Insect origin, rearing and parasitism

Four lepidopteran species were used in bioassays: Spodoptera frugiperda, Mamestra brassicae, Pseudoplusia includens and Anticarsia gemmatalis. All originated from laboratory colonies (S. frugiperda was derived from the DGIMI laboratory colony, P. includens and A. gemmatalis from the “Interações em Insetos » laboratory colonies and M. brassicae was provided by E. Jacquin-Joly, INRA-France). S. frugiperda and M. brassicae were maintained on a semi-synthetic maize diet, whereas P. includens and A. gemmatalis were maintained on a semi-synthetic bean-based diet, all under the same stable conditions (24±2°C; 75–65% relative humidity; 16 h light: 8 h dark photoperiod). The wasp Hyposoter didymator used in the experiments was derived from the DGIMI laboratory colony, reared on S. frugiperda at 26±2°C with a 16 h light: 8 h dark photoperiod.

For the bioassays, early third-instar caterpillars of similar weight (between 2 and 7 mg, depending on the host species) were individually introduced into a glass vial containing 10 two-day-old female H. didymator wasps. The host larvae were removed immediately after they had been stung, and they were maintained in an incubator at 24±2°C; 75–65% relative humidity; 16 h light: 8 h dark photoperiod for the rest of the experiment. We selected 10 parasitized caterpillars at random from the parasitized pool and dissected them to check for the presence of an egg, to ensure that oviposition had occurred.

Changes in host physiology induced by parasitization

We estimated the efficiency of parasitization and the rate of encapsulation by dissecting a batch of parasitized hosts 12–36 hours post-parasitism (p.p.) and recording the egg status of H. didymator (no egg, free egg or encapsulated eggs) for each of the hosts dissected. Another batch of hosts was dissected 72 hours p.p., for determination of the larval status (no larva, free larva or encapsulated larva) of each of the hosts dissected. The numbers of larvae dissected 12–36 p.p. and 72 h p.p. were 38 and 18, respectively, for S. frugiperda, 38 and 20 for P. includens, 54 and 61 for M. brassicae and 42 and 36 for A. gemmatalis.

We investigated the effect of parasitization on host weight gain, by weighing the parasitized caterpillars individually at 6 hours, 72 hours and 6 days p.p. The numbers of larvae (control/parasitized) used were 35/42 for S. frugiperda, 24/40 for P. includens, 37/35 for M. brassicae and 32/56 for A. gemmatalis.

Preparation and sequencing of HdIV genomic DNA

Genomic DNA was extracted from HdIV as previously described [27] from filter-purified HdIV particles collected from 300 dissected ovaries. The extracted HdIV dsDNA was then sequenced at the Génoscope, with the same shotgun and Sangers sequencing strategies used for the CcBV encapsidated genome, as previously described [12]. The sequences obtained were assembled with PHRED and PHRAP, as previously described [12]. In some cases, segment sequence assembly was further validated by comparing the segment sequence obtained with that of H. didymator genomic clones, when such sequences containing the corresponding proviral sequence were available, using a private BAC library available in our laboratory (Volkoff et al., unpub.).

HdIV segment analysis

We used the Blastn algorithm (http://www.ncbi.nlm.nih.gov/BLAST/) [28] to compare all of the identified HdIV segments, to assess potential sequence similarities between segments. We then checked for sequence similarity to segments of other IVs (TrIV, HfIV and CsIV) by using the Blastn algorithm to search the NCBI public nucleotide database. Synteny between the HdIV and HfIV segments was visualized with the ARTEMIS Comparison Tool (ACT) interface (www.sanger.ac.uk). Protein sequences were aligned with the ClustalW online tool [29].

RNA isolation and sequencing

For the analysis of HdIV transcripts in different lepidopteran species, we extracted total RNA from pools of five caterpillars, with the RNeasy purification kit (Qiagen). RNA was extracted 6 hours p.p. for the four lepidopteran host species, and then 24 hours and 72 hours p.p. for S. frugiperda. RNA samples were treated with the Turbo DNAse-free Kit (Ambion) and we checked that there was no contaminating DNA, by PCR with specific primers binding to the HdIV P30_Hd6 intron and the S. frugiperda ELF-1 (elongation-like factor 1) exon. Total RNA concentration was estimated with a Nanodrop ND-1000 spectrophotometer. Total RNA quality was assessed by electrophoresis in a 1% agarose gel and with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, California). For each host species and/or set of conditions, we carried out two or three independent extractions. Sequencing with Illumina technology (Hi-Seq 2000; 50 bp single reads) was then performed either by the GATC company (www.gatc-biotech.com/fr/) (3 samples, collected 6 hours p.p. for each of S. frugiperda, P. includens, M. brassicae and A. gemmatalis, and 1 sample each obtained at 24 and 72 hours p.p. for S. frugiperda) or by the MGX CNRS public sequencing facility (www.mgx.cnrs.fr/) (2 samples for S. frugiperda, obtained at 24 and 72 hours p.p.). More than 11 million 50 bp-long single reads were obtained for each sample (Table S1).

