TY - JOUR
AU - M, Geib, Scott
AB - Abstract Viral genome integration provides a complex route to biological innovation that has rarely but repeatedly occurred in one of the most diverse lineages of organisms on the planet, parasitoid wasps. We describe a novel endogenous virus in braconid wasps derived from pathogenic alphanudiviruses. Limited to a subset of the genus Fopius, this recent acquisition allows an unprecedented opportunity to examine early endogenization events. Massive amounts of virus-like particles (VLPs) are produced in wasp ovaries. Unlike most endogenous viruses of parasitoid wasps, the VLPs do not contain DNA, translating to major differences in parasitism-promoting strategies. Rapid changes include genomic rearrangement, loss of DNA processing proteins, and wasp control of viral gene expression. These events precede the full development of tissue-specific viral gene expression observed in older associations. These data indicate that viral endogenization can rapidly result in functional and evolutionary changes associated with genomic novelty and adaptation in parasitoids. polydnavirus, symbiosis, Fopius arisanus Introduction Viral endogenization events provide novel sources of genetic material to their host, thereby altering the pattern and process of evolution within these lineages. The discovery of viral integration into eukaryotic genomes was thought to be relatively rare, but increasingly it is clear that viruses have shaped the evolution of genomes, novel phenotypes, behaviors, and biodiversity itself (Horie et al. 2010; Feschotte and Gilbert 2012). The associations between viruses and parasitoid wasps (Hymenoptera) represent some of the most remarkable and extreme examples of the evolution of biological complexity and genetic innovation in nature. Parasitoid wasps lay their eggs into insect hosts, where progeny develop. Several lineages of wasps have co-opted viruses that are injected into hosts with eggs (Quicke 2015). In hosts, virions increase parasitism success through a variety of mechanisms including immune suppression, alteration of development, delivery of host-derived proteins, and/or molecular mimicry to disguise eggs from host recognition (Strand and Pech 1995; Reineke et al. 2006; Strand and Burke 2014). Two lineages of wasps belonging to the Braconidae and Ichneumonidae have associations with viruses known as bracoviruses and ichnoviruses (collectively polydnaviruses) that represent two independent ancient symbiogenesis events and are essential for parasitism in these wasps (Federici and Bigot 2003; Herniou et al. 2013; Strand and Burke 2014). Bracoviruses are produced in specific “calyx” cells within ovaries of braconid wasps belonging to a diverse monophyletic lineage (estimated 50,000 species) known as the microgastroid complex (Murphy et al. 2008; Rodriguez et al. 2013). The ancestor of bracoviruses derives from a family of insect-infecting DNA viruses known as the Nudiviridae, which integrated into the wasp genome ∼100 Ma (Murphy et al. 2008; Bézier et al. 2009). Compared with the circular double-stranded DNA genomes of their nudivirus ancestors, bracoviruses have undergone genome rearrangement, in which genes have been dispersed within wasp genomes and are comprised of two functional categories (Bézier et al. 2009,, 2013; Burke et al. 2014). The proviral category includes segments that are amplified, excised and circularized, and packaged into virions prior to delivery into host insects. The second category comprises the nudivirus-like genes involved in virus replication, that is, genes encoding the machinery and structural components necessary to make virions. The viral DNA polymerase gene conserved in all nudiviruses has been lost in all bracovirus-carrying wasps characterized to date, suggesting involvement of wasp DNA replication machinery in the production of viral DNAs (Bézier et al. 2009; Burke and Strand 2012a). Ichnoviruses likely derive from nucleocytoplasmic large DNA viruses and share many features with bracoviruses, including replication in calyx cells and genome rearrangement (Volkoff et al. 2010; Béliveau et al. 2015). One species of ichneumonid wasp, Venturia canescens (Gravenhorst, 1829), has lost its ichnovirus and acquired an association with