair protein

              Bombyx mori

              1.00E-28

              71

              NP_001177140.1

              Locus 14548

              JL943754

              580

              2.87

              Beta-1,3-glucan recognition protein 2

              Bombyx mori

              5.00E-40

              49

              NP_001037450.1

              Locus 15717

              JL943757

              333

              2.73

              Prophenoloxidase activating factor 3

              Bombyx mori

              3.00E-19

              57

              AAL31707.1

              Locus 13921

              JL943752

              602

              2.68

              Serine protease 33

              Mamestra configurata

              2.00E-36

              50

              ACR15983.2

              Locus 12724

              JL943748

              1288

              2.51

              Serine protease inhibitor 7

              Bombyx mori

              5.00E-94

              46

              NP_001139701.1

              Locus 43519

              JL943823

              256

              2.5

              Ceramidase

              Aedes aegypti

              2.00E-21

              62

              XP_001658093.1

              Locus 48995

              JL943836

              207

              2.5

              Toll receptor

              Tribolium castaneum

              2.00E-19

              59

              XP_971999.1

              Locus 1006

              JL943743

              1154

              2.41

              Lipocalin

              Bombus ignitus

              1.00E-89

              64

              ADA82597.1

              Locus 6114

              JL943851

              1293

              2.38

              Lysosomal acid lipase

              Tribolium castaneum

              1.00E-65

              39

              XP_972957.2

              Locus 28994

              JL943788

              911

              2.33

              Pattern recognition serine proteinase

              Manduca sexta

              3.00E-87

              51

              AAR29602.1

              Locus 20345

              JL943774

              322

              2.26

              Trypsin T6

              Heliothis virescens

              9 E-16

              54

              ABR88249.1

              Locus 17873

              JL943765

              412

              2.25

              NADP+

              Danio rerio

              1.00E-56

              76

              NP_998058.1

              Locus 7994

              JL943864

              1305

              2.24

              Serine protease inhibitor (pxSerpin 3)

              Plutella xylostella

              2.00E-99

              50

              BAF36821.1

              Locus 2093

              JL943776

              2890

              2.21

              Apolipophorins

              Manduca sexta

              0

              67

              Q25490.1

              Locus 20404

              JL943775

              251

              2.19

              Serine protease inhibitor (Serpin 13)

              Bombyx mori

              4.00E-14

              49

              NP_001139705.1

              Locus 41604

              JL943819

              282

              2.16

              Broad-Complex isoform Z2

              Bombyx mori

              1.00E-30

              98

              BAD24051.1

              Locus 35475

              JL943806

              363

              2.15

              Heat Shock Protein (HSP70)

              Acyrthosiphon pisum

              3.00E-12

              44

              XP_001950064.1

              Locus 29819

              JL943791

              461

              2.14

              Beta-1,3-glucan recognition protein 2a

              Helicoverpa armigera

              1.00E-53

              64

              ACI32826.1

              Locus 4518

              JL943827

              303

              2.13

              Serine protease 1

              Lonomia obliqua

              4.00E-22

              51

              AAV91432.2

              Locus 39662

              JL943817

              299

              2.12

              1-phosphatidylinositol-4,5-bisphosphate

              Bombyx mori

              6.00E-45

              86

              NP_001165393.1

              Locus 9824

              JL943870

              627

              2.11

              Peripheral-type benzodiazepine receptor

              Bombyx mori

              6.00E-34

              57

              NP_001040343.1

              Locus 19848

              JL943771

              1136

              2.1

              Peroxidasin

              Tribolium castaneum

              1.00E-134

              62

              XP_968570.1

              Locus 21658

              JL943781

              437

              2.04

              Hemolymph proteinase 8

              Manduca sexta

              4 E-49

              71

              AAV91006.1

              Locus 620

              JL943852

              1032

              -2

              Heat Shock Protein (HSP90)

              Plutella xylostella

              8.00E-140

              100

              BAE48742.1

              Locus 18755

              JL943766

              351

              -2.06

              Vasorin

              Culex quinquefasciatus

              1.00E-54

              85

              XP_001870087.1

              Locus 15108

              JL943755

              615

              -2.07

              Arylalkylamine N-acetyltransferase

              Antheraea pernyi

              1.00E-29

              38

              ABD17803.1

              Locus 19225

              JL943768

              253

              -2.12

              Flap endonuclease

              Carukia barnesi

              6.00E-25

              83

              ACY74444.1

              Locus 29249

              JL943789

              213

              -2.12

              Putative lysozyme

              Bombyx mori

              4.00E-22

              67

              ADA67927.1

              Locus 7618

              JL943861

              3056

              -2.14

              Fascin

              Tribolium castaneum

              0

              79

              XP_972494.1

              Locus 13139

              JL943751

              701

              -2.4

              Hemocyte protease-1

              Bombyx mori

              6 E-75

              60

              BAG70409.1

              Locus 11481

              JL943873

              364

              -3.25

              Trypsin

              Helicoverpa armigera

              5 E-16

              43

              ACB54939.1

              Locus 24418

              JL943783

              211

              -3.5

              Black (DOPA-deC-like)

              Papilio xuthus

              1.00E-29

              87

              BAI87832.1

              Locus 536

              JL943843

              527

              -3.65

              Catalase

              Takifugu obscurus

              1.00E-17

              35

              ABV24056.1

              Locus 3343

              JL943800

              289

              -3.78

              Immune-related Hdd1

              Hyphantria cunea

              3.00E-11

              39

              AAD09279.1

              Locus 13049

              JL943750

              452

              -5.81

              Putative defense protein Hdd11

              Hyphantria cunea

              8.00E-55

              67

              O96382.1

              Fold changes were generated by comparing average read depths for each contig.