HdIV genome annotation, read mapping and RPKM counting

For each HdIV genome segment, the open reading frames (ORF) present were predicted with the ARTEMIS interface (www.sanger.ac.uk) [30]. Only ORFs encoding proteins of more than 99 amino acids (a.a.), with an initiator methionine, were initially considered as putative genes. In cases of overlapping ORFs, we considered only the ORF displaying sequence similarity to known PDV genes or with limits consistent with RNAseq mapping (see below) data. For all putative genes, we carried out public database searches for sequence similarities with the NCBI Blastn, Blastx and tBlastx algorithms (http://www.ncbi.nlm.nih.gov/BLAST/) [28]. Searches for specific regulatory motifs in the upstream gene region were performed with the SCOPE and MEME online tools.

For all RNA samples sequenced, the corresponding raw data Tag-sorted FastQ Files containing the adaptor-cleaned single reads (between 47 and 50 bp/read) were used for mapping procedures on the 135 HdIV genes in BOWTIE [31], with classical parameters: a –v 1 –best –sam. Using the ARTEMIS interface, we determined the number of reads that could be mapped for all annotated HdIV genes. For each gene, the RPKM was calculated as defined by [32] using the following formula RPKM = 10⁹C/NL where C is the number of reads that could be mapped to gene exons, N is the total number of mappable reads in the experiment, and L is the sum of the lengths of the exons (in base pairs). We avoided RPKM overestimation for genes containing perfectly repeated sequences longer than 47 bp (i.e. the read length), by obtaining the final RPKM for these genes by dividing the read counts by the number of repeats identified within the gene sequence. We checked that all the reads mapped with HdIV were actually viral genes, by also mapping reads acquired from unparasitized S. frugiperda larval samples. None of the reads for this sample mapped onto HdIV genes, demonstrating the absence of sequence similarity between S. frugiperda and HdIV genes. We estimated a background threshold, by calculating the RPKM corresponding to the intergenic regions of each HdIV segment from the reads acquired for the S. frugiperda 72 h p.p. sample. The RPKM value obtained for HdIV intergenic regions was consistently below 0.03 (this may have been due to slight contamination of the RNA samples with HdIV DNA despite the DNAse treatment). As a consequence, we considered only HdIV genes with an RPKM value >0.1 to be effectively transcribed.

Statistical analysis

We compared the frequencies of encapsulated H. didymator eggs and larvae between the four hosts dissected at 12–36 h and 72 h p.p., by logistic regression analysis with a generalized linear model especially designed for the modeling of binomial data, using a logit link function to compare mean values [33]. For the weight gain of host larvae, we used a simple one-way ANOVA to compare the mean values obtained between parasitized and control (non-parasitized) larvae, at 6 h, 72 h and 6 d p.p.

For HdIV transcriptome analysis, we then clustered HdIV genes statistically into classes defined on the basis of their level of transcription during parasitization (classes A, B and C for high, medium and low RPKM levels, respectively), using the automatic Bayesian classification system available from the AutoClass@IJM webserver (http://ytat2.ijm.univ-paris-diderot.fr/) [34]. For the analysis of transcription during the parasitization of S. frugiperda, we used a two-way ANOVA to detect significant differences in transcription levels (RPKM values) for the HdIV genes, by analyzing the difference (as a fold-change) between the reference sample (mean value of S. frugiperda 6 h p.p.) and the other two sets of conditions tested (S. frugiperda 24 and 72 h p.p.). Finally, a two-way ANOVA was used to detect significant differences in the transcription level of each HdIV gene between the four host species (S. frugiperda 6 h p.p., P. includens 6 h p.p., M. brassicae 6 h p.p. and A. gemmatalis 6 h p.p.).

General features of the HdIV encapsidated genome

The main characteristics of the sequenced HdIV encapsidated genome, presented in Table 1, are very similar to those reported for other IV genomes (reviewed in [19]): 50 circular molecules ranging in size from 36 kb (segment Hd1) to 2.5 kb (segment Hd45), a genome size of 263 kb, a GC content of 43%, and 134 putative ORFs, corresponding to 31% of the genome (Figure 1; Table S2).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. Graphic representation of the HdIV genome and transcribed regions.