an endogenous nudivirus that makes virus-like particles (VcVLPs; Pichon et al. 2015). Whereas VcVLP nudivirus-like genes are dispersed in the V. canescens genome, unlike bracoviruses, no proviral genome segments are present. The virus-like particles (VLPs) produced do not contain DNA and function as liposomes that deliver virulence proteins into hosts (Pichon et al. 2015). Finally, nudiviruses have been endogenized in the brown planthopper Nilaparvata lugens (Stål, 1854) where viral genes have been re-arranged but have unknown function (Cheng et al. 2014). In parasitoid wasps, viral genome rearrangement is thought to be of key importance for the stability of the association because the viral genome is germline-inherited and can no longer be mobilized in its entirety to replicate in parasitoid hosts, preventing horizontal transfer and adaptation outside of wasps (Strand and Burke 2014). However, the specific events that led to these symbiogenic relationships and their timing are poorly understood (Strand and Burke 2012). Although parasitoid wasps represent one of the most diverse lineages of organisms on the planet, viral genome integration is considered to be relatively rare, with only three or four independent endogenization events currently known (Lawrence 2005; Quicke 2015). Because of the historical lack of high-throughput, inexpensive molecular techniques, the discovery of persistent viral associations has lagged behind other types of microbial symbiont associations of insects (but see Ng et al. 2011; Rosario et al. 2011,, 2013; Webster et al. 2015). The increased prevalence of insect genome sequencing projects may help to alleviate this problem. Fopius arisanus (Sonan, 1932) is a braconid wasp (subfamily Opiinae) and biological control agent of a broad range of tephritid fruit fly species, including the global pests Mediterranean fruit fly Ceratitis capitata (Wiedemann, 1824) and the Oriental fruit fly Bactrocera dorsalis (Hendel, 1912) (Haramoto and Bess 1970; Rousse et al. 2005). Native to the Indo-Pacific region, this species was introduced and established in Hawaii in the 1940s to control B. dorsalis, and has subsequently been introduced to a number of countries in Europe, North America, and several island nations (Rousse et al. 2005). To create foundational genomic resources for this species, the complete genome and transcriptomes for several wasp life stages have been recently generated (Calla et al. 2015; Geib et al. 2017). The F. arisanus genome is ∼154 Mb in size (scaffold N50 size 0.98 Mb) and includes 10,991 protein coding genes (Geib et al. 2017). Although F. arisanus belongs to a subfamily of braconids (Opiinae) that ancestrally lack bracoviruses, a subset of genes with homology to viral genes in the Nudiviridae were identified during annotation. This finding raised questions about whether these wasps produce virions used in parasitism, and whether this represents a recent symbiogenic event. In this study, we find that the Fopius arisanus Endogenous Nudivirus (FaENV) has been co-opted from pathogenic alphanudiviruses for the tissue-specific production and delivery of massive titers of VLPs into hosts that wasps parasitize. The genomic changes underlying this phenomenon include gene loss and changes in genome architecture and gene expression that occur very rapidly after a symbiogenic event, pointing towards their adaptive importance in parasitoid wasp biology. Results and Discussion FaENV Represents a Recent Acquisition of an Endogenous Nudivirus Viral genes identified in the F. arisanus genome shared homology with nudivirus genes. Morphological and genomic data indicate that nudiviruses are most closely related to extensively characterized viruses in the Baculoviridae. Most baculoviruses and nudiviruses are virulent pathogens of insects that establish systemic, lethal infections (Rohrmann 2013). However, two nudiviruses, Helicoverpa zea (Boddie, 1850) nudiviruses 1 and 2, infect the reproductive system of insect hosts and can establish persistent infections associated with integration into the host genome (Wu et al. 2010; Burand et al. 2012). Manual annotation revealed 55 virus-like genes in the F. arisanus genome that shared homology with nudivirus genes (supplementary table S1, Supplementary