              Table 3

              Developmental- and non-immune metabolism-related transcripts of P. xylostella, which were differentially expressed after D. semiclausum parasitization

              Sequence ID

              TSA Accession No.

              Nt. Length

              Fold

              change

              Protein

              Species

              E value

              Nt. ID

              (%)

              Accession no.

              Locus 29717

              JL943790

              504

              27.86

              Endonuclease-reverse transcriptase

              Bombyx mori

              1 E-46

              67

              ADI61832.1

              Locus 3567

              JL943808

              277

              20.01

              Glucose dehydrogenase precursor

              Pediculus humanus corporis

              1 E-15

              52

              XP_002429706.1

              Locus 784

              JL943863

              1943

              12.5

              Methionine-rich storage protein 1

              Plutella xylostella

              0

              100

              BAF45385.1

              Locus 50007

              JL943839

              234

              8.31

              Putative RecQ Helicase

              Heliconius melpomene

              1 E-11

              63

              CBH09254.1

              Locus 3712

              JL943813

              1744

              7.8

              Methionine-rich storage protein 2

              Plutella xylostella

              0

              100

              BAF45386.1

              Locus 1098

              JL943745

              724

              4.4

              Arylphorin-like hexamerin-2

              Plutella xylostella

              1 E-141

              100

              BAF32562.1

              Locus 9922

              JL943871

              1790

              4.31

              44 kDa zymogen (serine protease)