The 50 HdIV segments are shown in linear form, from the smallest (Hd45, 2548 bp) to the largest (Hd1, 36006 bp). Pairs of HdIV segments with similar sequences were designated, the two segments of each pair being named a and b. Colored boxes show the open reading frames (ORFs). Refer to the legend for color correspondences. Introns within ORFs are indicated by a line between two colored boxes (corresponding to exons). A “transcript coverage curve” is shown above each HdIV segment (i.e. the number of Illumina reads mapping to the segment sequence; for the sake of simplicity, only data from one of the 3 “72 h p.p.” replicates were used to draw the curve). Provisional GenBank accession numbers: Hd45:KJ586284; Hd44:KJ586285; Hd43:KJ586286; Hd42:KJ586287; Hd41:KJ586288; Hd40:KJ586289; Hd39: KJ586290; Hd38:KJ586291; Hd37:KJ586292; Hd36:KJ586293; Hd35:KJ586294; Hd34:KJ586295; Hd33: KJ586296; Hd32:KJ586298, Hd31:KJ586299; Hd30:KJ586300; Hd29:KJ586303; Hd28:KJ586304; Hd27:KJ586305; Hd26b:KJ586306; Hd26a:KJ586301; Hd25:KJ586307; Hd24:KJ586308; Hd23:KJ586309; Hd22:KJ586310; Hd21:KJ586311; Hd20b:KJ586297; Hd20a:KJ586312; Hd19:KJ586313; Hd18:KJ586315; Hd17b:KJ586316; Hd17a:KJ586314; Hd16:KJ586317; Hd15:KJ586318; Hd14:KJ586319; Hd13:KJ586320; Hd12:KJ586321; Hd11b:KJ586302; Hd11a:KJ586322; Hd10:KJ586323; Hd9:KJ586324; Hd8:KJ586325; Hd7:KJ586326; Hd6:KJ586328; Hd5:KJ586329; Hd4:KJ586330; Hd3:KJ586331; Hd2b:KJ586327; Hd2a:KJ586332; Hd1:KJ586333.

https://doi.org/10.1371/journal.pone.0104072.g001

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. General features of the HdIV, HfIV, TrIV and CsIV genomes.

https://doi.org/10.1371/journal.pone.0104072.t001

Interestingly, 10 HdIV segments contained identical regions of variable length (Figure 1). These common regions were probably derived from a common proviral sequence, –as demonstrated in some cases by sequence comparison with H. didymator BAC clones (data not shown). These 10 HdIV segments were grouped into five pairs of sequence, with the members of each pair differentiated by “a” and “b” designations (Hd2a/Hd2b with 6976 identical bp in common, Hd11a/Hd11b with 1394 identical bp, Hd17a/Hd17b with 3655 identical bp, Hd20a/Hd20b with 902 identical bp and Hd26a/Hd26b with 2878 identical bp). Consequently, in cases in which the overlapping region contained an open reading frame (ORF), the gene was present twice in the encapsidated form of the HdIV genome (Figure S1). Finally, sequencing also revealed that 11 of the HdIV segments (Hd1, Hd2ab, Hd3, Hd4, Hd5, Hd6, Hd7, Hd10 and Hd26ab) contained at least two internal direct repeat sequences (Table S2), suggesting possible nesting, as previously described for CsIV [17],[35],[36].

In total, the encapsidated HdIV genome contains at least 135 genes: 134 ORFs predicted from the sequenced viral genome, plus a previously described HdIV gene (GlyPro3 GenBank #AF132023.1) [22],[27],[37]–[40] not identified in the newly acquired HdIV sequence. The absence of this sequence indicates that at least one HdIV segment was not sequenced in this study, for unknown reasons. This finding raises questions about how we can ensure that the entire genome is sequenced in the case of complex genomes, such as those of PDVS (e.g. multipartite, with a high frequency of repeated regions). Wasp genome sequencing data (e.g. as the wasp genome contains the proviral form) will undoubtedly provide the answer to this question in the future.

