Functional annotation of Cotesia congregata bracovirus: identification of viral genes expressed in parasitized host immune tissues

Abstract

Bracoviruses (BVs) from the Polydnaviridae family are symbiotic viruses used as biological weapons by parasitoid wasps to manipulate lepidopteran host physiology and induce parasitism success. BV particles are produced by wasp ovaries and injected along with the eggs into the caterpillar host body, where viral gene expression is necessary for wasp development. Recent sequencing of the proviral genome of Cotesia congregata BV (CcBV) identified 222 predicted virulence genes present on 35 proviral segments integrated into the wasp genome. To date, the expressions of only a few selected candidate virulence genes have been studied in the caterpillar host, and we lacked a global vision of viral gene expression. In this study, a large-scale transcriptomic analysis by 454 sequencing of two immune tissues (fat body and hemocytes) of parasitized Manduca sexta caterpillar hosts allowed the detection of expression of 88 CcBV genes expressed 24 h after the onset of parasitism. We linked the expression profiles of these genes to several factors, showing that different regulatory mechanisms control viral gene expression in the host. These factors include the presence of signal peptides in encoded proteins, diversification of promoter regions, and, more surprisingly, gene position on the proviral genome. Indeed, most genes for which expression could be detected are localized in particular proviral regions globally producing higher numbers of circles. Moreover, this polydnavirus (PDV) transcriptomic analysis also reveals that a majority of CcBV genes possess at least one intron and an arthropod transcription start site, consistent with an insect origin of these virulence genes.

Importance: Bracoviruses (BVs) are symbiotic polydnaviruses used by parasitoid wasps to manipulate lepidopteran host physiology, ensuring wasp offspring survival. To date, the expressions of only a few selected candidate BV virulence genes have been studied in caterpillar hosts. We performed a large-scale analysis of BV gene expression in two immune tissues of Manduca sexta caterpillars parasitized by Cotesia congregata wasps. Genes for which expression could be detected corresponded to genes localized in particular regions of the viral genome globally producing higher numbers of circles. Our study thus brings an original global vision of viral gene expression and paves the way to the determination of the regulatory mechanisms enabling the expression of BV genes in targeted organisms, such as major insect pests. In addition, we identify sequence features suggesting that most BV virulence genes were acquired from insect genomes.

FIG 1

Eukaryotic features of CcBV genes. (A) Percentages of genes predicted to contain at least one intron among PDV genomes. Data for Glyptapanteles flavicoxis bracovirus (GfBV), Glyptapanteles indiensis bracovirus (GiBV) (20), Cotesia congregata bracovirus (CcBV) (14), Cotesia vestalis bracovirus (CvBV) (18), Microplitis demolitor bracovirus (MdBV), and Campoletis sonorensis ichnovirus (CsIV) (17) are shown. (B) CcBV gene transcription start site (TSS) predictions. Shown is a pictogram representation of a motif detected by MEME in the 5′ UTR of the 52 full-length mRNAs detected by 454 sequencing. A total of 10 bp of genomic sequence upstream of the mRNA was also included in the analysis. The height of the letters corresponds to their frequencies relative to the single-nucleotide background used when running MEME. (C) GC content among wasp genes, CcBV segment genes, and CcBV nudiviral genes. GC content (percent) was monitored in (i) 53 C. congregata genes present in the vicinity of the proviral form of CcBV or the nudiviral genes, (ii) 227 CcBV genes present in the CcBV integrated proviral form (222 predicted genes and 5 new genes), and (iii) 15 CcBV nudiviral genes. Error bars indicate standard deviations, and the asterisk indicates that the GC content differs significantly (P < 0.05 by pairwise t test).

FIG 2

Expression levels of CcBV genes in fat body of M. sexta larvae 24 h after parasitization by C. congregata. (A) RPKM values deduced from transcriptomic 454 sequencing for each CcBV gene detected in M. sexta fat body 24 h after parasitization. The M. sexta rpl3 gene was used as a reference (in gray). Genes encoding proteins with predicted signal peptides are symbolized by black boxes, whereas genes encoding potential intracellular proteins are indicated by white boxes. (B) Validation of expression levels by qRT-PCR analysis of 11 selected CcBV genes on cDNA used for 454 analysis. Gene expression was measured in triplicate on the same cDNA as that used for 454 library construction. The M. sexta rpl3 gene was used as a reference (in gray). (C) Validation of expression levels by qRT-PCR analysis of 11 selected CcBV genes in new samples. Gene expression was measured in five new independently parasitized individuals in triplicate. The M. sexta rpl3 gene was used as a reference (in gray). Error bars indicate standard deviations, and different letters indicate significant differences in transcript abundance. Gene expression levels were separated into three categories based on the statistical differences between gene expression levels deduced from qRT-PCR analysis on new cDNA samples: high expression levels (red bars) (≥25 RPKM), intermediate expression levels (green bars) (5 ≤ RPKM < 25), and low expression levels (blue bars) (<5 RPKM).