              Tenebrio molitor

              9 E-61

              35

              BAG14262.1

              Locus 2239

              JL943782

              2186

              3.46

              Methionine-rich storage protein

              Spodoptera exigua

              0

              59

              ABX55887.1

              Locus 352

              JL943805

              1023

              3.43

              Arylphorin-like hexamerin-1

              Plutella xylostella

              2 E-172

              89

              BAF32561.1

              Locus 5917

              JL943849

              2338

              3.4

              Phenylalanine hydroxylase

              Papilio xuthus

              0

              88

              BAE66652.1

              Locus 42829

              JL943822

              273

              3.33

              Syntaxin

              Culex quinquefasciatus

              3 E-21

              60

              XP_001865470.1

              Locus 1575

              JL943758

              1738

              3.12

              Phosphoribosylaminoimidazole carboxylase

              Bombyx mori

              0

              90

              NP_001040376.1

              Locus 33387

              JL943799

              375

              3.11

              Insulin receptor

              Bombyx mori

              3 E-34

              59

              NP_001037011.1

              Locus 47369

              JL943832

              347

              3

              Leucine-rich transmembrane protein

              Pediculus humanus corporis

              1 E-24

              60

              XP_002422869.1

              Locus 7126

              JL943859

              1075

              2.98

              Sugar transporter 4

              Bombyx mori

              2 E-104

              64

              NP_001165395.1

              Locus 35539

              JL943807

              592

              2.55

              Torso-like protein

              Tribolium castaneum

              2 E-31

              38

              NP_001107843.1

              Locus 36082

              JL943809

              735

              2.44

              Reverse transcriptase

              Aedes aegypti

              3 E-36

              39

              AAZ15237.1

              Locus 199

              JL943773

              1588

              2.4

              S-adenosyl-L-homocysteine hydrolase

              Plutella xylostella

              0

              100

              BAF36817.1

              Locus 5556

              JL943845

              1186

              2.33

              Yellow-fa

              Bombyx mori

              4 E-141

              69

              NP_001037424.1

              Locus 4588

              JL943829

              205

              2.24

              Endoprotease FURIN

              Spodoptera frugiperda

              4 E-28

              87

              CAA93116.1

              Locus 4894

              JL943835

              861

              2.24

              Lipase

              Helicoverpa armigera

              3 E-50

              54

              ACB54943.1

              Locus 4689

              JL943830

              2429

              2.21

              Cathepsin L precursor

              Tribolium castaneum

              0

              62

              NP_001164088.1

              Locus 3008

              JL943793

              950

              2.21

              Hemolymph proteinase 5

              Manduca sexta

              2 E-101

              62

              AAV91003.1

              Locus 20931

              JL943777

              661

              2.17

              Gamma-glutamyl transferase

              Bombyx mori

              1 E-62

              56

              NP_001165385.1

              Locus 21405

              JL943780

              705

              2.14

              Juvenile hormone binding protein

              Manduca sexta

              2 E-27

              35

              AAB25736.2

              Locus 3797

              JL943815

              1653

              2.13

              Imaginal disk growth factor

              Plutella xylostella

              0

              100

              BAF36822.1

              Locus 5088

              JL943841

              1476

              2.12

              Cathepsin B-like cysteine proteinase

              Spodoptera exigua

              1 E-146

              81

              ABK90823.1

              Locus 1887

              JL943767

              1872

              2.1

              Cathepsin D isoform 1

              Tribolium castaneum

              7 E-165

              73

              XP_966517.1

              Locus 47019

              JL943831

              207

              2.09

              DNA-binding protein Ewg putative

              Pediculus humanus corporis

              3 E-22

              93

              XP_002430412.1

              Locus 16109

              JL943872

              515

              2.08

              Nesprin-1

              Pediculus humanus corporis

              3 E-26

              36

              XP_002427810.1

              Locus 41758

              JL943820

              222

              2.06

              Neuroglian

              Mythimna separata

              4 E-29

              78

              BAI49425.1

              Locus 6830

              JL943856

              427

              2.04

              Cytochrome P450

              Plutella xylostella

              2 E-67

              96

              ABW34440.1

              Locus 17556

              JL943762

              1272

              2.03

              Arginase

              Bombyx mori

              9 E-144

              72

              BAH19308.1

              Locus 25700

              JL943784

              286

              -2.03

              Cytochrome P450 monooxygenase

              Helicoverpa zea

              7 E-16

              74

              AAM54723.1

              Locus 7042

              JL943858

              809

              -2.05

              Tyrosine transporter

              Aedes aegypti

              2 E-18

              76

              XP_001658764.1

              Locus 4738

              JL943833

              1359

              -2.07

              Collagen

              Bombyx mori

              2 E-23

              49

              CAA83002.1

              Locus 3369

              JL943802

              471

              -2.09

              Trypsin alkaline B

              Manduca sexta

              7 E-37

              71

              P35046.1

              Locus 7004

              JL943857

              696

              -2.13

              Cuticular protein glycine-rich 20

              Bombyx mori

              3 E-15

              52

              NP_001166784.1

              Locus 17043

              JL943761

              237

              -2.13

              Voltage & ligand gated potassium channel

              Culex quinquefasciatus

              2 E-15

              71

              XP_001853758.1

              Locus 16717

              JL943760

              1585

              -2.14

              Multidrug resistance protein 2

              Culex quinquefasciatus

              1 E-93

              41

              XP_001866984.1

              Locus 5571

              JL943846

              389

              -2.19

              Sugar transporter

              Aedes aegypti

              1 E-11

              48

              XP_001652873.1

              Locus 13022

              JL943749

              216

              -2.36

              Ecdysis-triggering hormone

              Manduca sexta

              2 E-15

              65

              AAD45613.1

              Locus 6441

              JL943853

              661

              -2.43

              Retinol dehydratase

              Spodoptera frugiperda

              1 E-53

              58

              AAC47136.1

              Locus 15939

              JL943759

              279

              -2.46

              Cyclin-dependent kinase 2-like

              Saccoglossus kowalevskii

              3 E-23

              68

              XP_002740677.1

              Locus 487

              JL943834

              1615

              -2.56

              Glucosinolate sulphatase

              Plutella xylostella

              0

              64

              CAD33828.1

              Locus 6512

              JL943854

              484

              -2.57

              Phosphohistidine phosphatase

              Bombyx mori

              3 E-45

              62

              NP_001040265.1

              Locus 7636

              JL943862

              454

              -2.7

              Reverse transcriptase

              Ostrinia nubilalis

              3 E-39

              48

              ABO45231.1

              Locus 33235

              JL943798

              220

              -2.74

              Juvenile hormone epoxide hydrolase

              Bombyx mori

              5 E-18

              64

              NP_001159617.1

              Locus 36575

              JL943812

              209

              -2.83

              Lipase

              Bombyx mori

              8 E-12

              52

              ADA67928.1

              Locus 5577

              JL943847

              1813

              -2.9

              Chitinase

              Plutella xylostella

              0

              100

              ACU42267.1

              Locus 4357

              JL943824

              1009

              -3.21

              Cuticular protein RR-1 motif 10

              Bombyx mori

              1 E-56

              68

              NP_001166738.1

              Locus 21088

              JL943778

              247

              -3.25

              Carboxypeptidase M

              Aedes aegypti

              1 E-37

              83

              XP_001661307.1

              Locus 2898

              JL943787

              485

              -4.27

              Acheron

              Manduca sexta

              1 E-68

              88

              AF443827_1

              Locus 5445

              JL943844

              1708

              -8.82

              Urbain

              Bombyx mori

              4 E-24

              37

              NP_001139414.1

              Locus 21299

              JL943779

              290

              -15.5

              37-kDa serine protease

              Bombyx mori

              6 E-41

              81

              NP_001128675.1

              Locus 17795

              JL943764

              363

              -24.01

              Cuticle protein

              Aedes aegypti

              3 E-29

              67.78

              XP_001659461.1

              Locus 8908

              JL943867

              625

              -26.71

              Ecdysteroid regulated protein

              Manduca sexta

              1 E-12

              71

              AAA29312.1

              Fold changes were generated by comparing average read depths for each contig.

              In addition to post-transcriptional effects of PDV infection, there may also be variations in different host-parasitoid interactions with regard to host transcripts affected by parasitization/PDV infection. In a very recent study, it was shown that in Spodoptera frugiperda larvae injected with Hyposoter didymator ichnovirus (HdIV) or Microplitis demolitor bracovirus (MdBV), the differentially expressed transcripts in hemocytes and fat body largely differed depending on the PDV injected suggesting that the tissues responded differently to the different viruses [36]. However, there were a number of host genes that responded similarly when infected with HdIV or MdBV. Based on this study, HdIV affected transcript levels in both hemocytes and fat body, whereas MdBV mostly affected gene expression in the fat body.