We found that 65 of the total of 135 predicted HdIV genes could be grouped into the six multigene families classically found in Campopleginae IV genomes [13],[17]: the “Cysteine motif proteins” (Cys), “Repeat element” (Rep), “Viral innexins” (Vinx), “Viral ankyrins” (Vank), “Polar-Residue-Rich Proteins” (PRRP) and “N-genes” families (Table 1). In HdIV, the most abundant gene families are the Rep family (26 genes), as for other Campopleginae IVs, and the Vinx family (14 genes). In addition, the HdIV genome contains 12 other gene families encoding proteins of unknown function (Table 1). These families have been named HdIV_F0 (corresponding to the previously described GlyPro family [27]) to HdIV_F11, and each of these families contains two to four gene copies (Table 1). Lastly, HdIV genome contains 37 single-copy genes (Table 1), including four that have already been described (D8_Hd50 (GenBank #AF464931.1), K19_Hd29 (GenBank #AF241775.1), SerThr_Hd7 (S6 mRNA, GenBank #AF464930.1) and P30_Hd6 (Orf1, GenBank #AF479654.1)) [38],[40]. Nineteen of the 135 HdIV genes contained at least one intron (Figure 1), a classical characteristic of PDV genes. These genes included the 9 Cys genes, the 3 members of the GlyPro family, P30_Hd6, U1_Hd6, SerThr_Hd7, D8_Hd45, K19_Hd29, U1_Hd41 and U1_Hd19. As reported in previous studies [27],[38], the first exon, corresponding to a putative signal peptide, was found to be conserved in several of these genes (GlyPro family, P30_Hd6, U1_Hd6 and SerThr_Hd7).

Blastn similarity searches against the NCBI public database revealed strong similarities between HdIV and the IV carried by the wasp Hyposoter fugitivus, HfIV: 44 of the 50 HdIV segments displayed nucleotide sequence identity to 42 of the 56 HfIV segments (Table S2), in their gene or intergenic regions. By contrast, except for genes belonging to multigene families conserved in IV, no significant sequence similarity was found to segments from other sequenced IV genomes. This conservation of the viral segment sequences within a given wasp genus, with differences probably related to diversification of the wasp genus, has also been described for bracoviruses [14].

Interestingly, comparisons between HdIV and HfIV led to the identification of four new coding sequences in HfIV, one in each of the 4 HfIV segments C1, C8, C15 and C17 (named U1_HfC1, U1_HfC8, U1_HfC15 and U1_HfC17 indicated in Figure S2, A). Portions of the intergenic regions of the HdIV and HfIV segments were very similar (more than 65% nucleotide identity), probably reflecting a common ancestral origin for the segments. The newly identified HfIV genes each had two exons, the first of which presented a high degree of sequence similarity to the first exon of the abovementioned HdIV genes (Figure S2, B). This first-exon sequence is thus conserved in diverse IV genes carried by Hyposoter wasps, possibly due to a currently unknown mechanism for the de novo acquisition of signal peptides. Conversely, the second exon was found to be divergent both within and between species, suggesting a possible divergence of the functions of the corresponding proteins. Such divergence between the two Hyposoter species may reflect the adaptation of the two wasp species to different host ranges. Indeed, H. didymator is known to parasitize mostly lepidopteran species from family Noctuidae and a few species from the Pieridae, Nymphalidae and Lasiocampidae families [41], whereas H. fugitivus mostly parasitizes species from the Lasiocampidae, Arctiidae, Saturniidae, Notodontidae and Lymantriidae families [42].

Thus, the known HdIV encapsidated genome contains 37 single-copy genes and 98 genes clustered into 18 gene families: 65 belong to the six families conserved in all IVs, and the other 33 belong to 12 other families named HdIV_F0 to HdIV_F11 (Table 1). One member of each of the HdIV_F1, F5 and F8 families was identified in HfIV, and one member of the HdIV_F1 family was identified among the DsIV RNAseq contigs. Sequence comparisons between HdIV and other known IV genes also indicated that 10 single-copy HdIV genes displayed sequence similarity to HfIV and one (K19_Hd27) displayed sequence similarity to DsIV (Table S2). Thus, 9 HdIV gene families and 28 single-copy genes have been described only in HdIV, suggesting that at least some of these genes and gene families were acquired more recently in the Hyposoter lineage.

Analysis of HdIV transcription during S. frugiperda parasitization

Recent studies on the interactions of H. didymator with its permissive host S. frugiperda have shown that HdIV infection is essential for successful parasitism, particularly for the early larval stages of the parasitoid (48 h–72 h p.p.) [43]. The availability of the HdIV genome sequence made it possible to determine which HdIV genes were transcribed in the insect host and, potentially, to identify those involved in inducing the changes to host physiology required for parasitoid development. We identified HdIV genes expressed during the parasitization of S. frugiperda, by analyzing the HdIV transcriptome in whole host larvae and at three stages of parasitism, corresponding to the embryonic (6 h and 24 h p.p.) and early larval (72 h p.p.) development stages of H. didymator.