Material online). Reproductive tract associated viruses have been described by microscopy in several opiine species. These include the entomopoxvirus, rhabdovirus, and rod-shaped viruses found in Diachasmimorpha longicaudata (Shestakov, 1940), and the corona- and cypo-like viruses in Psyttalia concolor (Szépligeti, 1910) (Lawrence and Akin 1990; Jacas et al. 1997; Lawrence and Matos 2005; Luo and Zeng 2010). The presence of these other viruses and the lack of nudiviruses in these species demonstrates that the endogenization event in F. arisanus is not shared among all species within the subfamily Opiinae. In order to determine how widespread nudivirus-like genes are within the genus Fopius, we designed a PCR assay with degenerate primers targeting the most conserved gene, lef-8, for all currently sequenced viruses in the alphanudivirus clade. We tested 11 individual specimens representing five species of wasps from the genus Fopius collected worldwide (supplementary table S2, Supplementary Material online). Positive amplicons were attained for FaENV from F. arisanus, as well as F. bevisi (Brues, 1926), F. ceratitivorus Wharton, 1999, and F. desideratus (Bridwell, 1919). Alignment of sequences derived from amplicons revealed a total of 60/364 variable sites as expected from divergence among species over time (supplementary fig. S1, Supplementary Material online, accession numbers MH558032–MH558034). No amplicons were detected from Fopius sp. 1 (belonging to the marangensis species group) or F. caudatus (Szépligeti, 1913). This indicates that the presence of FaENV is limited to a subset of species within the genus, most parsimonious with an acquisition of FaENV in the ancestor of the four positive species. However, further testing across a broad range of Fopius species would be needed to assess the exact evolutionary history of the virus within the lineage. Diachasma alloeum (Muesebeck, 1956) is an opiine wasp species for which the full genome has been sequenced. tblastn searches of the genome revealed the absence of nudivirus-like genes in this genome, consistent with degenerate PCR results. Conservation of synteny confirmed the absence of nudivirus-like genes in D. alloeum in three specific locations that contain FaENV genes in F. arisanus (supplementary fig. S2, Supplementary Material online). We also examined the nudivirus-like protein-coding sequences to determine whether they were more similar in GC content to pathogenic viral ancestors or the wasp genome in which they have been integrated. GC content of FaENV genes ranged from 34 to 44% with an average of 39%, which is significantly lower than native F. arisanus genes (average 46% G + C, range 30–68%, t18,981 = 14.0, P < 0.0001). The best BLAST hits for nudivirus-like genes were mostly to Oryctes rhinoceros nudivirus (OrNV, Coleptera), Kallithea virus (KaV) of Drosophila melanogaster (Meigen, 1830), VcVLP, and Nilaparvata lugens endogenous nudivirus (NlENV), with amino acid identity of 24–59% (Wang, van Oers, et al. 2007; Cheng et al. 2014; Pichon et al. 2015; Webster et al. 2015). These pathogenic viruses have a GC content that is more similar to FaENV genes, for example, OrNV and KaV have average 42.4% and 34.5% G + C in coding regions, respectively. Taken together, these data demonstrate that FaENV is a recent acquisition that is younger than the genus Fopius. Fopius arisanus Endogenous Virus Produces VLPs and Derives from an Alphanudivirus Ancestor Given the recent acquisition of this virus, we sought to analyze how FaENV is functionally and phylogenetically related to other viruses, and to characterize the initial stages of viral adaptation to a symbiotic lifestyle. Dissection of reproductive tissues from adult females revealed that ovaries are the site of VLP production (fig. 1, supplementary fig. S3, Supplementary Material online). Specifically, a region of the ovary closest to mature eggs and the oviducts used for egg laying (the calyx) was distended and had an iridescent blue appearance characteristic of dense aggregations of virus (fig. 1B, Lawrence et al. 2000). Electron microscopy of ultra-thin sections from the calyx region of F. arisanus ovaries revealed that VLPs are present in extremely high density in the lumen of the calyx and