              In our study, we found that antimicrobial peptides such as gloverin, moricin, lysozyme II and cecropin were up-regulated after parasitoid attack, compared to transcription levels in control unparasitized larvae. Among these, lysozyme II and gloverin had the highest transcription levels in parasitized P. xylostella larvae relative to other antimicrobial peptides (Figure 5; Table 2). Transcription levels for cecropin 1 and pxCecropin E increased by more than 3-fold after D. semiclausum attack (Table 2). It has been reported that immune-related genes were also up-regulated in P. xylostella larvae in response to microbial challenge (e.g. pxCecropin 5.94 fold, hemolin 3.18 fold and cecropin E 4.99 fold) [37]. Barandoc et al. measured pxCecropin expression levels by quantitative RT-PCR (qRT-PCR) in parasitized P. xylostella larvae and showed that pxCecropin was suppressed by C. plutellae BV [12]. Some antimicrobial peptides, such as moricin and gloverin, were previously found not to be induced by bacterial challenge of lepidopteran larvae, while lysozyme and cecropins are well-known inducible antimicrobial peptides [12, 38]. Recently, it has been reported that gloverin expression was changed after bacterial infection in P. xylostella [39]. Our data shows that immune responses after parasitoid attack appear to be different from microbial challenge responses, because gloverin and moricin-like peptide were up-regulated about 7 and 11 fold, respectively, after D. semiclausum attack.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig5_HTML.jpg
              Figure 5

              Relative gene expression values based on average read depth for selected antimicrobial peptide classes in non-parasitized (control) and parasitizedP. xylostellalarvae.

              Other related genes which were up-regulated in parasitized larvae included a serine protease and serine protease inhibitor (pxSerpin 3) (Table 2). Serine proteases are major immune regulatory proteins, which are found in a wide range of species from insects to mammals. In contrast, a proteomics analysis showed that pxSerpin 2 was suppressed in P. xylostella larval plasma during parasitism by C. plutellae [7]. Beck et al. demonstrated that the ovarian calyx fluid of the ichneumonid endoparasitoid Venturia canescens has the potential to suppress the host immune system due to a putative serpin activity [40]. Here, we found that serine protease inhibitors (JL943746, JL943748, JL943775, JL943864) were over-expressed two- to seven-fold, after parasitism in P. xylostella. Thus, the induction of serine protease inhibitors upon immune challenge in parasitized larvae could be part of an endoparasitoid immune suppressive strategy. Many insect protease inhibitors are known to inactivate enzymes isolated from entomopathogenic fungi, and their involvement in insect-pathogen interactions has been widely postulated [41]. Aguilar et al. reported that five serine proteases involved in a single metabolic cascade were up-regulated in Anopheles gambiae upon microbial challenge, but they suggested a role for the proteases in protecting the mosquito from detrimental effects of an uncontrolled spread of immune reaction [42].

              Serine proteases also play a significant role in the activation of the prophenoloxidase (proPO) cascade. In insects, proPO is activated upon injury or invasion, which results in localized melanization of the wound area and/or melanotic capsules capturing invading microorganisms and parasites [43]. In the current study, proPO activating protease (PAP) transcription was up-regulated in parasitized larvae of P. xylostella. In a genome-wide microarray study of D. melanogaster, several genes encoding enzymes of the melanization cascade were found to be up-regulated by L. boulardi parasitization [44], consistent with this study. Asgari et al. (2003) reported that venom protein (Vn50) from Cotesia rubecula is homologous to serine protease homologs [43]. It is likely that the injection of this protein (or putative homologs thereof) by parasitoid wasps into the host body, may interfere with the proteolytic cascade that leads to the activation of proPO. cDNA microarray analysis of S. frugiperda hemocytes and fat body 24 hours after injection of HdIV revealed differential expression of several host genes [45]. Among these, eight immune-related genes showed differential expression in hemocytes with proPO-1 and proPO-2 showing up-regulation, while PAP transcript levels declined. Other immune-related genes that were differentially expressed in the hemocytes were galectin, which showed up-regulation, whereas scavenger R, immulectin-2, lysozyme and calreticulin showed down-regulation [45]. A recent study confirmed up-regulation of proPOs in S. frugiperda larvae injected with HdIV; however, in the same host injected with MdBV, proPO transcript levels declined [36] which suggested differential responses of the host to different PDVs.

              Transcripts of most proteins, which are involved in the Toll pathway such as Relish, Dorsal, Pelle, Cactus and Toll receptor, were found in our deep sequencing analysis, but only transcription levels of proteins that showed similarity to the Toll receptor were up-regulated (2.5 fold; Table 2). In D. melanogaster larvae parasitized by Asobara tabida or L. boulardi, components of the Toll/Imd (Immune deficiency) pathways were up-regulated, and antimicrobial peptide expression was increased [44, 46]. In addition, in Drosophila, Toll and Imd pathways are required for activation and stimulation of NF-κBs signal transduction and also responsible for innate immune response in parasitized Drosophila [47]. NF-κB proteins are a family of proteins in eukaryotes that are involved in the control of a large number of cellular and organismal processes, such as immune responses, developmental processes, cellular growth, and apoptosis. Furthermore, NF-κB signalling is important in immune inducibility of pathogen-associated-molecular-patterns, and it is widely assumed that it plays a conserved role in invertebrate immune regulation [48, 49].