We used an automatic Bayesian classification system [34] based on mean RPKM values and taking the three sampling periods into account, to classify the 135 HdIV genes into three classes (A (RPKM>18), B (3<RPKM<18) and C (RPKM<3)) as a function of their level of transcription during parasitization (Figure 2). Class A (RPKM>18) contained only 20 of the 135 HdIV genes predicted by in silico analyses (Figure 2, Time-Class A). Five of the seven genes with the highest RPKM values, were intron-containing genes specific to H. didymator (P30_Hd6, the 3 members of the GlyPro family and U1_Hd6). RNAseq thus confirmed previous results concerning transcription obtained by the less sensitive northern-blot method for the three members of the GlyPro family and P30_Hd6 [38],[39]. All five HdIV-specific genes were found to encode secreted proteins characterized by repeated amino-acid motifs (glycine- and proline-rich for the GlyPro family, serine- and threonine-rich for P30_Hd6), but their function remains unknown [27],[38]. Class A also included at least one gene from each of the six conserved IV multigene families (2 Rep, 2 Vank, 2 Vinx, 1 Cys-motif, 2 PRRP and 1 N-gene). Our results thus also confirm previous descriptions of high transcript levels for Rep2_Hd17ab, Rep1_Hd16, Vank1_Hd24 and Vank1_Hd43 obtained with RT-qPCR techniques [22],[37] and for SerThr_Hd2b, K19_Hd27 and D8_Hd45 by northern-blot analyses [27],[38]. The homologs of several HdIV Class A genes (vank1_Hd24, Vinx1_Hd38, PRRP1_Hd26ab, N-gene_Hd29, K19_Hd27 and F1U2_Hd38) were also the genes with the highest levels of transcription in the indirect study of DsIV gene transcription during Plutella xylostella parasitization by D. semiclausum (DsIV vankyrin 1 (GenBank #JI257593), viral innexin 1 (GenBank #JI257597), polar residue-rich protein (GenBank #JI257606), unknown protein (GenBank #JI257608), unknown protein (GenBank #JI257609), and unknown protein (GenBank #JI257611) genes) [26]. Class B (3<RPKM<18) contained 24 HdIV genes (Figure 2, Time-Class B) and included at least one member of each of the six multigene families conserved in IVs (2 Vank, 2 Vinx, 8 Rep, 2 Cys-motif, 3 PRRP, 2 N-gene genes), and members of the Hd_F3 and Hd_F6 families, together with two single-copy genes (U1_Hd7 and U1_Hd19). Class C (RPKM<3) contained 91 HdIV genes (Figure 2, Time-Class C). However, based on the RPKM threshold for background (determined for HdIV segment intergenic regions, see Materials & Methods), only 50 HdIV genes were considered to be truly transcribed (RPKM>0. 1) in parasitized S. frugiperda larvae. However, we cannot exclude the possibility that the transcription of the remaining 41 genes was not detected because they are actually transcribed in specific host tissues and their transcripts are thus rare in analyses of RNA from the entire larva. Alternatively, they may be transcribed exclusively in other insect host species.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Profile of HdIV transcript levels during the time-course of S. frugiperda parasitization.

Each row represents the mean RPKM value for each of the 135 HdIV genes analyzed in S. frugiperda at 6 h, 24 h and 72 h p.p. The color scale (black to yellow) indicates RPKM level. The RPKM values were used to cluster genes into classes (A, B and C) with the AutoClass algorithm available from the AutoClass@IJM website. Asterisks indicate significant (p>0.05) RPKM fold-changes with respect to the arbitrary chosen reference (Sf 6 h p.p. sample). White asterisks indicate a significant decrease and black asterisks indicate a significant increase in transcript levels (fold-changes ranging from 2 to 67).

https://doi.org/10.1371/journal.pone.0104072.g002

Our results clearly indicate that a small subset of HdIV genes are more strongly transcribed than the others. This difference may be accounted for by a corresponding larger number of gene copies within the viral particles, and delivered to the caterpillar. However, not all the genes present in a given HdIV molecule had a high RPKM (for instance Vank1_Hd24 in class A and Vinx1_Hd24 in class C; Figure 2), indicating that the differences between HdIV transcript levels cannot be accounted for entirely by differences in segment molarity. We investigated these observed differences further, by searching for regulatory motifs in the upstream regions of a subset of genes with RPKM values greater than 10 (24 genes present in 19 segments). However, this analysis revealed no particular characteristic upstream sequences linking high levels of expression with a given regulatory mechanism.

The time-course analysis of the HdIV transcriptome during S. frugiperda parasitization showed that, for most of the HdIV genes, transcript levels were already high 6 h p.p. Cell infection and HdIV transcription are therefore rapid in S. frugiperda larvae. Nevertheless, increases or decreases in the levels of transcripts for some genes were observed over the time course (at 24 and 72 h p.p. versus the reference time point 6 h p.p.) (Figure 2). Such changes were observed for the genes from the Hd38 and Hd36 segments (F1U1_Hd36 and F1U2_Hd38 for the most transcribed), several Vank genes (Vank1_Hd11ab, Vank1_Hd28 and Vank1_Hd24), PRRP2_Hd25 and Rep4_Hd4, which were expressed more strongly at 24 or 72 h p.p. than at 6 h p.p. Conversely, Rep1_Hd16, U1_Hd19 and Rep3_Hd17ab were less strongly expressed at 24 h p.p. than at 6 h p.p. Such changes in transcript level during the course of parasitization may indicate, for these particular HdIV genes, the existence of transcriptional regulation by host cellular/development factors.