are produced by the nuclei of cells lining the interior of the lumen (calyx cells; fig. 1C, supplementary fig. S4, Supplementary Material online). VLPs are enveloped with a single lipid bilayer and contain cylindrical capsids 37 nm in diameter. Negative staining revealed that capsids of these VLPs can be extremely long and are fragile; the longest capsid visualized was 2.28 μm long. This is well above the maximum length observed for nudiviruses (350 nm, Jehle 2010, supplementary fig. S5, Supplementary Material online). Capsids appeared to lack closed ends and the electron-dense interior usually indicative of nucleic acid was absent. VLPs are released into the calyx lumen via cell lysis along with extra coils of membranous material that are possibly derived from the nuclear membrane. Fig. 1. View largeDownload slide Fopius arisanus and its endogenous nudivirus FaENV. (A) A female wasp parasitizing tephritid fruit fly eggs. (B) The calyx region of F. arisanus wasps has an iridescent appearance. (C) An image of very tightly packed virus-like particles in the calyx region of F. arisanus. Fig. 1. View largeDownload slide Fopius arisanus and its endogenous nudivirus FaENV. (A) A female wasp parasitizing tephritid fruit fly eggs. (B) The calyx region of F. arisanus wasps has an iridescent appearance. (C) An image of very tightly packed virus-like particles in the calyx region of F. arisanus. To identify the closest relatives of FaENV, a phylogenetic tree was constructed using amino acid data concatenated from 19 universally conserved genes of nudiviruses and related baculoviruses (fig. 2, supplementary fig. S6, Supplementary Material online). Representatives included: select taxa within the Baculoviridae; taxa within the Nudiviridae for which genome sequence data were available (genera Alphanudivirus and Betanudivirus; members of Hytrosaviridae; and nudivirus-like sequences from three braconid wasp species representing bracoviruses, the ichneumonid wasp V. canescens, and the planthopper N. lugens, supplementary table S3, Supplementary Material online). Sixteen of the 21 universally conserved genes were identified in the F. arisanus genome (Herniou et al. 2004; Bézier et al. 2015, supplementary fig. S6, Supplementary Material online). Maximum likelihood analysis of evolutionary relationships revealed that baculovirus, nudivirus and hytrosavirus taxa were placed in accordance with previous studies (Thézé et al. 2011), and that FaENV belongs to the pathogenic alphanudiviruses with high support. The phylogeny of the alphanudiviruses does not reflect the evolutionary relatedness of their insect hosts in the Orthoptera, Coleoptera, Lepidoptera, Diptera, and Hemiptera (Herniou et al. 2004; Wang, Kleespies, et al. 2007). FaENV is distantly related to OrNV (coleopteran nudivirus) and three dipteran viruses Drosophila innubila nudivirus DiNV (Hill and Unckless 2018), ToV and KaV. Because of the current paucity of near relatives of FaENV, phylogenetic reconstruction resulted in a monophyletic group for nudivirus-like sequences in F. arisanus, V. canescens, and N. lugens (albeit with poor support). Despite this, nudivirus-like sequences in all three species are considered independently derived because such sequences are absent in species related to all three taxa. Fig. 2. View largeDownload slide FaENV derive from the genus Alphanudivirus and represents an independent endogenization event in insects. A maximum likelihood phylogeny constructed from a concatenated alignment of 19 core genes is shown, with numbers on nodes indicating support from 1,000 bootstrap replicates. Shaded boxes indicate virus families or genera from which four endogenous virus integration events are derived, and numbers in circles indicate independent cases of endogenization in insects: 1) Fopius arisanus endogenous nudivirus; 2) Venturia canescens virus-like particles; 3) Nilaparvata lugens endogenous nudivirus; and 4) bracoviruses from the Polydnaviridae. The sequences used for alignment were obtained from Microplitis demoltior bracovirus (MdBV), Cotesia congregata bracovirus (CcBV), Chelonus insularis bracovirus (CiBV), Tipula oleracea nudivirus (ToNV), Heliothis zea nudivirus 1 (HzNV-1), Penaeus monodon nudivirus (PmNV), Venturia canescens virus-like