              It has been suggested that PDV-expressed vankyrin proteins may interfere with NF-κB-mediated signalling during immune response and development in parasitized larvae [15, 50]. Fath-Goodin et al. (2009) reported that CsIV vankyrin genes also encode proteins with sequence homology to the inhibitory domains of NF-κB transcription factor inhibitors [13].

              The results of our transcriptome analysis also indicated that genes known to be involved in insecticide resistance/detoxification are up-regulated following parasitism (Table 2). In agreement, it has previously been reported that cytochrome P450 (CYP) and glutathione-S-transferase (GST) activities increased in parasitized P. xylostella larvae [29]. Takeda et al. (2006) suggested that parasitoid larvae contributed to CYP activity enhancement since the parasitoid hatched two days after oviposition and the CYP activity was significantly increased three days after parasitization [29].

              Our analyses also detected a considerable number of other immune-related genes whose transcription levels altered after parasitization by D. semiclausum. However, only a small group affected by parasitoid attack were found to be altered enough to be statistically biologically relevant (i.e. showing greater than two-fold change).

              Transcription levels of host development-related genes

              Generally, larval endoparasitoids lay their eggs into the host hemocoel, and their progenies develop by consuming host hemolymph and tissues. As a consequence, the parasitoid's larval growth also fully depends on the host's development [1, 51, 52]. In P. xylostella larvae parasitized by D. semiclausum, development is arrested at the prepupal stage [53]. Developmental arrest before pupation is one of the most common effects of PDVs and/or other maternal factors injected by many endoparasitoids into their hosts [14, 5456]. In these interactions, the parasitoid larvae and PDVs are responsible for increasing the juvenile hormone (JH) titre in host larvae and preventing ecdysteroid levels from rising sufficiently to allow host pupation [5761].

              Based on our transcriptome data, parasitism by D. semiclausum leads to down-regulation of genes associated with ecdysteroid activities; for example, the transcription level of ecdysteroid regulated protein was down-regulated more than 26 times in parasitized larvae (Table 3). Considering that ecdysteroids are required to trigger expression of ecdysteroid regulated protein [62], and that parasitism in general leads to reductions in ecdysteroid titres [54, 6366], down-regulation of ecdysteroid regulated protein is expected.

              Juvenile hormone binding protein (JHBP) may protect JH from non-specific degradation and adsorption by preventing exposure of JH to epoxide hydration by JH epoxide hydrolase (JHEH), which generates the hormonally inactive JH diol [67]. In agreement, JHBP transcript levels were up-regulated more than 2 times and interestingly, transcription levels of JHEH were down-regulated more than 2 times (Table 3). In all the reported host-parasitoid systems, it seems that JH is maintained at high levels during parasitoid larval development [58, 66, 68]. Therefore, an increase in JHBP, and corresponding decrease in JHEH transcription levels, after parasitism, seems logical to maintain higher JH levels during parasitism.

              As indicated above, previous studies have shown that PDVs may inhibit translation of specific storage or growth-associated proteins despite up-regulation (or steady-state) of transcript levels of the encoding genes, following parasitism or injection of PDVs [6971]. In this study, we found that arylphorin and methionine-rich storage proteins were over-transcribed in parasitized larvae (Table 3); however, their translation may be affected similarly to mechanisms in the reports discussed above, which needs to be experimentally shown.

              Quantitative RT-PCR validation of transcriptome analysis

              To validate our deep sequencing data, nine differentially regulated P. xylostella genes were selected from immune- and development-related genes (Tables 2 and 3) for qRT-PCR analysis, using the same RNA samples as for deep sequencing. These were cuticle protein, ecdysteroid regulated protein, JH epoxide hydrolase, insulin receptor, methionine-rich storage protein 1, gloverin, hemolin, pxSerpin 3 and Toll receptor. The qRT-PCR results confirmed the data obtained from deep sequencing analysis showing similar trends in up- or down-regulation of host genes (Figure 6). For example, based on deep sequencing analysis, gloverin, Toll receptor, and pxSerpin 3 were up-regulated 6.9, 2.5 and 2.2 fold, respectively (Table 2), and showed 5.5, 2.6 and 2.7 fold changes, respectively in qRT-PCR analyses (Figure 6).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig6_HTML.jpg
              Figure 6

              qRT-PCR analysis of nine selected genes fromP. xylostellawhich showed differential expression after parasitization based on deep sequencing analysis. Error bars indicate standard deviations of averages from three replicates. Fold changes are shown in brackets.

              Since the samples analysed above were pools of RNA from various time points, and it is known that expression of host genes may vary at different periods after parasitization, we further isolated RNA from 2nd instar P. xylostella larvae at 16, 24 and 48 hrs after parasitization, and analyzed three associated genes by qRT-PCR. These were hemolin, gloverin and the ecdysteroid regulated protein. For each time point, a mixture of 10 larvae was used. As expected, there were fluctuations in the expression levels of these genes following parasitization (Figure 7). For example, gloverin expression was highly induced at 16 hrs after parasitization, subsequently declined at 24 hrs, and then further reduced to the same level as unparasitized larvae at 48 hrs post-parasitization (Figure 7). Hemolin was only up-regulated at 48 hrs after parasitization. Expression of ecdysteroid regulated protein was initially down-regulated at 16 hrs after parasitization, but increased at 24 hrs before subsequently declining by 48 hrs post-parasitization (Figure 7).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig7_HTML.jpg
              Figure 7

              qRT-PCR analysis of expression levels of three selected genes in 2 nd instarP. xylostellalarvae at three time points after parasitization withD. semiclausum, which showed differential expression after parasitization based on deep sequencing analysis. Error bars indicate standard deviations of averages from three replicates.