The highly transcribed genes, particularly those mentioned above as being conserved in the Campopleginae lineage (on the basis of their sequence and transcription pattern), may be essential for changes in host physiology successful parasitism. However, our knowledge of the role of the corresponding proteins remains limited, essentially because these proteins display no similarity to any known protein of known function. For those belonging to multigene families conserved in IVs, the related proteins have been studied in other biological models and the available data suggest that many play a role in immunosuppression. For instance, the CsIV viral innexins can form functional GAP junction hemi-channels and thus interfere with cell-cell interactions in the lepidopteran host, particularly during the encapsulation process [44]. Thus, HdIV Vinx (Vinx1_Hd38 and Vinx1_Hd36), the transcript levels of which increased during the parasitization time course (Figure 2, Time-Class A), may be involved in disruption of the encapsulation process, as previously observed in parasitized S. frugiperda [9]. Similarly, the Cys1_Hd20a protein may be involved in alterations to cellular immunity. Effectively, two secreted CsIV cysteine-motif proteins, VHv1.1 and VHv1.4, decrease host hemocyte cell adhesion capacity by an unknown mechanism [45]. The HdIV transcriptome analysis also showed that four Vank genes (Vank1_Hd24, Vank1_Hd43, Vank1_Hd28 and Vank1_Hd12ab) were highly transcribed during parasitism. The Vank family is present in all PDVs (BV and IV), suggesting that the corresponding proteins are essential for parasitism success (reviewed by [46]). Experiments in vitro and in cells have shown that BV vankyrins interact with the insect host transcription factor NF-kB and that, like the IkB protein, they maintain NF-kB in its inactive form [47],[48]. Signaling through NF-kB regulates diverse cellular and physiological processes that could consequently be affected by vankyrins. For instance, two CsIV Vanks expressed in a heterologous in vivo Drosophila system have been shown to impair the cellular immune response, humoral inflammation and embryonic development of Drosophila, with phenotypes similar to those observed in NF-kB-deficient flies (Dorsal and Dif (encoding NF-kB factors) silenced (RNAi) or mutant Drosophila) [49]. These previous investigations in the Drosophila model (which is not naturally parasitized by PDV-associated wasps) have highlighted the potential function of the Vanks, but these findings require validation in natural lepidopteran models. Some IV Vanks are also known to inhibit lepidopteran cell apoptosis [50],[51]. By preventing apoptosis of the IV infected cell, IV Vanks may ensure the maintenance of the non-replicative IV genome and gene transcription throughout parasitism. Additional in vivo investigations of the function of HdIV proteins are required to understand the as yet unknown role of these proteins in parasitism, and our results suggest that proteins encoded by genes abundantly transcribed in the S. frugiperda host should be given priority, although other genes are probably also required for successful parasitism.

Assessment of the permissiveness of different lepidopteran host species

Analysis of the HdIV transcriptome during the parasitization of S. frugiperda showed that less than one third of the HdIV genes had high RPKM values. This raises questions about the importance of the remaining HdIV genes, particularly those with RPKM values below the threshold of 0.1. H. didymator can develop in several noctuid species. It is therefore possible that HdIV genes are differentially transcribed in different host species. We tested this hypothesis, by determining HdIV transcript levels after the parasitization of permissive and non-permissive lepidopteran hosts.

We first assessed the permissiveness of different host species to H. didymator development. We assessed oviposition by H. didymator in S. frugiperda and three other species of noctuids: P. includens, M. brassicae and A. gemmatalis. We found that the females readily laid eggs in larvae of all these species but that only S. frugiperda and P. includens larvae were permissive for the development of H. didymator offspring (wasp pupae obtained for more than 70% of the parasitized hosts).