FIG 3

Expression levels of CcBV genes in hemocytes of M. sexta larvae 24 h after parasitization by C. congregata. (A) RPKM values deduced from transcriptomic 454 sequencing for each CcBV gene detected in M. sexta hemocytes 24 h after parasitization. The M. sexta rpl3 gene was used as a reference (in gray). Genes encoding proteins with predicted signal peptides are symbolized by black boxes, whereas genes encoding potential intracellular proteins are indicated by white boxes. (B) Validation of expression levels by qRT-PCR analysis of 11 selected CcBV genes in new samples. Gene expression was measured in triplicate on samples from five independently parasitized individuals. The M. sexta rpl3 gene was used as a reference (in gray). Error bars indicate standard deviations, and different letters indicate significant differences in transcript abundance. Gene expression levels were separated into two categories based on the statistical differences between gene expression levels deduced from qRT-PCR analysis of new cDNA samples: high expression levels (red bars) (≥25 RPKM) and low expression levels (green bars) (<5 RPKM).

FIG 4

RT-PCR expression analysis of genes below the limit of detection by 454 analysis. Genes tested were cystatin-1, showing high expression levels by 454 analysis; bv8-3, showing low expression levels by 454 analysis; and genes below the 454 limit of detection: ben-2, crp4, ptph, DNApolB2, CcBV_4.4, and crp2. Host tissues examined were from five independently parasitized M. sexta larvae. Expression of the host rpl3 gene was used as an internal control.

FIG 5

Localization in CcBV segments of genes expressed in fat body. Each segment is represented by its predicted genes but is not to scale in terms of segment or gene length. Segment names and RU numbers are positioned to the right of each segment. Gene names are indicated below each bar. Expression levels are log normalized to facilitate visualization and are represented by colored bars.

FIG 6

Localization in CcBV segments of genes expressed in hemocytes. Each segment is represented by its predicted genes but is not to scale in terms of segment or gene length. Segment names and RU numbers are positioned to the right of each segment. Gene names are indicated below each bar. Expression levels are log normalized to facilitate visualization and are represented by colored bars.

FIG 7

Relationships between gene localization in the proviral genome of CcBV, circle abundance, and gene expression levels in fat body (A) and hemocytes (B) of M. sexta larvae 24 h after parasitization by C. congregata. CcBV DNA segments are represented by boxes, with the segment numbers indicated below. The 35 segments are organized into nine proviral loci (PL) that correspond to wasp genomic regions containing integrated CcBV segments. Segments are coamplified during particle production in the wasp ovaries in 12 different molecules constituting replication units (RUs) (15). The position of expressed CcBV genes is represented in each segment by either a red, green, or blue bar, which corresponds to the level of expression in host tissues (high, intermediate, or low expression levels, respectively). Only positions of genes detected by 454 analysis are indicated. Genes with identical sequences are indicated by dashed bars. Segments corresponding to high-abundance circles are shown in black, and those corresponding to low-abundance circles are shown in gray.

FIG 8

Relative abundance of CcBV circles in viral particles produced by C. congregata female wasps. Circle abundance in a purified CcBV preparation was monitored by qPCR. Circle abundance is represented as a factor of the less represented circle (C28). Segments corresponding to high-abundance circles are shown in black, and those corresponding to low-abundance circles are shown in gray. Braces indicate circles that belong to the same replication unit (RU).

FIG 9

Phylogenetic analyses, expression profiles in fat body, and transcription factor binding site predictions for the CcBV BV5 multigenic family. (A) Phylogenetic trees of genes from the BV5 gene family, with their respective log-normalized RPKM expression levels represented by colored circles. Bootstrap values of >70 are represented by asterisks. Long branches are interrupted by dashed lines for clarity. (B) Transcription factor binding site predictions for the BV5 gene family using Matinspector from the Genomatix software suite. Analyzed sequences correspond to 1,000 bp upstream of the ATG start codon of each gene. Colored motifs represent motif family predictions. A complete description of motif names is available (http://www.genomatix.de/). Sequences within a black rectangle possess a majority of identical motifs, represented by asterisks. Red lines correspond to regions with predicted CDSs or known expressed mRNAs.