              Diadegma semiclausum ichnovirus genes

              PDV genes are divided into three groups based on whether they are expressed in the carrier wasp (class I), the infected host larvae (class II) or both (class III) [72]. Among these genes, class II genes have received the greatest attention and have been studied more than other groups [73]. The genomes of some IVs, such as those found in the wasps C. sonorensis, C. chlorideae, Hyposoter fugitivus, H. didymator, and Tranosema rostrale, have been sequenced and resultant data are available on public databases [21, 72, 74]. Six conserved gene families: repeat element, cysteine motif, viral innexin, viral ankyrin, N-family and the polar-residue-rich proteins (a newly defined putative family), have been reported in most IV genomes [72].

              Here, expression of a range of DsIV genes were detected in parasitized larvae. In our analysis, 19 unique sequences were identified from five PDV gene families including vankyrin, viral innexin, repeat elements, cysteine-rich motif, and polar residue rich protein families (Table 4). In addition, five other putative virus protein sequences with unknown function were identified in parasitized larvae, and showed more than 50% similarity with some parts of IV reference genomes, but no putative specific protein domain homologies were detected in their sequences (Table 4). The online open reading frame finder tool at the NCBI website http://​www.​ncbi.​nlm.​nih.​gov/​projects/​gorf was used for prediction of full-length sequences in DsIV genes and only four of these genes are reported here as full-length (Table 4).
              Table 4

              D. semiclausum IV transcripts which were detected in parasitized P. xylostella larvae

              Protein

              Length (nt)

              Accession

              Number

              Similarity (Protein/virus/Accession No.)

              length (aa)

              E value

              Nt. ID %

              Conserved Domains

              Vankyrin 1

              671*

              JI257593

              vankyrin-b17 (HfIV) AAS90270.1

              170

              3.56E-52

              61

              Yes

              Vankyrin 2

              519*

              JI257594

              vankyrin-d8.3 (HfIV) BAF45734.1

              159

              6 E-64

              80

              Yes

              Vankyrin 3

              257

              JI257595

              hypothetical protein 2 (HdIV) AAR99845.1

              126

              3.58E-23

              61

              Yes

              Vankyrin 4

              361

              JI257596

              vankyrin-b1(HfIV) AAX24120.1

              167

              2 E-39

              76

              Yes

              Viral Innexin 1

              1370

              JI257597

              viral innexin-b5.1 (HfIV) BAF45654.1

              354

              5 E-95

              60

              Yes

              Viral Innexin 2

              576

              JI257598

              viral innexin-c16 (HfIV) AAS58041.1

              371

              2 E-81

              75

              Yes

              Repeat element 1

              833

              JI257599

              repeat element protein 7 (HdIV) AAR89179.1

              224

              4.32E-73

              67

              Yes

              Repeat element 2

              697

              JI257600

              repeat element protein-d10.1 (HfIV) BAF45740.1

              244

              1.53E-72

              64

              Yes

              Repeat element 3

              947

              JI257601

              repeat element protein-c18.1 (HfIV) BAF45697.1

              244

              9.52E-94

              71

              Yes

              Repeat element 4

              859*

              JI257602

              repeat element protein-e2.3 (HfIV) BAF45758.1

              209

              3.40E-76

              67

              Yes

              Repeat element 5

              656

              JI257603

              repeat element protein (HdIV) AAO16959.1

              225

              1.21E-73

              59

              Yes

              Repeat element 6

              500

              JI257604

              repeat element protein-d11.2 (HfIV) BAF45744.1

              244

              1E-68

              72

              Yes

              Cysteine rich motif

              630

              JI257605

              cysteine motif gene-d9.1(HfIV) BAF45736.1

              311

              2.99E-55

              64

              Yes

              Polar residue rich protein

              671

              JI257606

              polar residue rich protein-b13.2 (HfIV) BAF45664.1

              159

              1.24E-12

              42

              No

              Unknown Protein

              256

              JI257607

              c7-2.1 (TrIV) BAF45599.1

              119

              2.90E-17

              68

              Yes

              Unknown Protein

              2143*

              JI257608

              c12.1 (HfIV) AAS68099.1

              432

              2E-107

              50

              No

              Unknown Protein

              418

              JI257609

              P12 (HdIV) AAS83461.1

              106

              6E-18

              58

              No

              Unknown Protein

              584

              JI257610

              c10.1 (HfIV) AAS90272.1

              385

              9.15E-50

              51

              No

              Unknown Protein

              208

              JI257611

              b5.3 (HfIV) BAF45655.1

              115

              9.57E-13

              69

              No

              * Full length

              Hf: Hyposoter fugitives

              Hd: Hyposoter didymator

              Tr: Tranosema rostrale

              Presence of multiple sequences for each PDV gene family is common in other IVs and we identified four sequences with vankyrin domain and two viral innexin transcripts, which showed high similarity with H. fugitivus IV innexins (Table 4). Repeat element domains were also identified in six sequences with translated lengths of between 209-244 amino acids. Transcription levels (or gene expression values) were found to be different among different members within each gene family. Vankyrin 1 and repeat element 1 had the highest transcription levels in their respective families, and also relative to other DsIV genes (Figure 8).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig8_HTML.jpg
              Figure 8

              Relative gene expression values based on average read depth for all detectedD. semiclausumichnovirus genes. RPKM normalized values were used to generate the data.