We assessed the permissiveness of the different host species further, by evaluating several host physiological traits known to be affected by HdIV. The first of the traits measured evaluated the efficiency of the host immune response, through measurement of the H. didymator encapsulation rate at various time points after parasitism (12 h, 36 h and 72 h p.p.). As expected, neither S. frugiperda nor P. includens could encapsulate the parasitoid eggs or larvae (Figure 3 A, B, D and E). Conversely, more than 40% of the parasitoid eggs recovered from M. brassicae and A. gemmatalis were encapsulated (Figure 3 A and C), and more than 80% of the parasitoid larvae that escaped encapsulation at the egg stage were also found to be encapsulated (Figure 3 D and F). The second trait measured was used to evaluate the impact of parasitization on host growth rate. Indeed, previous studies have shown that HdIV plays a major role in the lower weight gain observed in parasitized S. frugiperda larvae than in control larvae [9]. As for S. frugiperda, parasitized P. includens larvae gain significantly less weight than non-parasitized larvae (Figure 3 G). However, in P. includens, this difference was smaller and occurred later (Figure 3 G). Parasitized A. gemmatalis displayed a significantly smaller weight gain than controls 3 and 6 days p.p. For M. brassicae, a significant decrease in growth rate was observed only 3 days p.p. (Figure 3 G).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Encapsulation response to parasitization by H. didymator in four lepidopteran host species.

A. Percentage of larvae from which H. didymator eggs were recovered following host dissection 12–36 h p.p. B. and C. Examples of H. didymator eggs recovered between 12–36 h p.p from a permissive host (S. frugiperda or P. includens) and a non-permissive host (M. brassicae or A. gemmatalis), respectively. D. Percentage of H. didymator larvae recovered following host dissection 72 h p.p. E. and F. Example of an H. didymator larva recovered at 72 h p.p. from a permissive host and a non-permissive host, respectively. Note that, in C. and F., a layer of host immune cells surrounds the parasitoid; this is known as encapsulation. In A. and D., different lowercase letters indicate significantly different (p<0.05) results. G. Weight of lepidopteran larvae at 3 times p.p. depending on the species. C: control host larvae, P: host larvae parasitized by H. didymator. Asterisks indicate that, for one species and one time, body weight differed significantly between control and parasitized hosts (p<0.05). Sf: S. frugiperda, Pi: P. includens, Mb: M. brassicae and Ag: A. gemmatalis.

https://doi.org/10.1371/journal.pone.0104072.g003

These data confirm that P. includens is a permissive host, whereas M. brassicae and A. gemmatalis are both non-permissive species. Our results indicate that, under laboratory conditions, the main barrier to successful H. didymator parasitism on M. brassicae and A. gemmatalis is the host cellular immune response. We previously showed that, in S. frugiperda, the egg is protected against encapsulation by as yet unidentified surface proteins, whereas HdIV is involved in the immune protection of the parasitoid larvae [43]. In the case of M. brassicae and A. gemmatalis, none of these effectors are effective, resulting in high encapsulation rates for both eggs and parasitoid larvae. H. didymator uses a combination of virulence strategies to escape the host immune system [43], so future investigations will be required to investigate the effects on each of these strategies in non-permissive hosts. However, the observed lower weight gain in parasitized two non-permissive hosts strongly suggests that these species are nevertheless affected by HdIV, the main source of virulence factors in the S. frugiperda/H. didymator interaction [9].

Comparative analysis of HdIV transcription in different lepidopteran host species

We analyzed the profile of HdIV gene transcription following the parasitization of P. includens, M. brassicae and A. gemmatalis. The transcription profiles in these different lepidopteran host species were then compared. No major variations in HdIV transcription profile over time were observed in the time analysis of S. frugiperda parasitism, so all transcriptome analyses were conducted at 6 h p.p.

Illumina sequencing resulted in the detection of HdIV transcripts in all the parasitized host species, with 97, 56 and 69 HdIV genes having RPKM values >0.1 for P. includens, M. brassicae and A. gemmatalis, respectively (88 genes in S. frugiperda 6 h p.p.). Thus, HdIV particles can infect cells from different lepidopteran species but this infection is not necessarily associated with successful parasitoid development. HdIV infection may account for the significant decreases in weight gain on parasitization observed for the non-permissive hosts.