              Hierarchical cluster analysis of vankyrin gene sequences of all reported PDV vankyrins (protein sequences at NCBI) using a neighbour-joining algorithm, classified BVs and IVs into two major groups (Figure 9). All four DsIV vankyrins that were identified in this study have high similarities with other IVs and were distributed into four separate clusters indicating that these members of the ankyrin family possibly originated from different segments of the DsIV genome or at least there has been some ancient gene duplication and/or differential selection even if encoded on the same circle. In addition, the separate clustering of vankyrin genes suggests that they are not closely related, and therefore did not likely undergo recent duplication to form similar paralogs.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-446/MediaObjects/12864_2011_3598_Fig9_HTML.jpg
              Figure 9

              Hierarchical Cluster analysis of vankyrin gene sequences of all reported PDV vankyrins, using neighbour-joining algorithm. The accession code for each vankyrin is provided in brackets. The yellow and purple backgrounds show the Ichnovirus and provisional group of banchine virus genes, respectively, and the rest are Bracovirus vankyrins.

              Conclusion

              Overall, this study provides the first comprehensive analysis of the impact of a parasitoid wasp on its host at the transcriptomic level, using RNA deep sequencing technique. The results showed differential expression of a large number of P. xylostella genes, including immune-related genes, upon parasitization by D. semiclausum. In addition, although presence of DsIV particles has been reported in parasitized larvae, our results provide evidence for expression of 19 DsIV genes expressed in the host, which have not been previously reported. Analysis of these sequences indicated the presence of conserved genes that belong to major IV class II genes. The transcriptome profiling data sets obtained in this study provide a basis for future research in this under-explored host-parasitoid interaction. In addition, the identified immune-, development- and detoxification-related genes may be targets for P. xylostella control and allow manipulation of host-parasite interactions.

              Methods

              Insects and parasitization

              P. xylostella and the parasitoid wasp (D. semiclausum) were raised on cabbage plants and host larvae, respectively, at 25°C. Twenty five 3rd and 4th instar P. xylostella larvae each were exposed to wasps until parasitization was observed. Individual larvae that had been attacked by the parasitoid were collected and fed on fresh cabbage leaves. Larval samples were taken at four different time intervals after parasitization (6, 12, 24 and 48 hrs post-parasitization) and the samples were kept at -80°C until RNA isolation. The same numbers of mixed larval instars (3rd and 4th) of non-parasitized larvae were collected as the control treatment. It is worth mentioning that P. xylostella larvae parasitized at 3rd instar continue to develop to 4th instar.

              Sample preparation, deep sequencing and de novo transcriptome assembly

              Total RNA was extracted from all larval samples using Tri-Reagent™ (Molecular Research Center Inc.). RNA extracted from larvae at various time points post-parasitization were pooled and therefore temporal expression data was lost. This was also performed for non-parasitized samples. The pooled RNA sample concentrations were measured using a spectrophotometer and integrity was ensured through analysis on a 1% (w/v) agarose gel. The samples with total concentration of 3.9 and 4.1 μg/μl for parasitized and non-parasitized larvae, respectively, were used for cDNA library production.

              The cDNA library was prepared by using 5 μg of starting material for the Illumina mRNA Sequencing Sample preparation procedure (kit RS-930-1001). This involved purification and fragmentation of mRNA, first strand cDNA synthesis, second strand cDNA synthesis, end repair, addition of "A" bases to 3' ends, ligation of adapters, purification of ligated products, and PCR amplification to enrich cDNA templates. The library was validated, quantified and subjected to deep sequencing using a Genome Analyzer IIx Next generation sequencer on a 66 cycle single-end sequencing run, following the supplier's instructions (Geneworks, Adelaide). The GAII analyzer data were output as sequence tags of 65 bases. Sequence.txt files (in FASTQ format) were generated using Illumina Pipeline version 1.5.1. The CLC Genome Workbench (version 4.0.2) [75] algorithm for de novo sequence assembly was used to assemble contigs from a pooling of all the short-read data, using default parameters (similarity = 0.8, length fraction = 0.5, insertion/deletion cost = 3, mismatch cost = 3).

              RNA sequence analysis

              The contigs arising from the de novo assembly were then used as a reference set of transcripts for RNAseq analysis. Short-read sequence data from parasitized and non-parasitized larvae were separately mapped against the reference set of assembled transcripts using the CLC Genome Workbench RNAseq function (min. length fraction = 0.9, maximum mismatches = 2). The relative transcript levels were output as RPKM (Reads Per Kilobase of exon model per Million mapped reads) values, which take into account the relative size of the transcripts and only uses the mapped-read datasets (i.e. excludes the non-mapped reads), to determine relative transcript abundance. In this way, the output for each dataset can be directly compared as the number of mapped reads per dataset and transcript size has already been taken into account.