A comparison of two permissive hosts, P. includens and S. frugiperda, confirmed our unexpected finding that most HdIV genes were expressed at very low levels (or apparently not at all) and led us to draw three main conclusions: (i) in general, the genes for which transcripts are the most abundant in S. frugiperda are also the most strongly transcribed in P. includens; (ii) there are significant differences in gene transcript levels between the two permissive host species, but only for a small number of genes; (iii) these observed differences did not necessarily correspond to different genes from the same multigene family (i.e. none of the observed increases in expression for a particular gene was correlated with a decrease in expression for another gene from the same gene family). Thus, most HdIV genes produced similar amounts of transcript in the two permissive hosts, P. includens and S. frugiperda (Figure 4, Sf and Pi). Nevertheless, significant differences in transcript levels were observed for a number of genes. For instance, Vank3&Vank4_Hd11a, Cys1_Hd20b, Cys1_Hd13, PRRP3_Hd25, Rep3_Hd17b, Rep3_Hd17a, Rep3_Hd10, Rep2_Hd4, Rep5_Hd5, K-19_Hd27, F3U2_Hd2ab, F3U4_Hd2ab, F4U1_Hd31, F4U1_Hd34, F5U2_Hd31, U1_Hd41, U1_Hd20ab and U1_Hd16 had significantly higher RPKM values in P. includens than in S. frugiperda. Conversely, a few HdIV genes, such as Vinx1_Hd38 and Ngene1_Hd15 in particular, generated fewer transcripts in P. includens than in S. frugiperda. Overall, the data obtained suggest that some of the global physiological changes observed in the two permissive hosts may be mediated by different viral effectors. However, too little is currently known about the function of HdIV proteins to test this hypothesis. Finally, we observed no major variation or inversion of patterns of RPKM values within any of the HdIV multigene families as a function of host species (Figure 4, Sf and Pi). Thus, based on this analysis of only two permissive lepidopteran species, there seems to be no clear relationship between the existence of multigene families in HdIV and the possibility of the associated parasitoid being able to exploit a large range of host species.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. The HdIV transcriptome in different lepidopteran host species (6 h p.p.).

RPKM levels are indicated for genes belonging to the Vank family (A), the Cys-motif family (B), the Vinx family (C), the PRRP family (D), the Rep family (E), the N-gene family (F), the GlyPro family (G), the known single-copy gene (H), the F1 to F11 family (I) and the newly characterized single-copy gene (J). For each HdIV gene, different lowercase letters indicate a significant difference (p<0.01).

https://doi.org/10.1371/journal.pone.0104072.g004

The above observations for the comparison of the two permissive hosts also applied to the comparison of the two non-permissive hosts M. brassicae and A. gemmatalis (transcription levels generally higher in A. gemmatalis regardless of the gene considered; Figure 4 Mb and Ag). However, a comparison of permissive and non-permissive hosts showed that HdIV gene transcription rates were much lower in the non-permissive hosts (generally by a factor of log10; Figure 4). In the non-permissive hosts tested 6 h p.p., a smaller number of HdIV genes were found to be transcribed and overall transcript levels were lower than those in permissive hosts (Figure 4). Relevant examples are provided by Cys1_Hd20a, PRRP2_Hd26ab and Rep1_Hd16, which had RPKM values below 2 in M. brassicae but over 20 in the permissive hosts. One explanation for the failure of parasitism in M. brassicae and A. gemmatalis is that low transcript levels result in protein levels that are too low to induce all the changes in host physiology required for the successful development of the parasitoid larva. There may be several reasons for the observed lower levels of transcripts in the non-permissive hosts at early stages of parasitism. Firstly, assuming that H. didymator females inject the same volume of calyx fluid during oviposition, regardless of the size of the host, the density of viral particles injected into the host may be lower in non-permissive hosts, as M. brassicae and A. gemmatalis are slightly larger than the two permissive hosts (Figure 3G). Alternatively, HdIV particles may infect the host cells of non-permissive species less efficiently, either because they penetrate a smaller number of cells or because they are eliminated by an antiviral immune response mediated by autophagy or apoptotic mechanisms, as already described in insects [52]–[54]. A third, non-mutually exclusive, alternative is that putative host regulatory factors (enhancers) are absent from these species.

Our high-throughput transcriptome results complement existing knowledge acquired through experiments on the wasp C. sonorensis, which showed only that non-permissive hosts were not able to sustain CsIV infection during parasitism [55],[56]. Based on this previous work, we could assume that the low level of HdIV gene transcription measured at 6 h p.p. in the two non-permissive hosts (M. brassicae and A. gemmatalis) in our system may decrease still further during parasitization, accounting for the recovery and normal development of non-permissive hosts (Figure 3G, Mb and Ag 6 d p.p.) and efficient immune responses directed against wasp larvae escaping encapsulation (Figure 3D, Mb and Ag).

One of the key conclusions of this study is that, despite the variations of expression observed for a subset of HdIV genes, the global HdIV gene transcription profile does not differ between parasitized host species. This may be because there is no need for a diversification of the molecular mechanisms driving HdIV gene expression to allow H. didymator to exploit a large range of host species. However, our study concerned only a limited number of H. didymator hosts, and many other lepidopteran host species need be analyzed before any firm conclusions can be drawn.

Contributions

Conceived and designed the experiments: ANV TD FLC. Performed the experiments: TD VJ PM GG. Analyzed the data: TD FC EW ANV. Contributed reagents/materials/analysis tools: VJ PM. Contributed to the writing of the manuscript: ANV TD FLC ID EW.