              Reads from parasitized and non-parasitized larvae were cleaned and combined, before de novo transcriptome assembly was carried out using Oases 0.1.18 [32]. The individual sets of reads were then mapped back to the transcripts using BWA 0.5.8a [76]. The average read depth (proportional to expression level) for each transcript was then calculated using SAMtools 0.1.8 [77]. The transcripts that had a greater than two-fold average read depth difference between the parasitized and non-parasitized sets were counted as being statistically biologically relevant and were selected for annotation. We used both CLC and Oases to compare assembly of contigs. In general, Oases produced similar contigs to CLC, although contig lengths produced by Oases were in some instances longer.

              BLAST homology search and annotation

              BLASTX algorithm [78] with an E-value cut off of 10-6 was applied to the National Centre for Biotechnology Information (NCBI) non-redundant protein sequence database, to determine the homology of sequences with known genes. In the absence of P. xylostella and D. semiclausum genome sequences, we discarded annotations that showed similarity to hymenopteran genes and tried to use annotations that showed the highest similarity to lepidopteran genes. Gene ontology and annotation were performed on all assembled contigs greater than 200 bp length by BLAST2GO software http://​www.​blast2go.​org[79]. For gene ontology mapping, Blast2GO (which performs four different mapping strategies) was used, and the program defaults were applied for all annotation steps [79]. BLAST2GO allows the selection of a significance level for the False Discovery Rate (FDR), which was used as a cut-off at the 0.05% probability level. The data from InterPro terms [80], enzyme classification codes (EC), and metabolic pathways (KEGG, Kyoto Encyclopedia of Genes and Genomes) were merged with GO terms for a wide functional range cover in annotation.

              For some of the identified D. semiclausum ichnovirus (DsIV) genes, ORFs were predicted and identified by using ORF finder at NCBI http://​www.​ncbi.​nlm.​nih.​gov/​gorf/​gorf.​html. Predicted ORFs with highest BLASTp E-values in internal comparisons involving other IV genes, were accepted for further analyses.

              Quantitative RT-PCR (qRT-PCR) validation of deep sequencing data

              Quantitative RT-PCR technique was used on the same RNA samples which were used for transcriptome profiling to verify deep sequencing results using three replicates, each obtained from a pool of 10 larvae. In addition, to observe gene expression levels at different time points after parasitization for a selected group of genes, another experiment was performed by parasitizing 2nd instar P. xylostella larvae. For each time point after parasitization, a pool of 10 larvae was used. The RNA samples were extracted from larvae at 16, 24 and 48 hrs after parasitization.

              First strand cDNA was synthesized from 1 μg of RNA using M-MuLV reverse transcriptase (New England BioLabs). The qPCR reaction consisted of 2 μL of diluted cDNA (10 ng), 5 μL of Platinum SYBR Green SuperMix-UDG with ROX (Invitrogen), and 1 μM of each primer (Table 5) in 10 μL total volume. Reactions were performed in triplicates in a Rotor-Gene thermal cycler (QIAGEN) under the following conditions: 50°C for 2 min; 95°C for 2 min; and 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s, followed by melting curve generation (68°C to 95°C). Melting curves for each sample were analyzed to check the specificity of amplification. Gene copy numbers were calculated using the Rotor-Gene software, and an endogenous actin reference gene was used for normalization.
              Table 5

              Primers used for qRT-PCR analyses to validate deep sequencing data

              Gene

              Forward primer

              Reverse Primer

              Storage protein 1

              CAAGACACGCTACGACGC

              GTCGGCATGACGAAGTAC

              Insulin receptor

              GTACCCCTCGATCTCGCG

              CCCACGTCAAGGGAACCC

              JH epoxide hydrolase

              AGGATCTACGCGGAGGGC

              TGGTACACCACTTCGTAC

              Ecdysteroid regulated protein

              AACCCGAAGAGCCGAAGC

              CTCTGTAGTCGCTGCTAC

              Cuticle protein

              CAGGATGACGAGTCTGGC

              GTCTGCCTCGTATTCTAC

              Toll receptor

              CCTCCGGCAACGCCCTAG

              CGCACAGAAATTCAGAGG

              Hemolin

              AGCTCCAGAGACTACGCC

              GTGTTGTAGGAACCATTG

              Gloverin

              AGCTAGCCCGGCATCCGC

              GACGGTAGCCCGCCTTAC

              pxSerpin 3

              GAATAGCTTCTACTACGC

              TGATAGCGAATTCGGTAC

              Actin

              ATGGAGAAGATCTGGCAC

              GGAGCCTCCGTGAGCAGC

              Declarations

              Acknowledgements

              This work was supported by a UQ Research Higher Degree scholarship to Etebari, an ARC Discovery grant (DP110102112) to Asgari and a Horticulture Australia Ltd grant (VG08048) to Glatz and Asgari.

              Authors’ Affiliations

              (1)
              School of Biological Sciences, The University of Queensland
              (2)
              Australian Institute for Bioengineering and Nanotechnology, The University of Queensland
              (3)
              AgriScience Queensland, Department of Employment Economic Development and Innovation, Ecosciences Precinct
              (4)
              South Australian Research and Development Institute (SARDI), Entomology

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              © Etebari et al; licensee BioMed Central Ltd. 2011

              This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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