particles (VcVLP), Nilparvata lugens endogenous nudivirus (NlENV), Drosophila innubila nudivirus (DiNV), Kallithea virus (KaV), Tomelloso virus (ToV), Oryctes rhinoceros nudivirus (OrNV), Fopius arisanus endogenous nudivirus (FaENV), Gryllus bimaculatus nudivirus (GbNV), Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), Cydia pomonella granulovirus (CpGV), Neodiprion sertifer nucleopolyhedrovirus (NeseMNPV), Culex nigripalpus nucleopolyhedrosis virus (CuniNPV), Glossina pallidipes salivary gland hytrosavirus (GpSGHV), and Musca domestica salivary gland hytrosavirus (MdSGHV), supplementary table S3, Supplementary Material online. Fig. 2. View largeDownload slide FaENV derive from the genus Alphanudivirus and represents an independent endogenization event in insects. A maximum likelihood phylogeny constructed from a concatenated alignment of 19 core genes is shown, with numbers on nodes indicating support from 1,000 bootstrap replicates. Shaded boxes indicate virus families or genera from which four endogenous virus integration events are derived, and numbers in circles indicate independent cases of endogenization in insects: 1) Fopius arisanus endogenous nudivirus; 2) Venturia canescens virus-like particles; 3) Nilaparvata lugens endogenous nudivirus; and 4) bracoviruses from the Polydnaviridae. The sequences used for alignment were obtained from Microplitis demoltior bracovirus (MdBV), Cotesia congregata bracovirus (CcBV), Chelonus insularis bracovirus (CiBV), Tipula oleracea nudivirus (ToNV), Heliothis zea nudivirus 1 (HzNV-1), Penaeus monodon nudivirus (PmNV), Venturia canescens virus-like particles (VcVLP), Nilparvata lugens endogenous nudivirus (NlENV), Drosophila innubila nudivirus (DiNV), Kallithea virus (KaV), Tomelloso virus (ToV), Oryctes rhinoceros nudivirus (OrNV), Fopius arisanus endogenous nudivirus (FaENV), Gryllus bimaculatus nudivirus (GbNV), Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), Cydia pomonella granulovirus (CpGV), Neodiprion sertifer nucleopolyhedrovirus (NeseMNPV), Culex nigripalpus nucleopolyhedrosis virus (CuniNPV), Glossina pallidipes salivary gland hytrosavirus (GpSGHV), and Musca domestica salivary gland hytrosavirus (MdSGHV), supplementary table S3, Supplementary Material online. Genomic Rearrangement of FaENV in F. arisanus In contrast to the nudivirus ancestors of FaENV that have compact circular dsDNA genomes, FaENV genes are dispersed within the wasp genome. FaENV genes were located on 11 genomic scaffolds, in nine clusters of genes containing two to nine genes each, in addition to seven genes located singly on scaffolds containing other wasp-derived genes (fig. 3). Two scaffolds contained two clusters each, spaced apart by 249 kb (Clusters 2 and 3) and 86 kb of sequence (Clusters 7 and 8). Each cluster of nudivirus-like genes is flanked by or partially overlapping native F. arisanus genes; three of these flanking regions were chosen at random and verified by PCR with gene-specific primers (fig. 3). Very few paralogous nudivirus-like genes were identified in the wasp genome (supplementary table S1, Supplementary Material online, fig. 3). Exceptions include pif-4, GbNV gp28-like and odv-e66. Three odv-e66 paralogs are located in tandem, whereas a fourth is located among other nudivirus-like genes, suggestive of a single local expansion event. This pattern suggests that a single integration event occurred and subsequently genes moved alone or in clusters to dispersed loci within the wasp genome; undergoing at least 15 genomic rearrangement events to create the current architecture. Viral genome rearrangement to dispersed loci in the wasp genome prevents viral DNAs from replicating as an autonomous unit to produce virions and undergo horizontal transmission between cells and organisms (Burke and Strand 2012b). Fig. 3. View largeDownload slide Genome architecture of endogenous virus elements in the Fopius arisanus genome. Regions of scaffolds containing more than one nudivirus-like gene are shown. Nudivirus-like genes and native F. arisanus genes are displayed in red and blue, respectively. Coding sequence exons and their transcriptional direction are indicated as arrows joined by lines (introns, if present), whereas untranslated regions are shown as boxes. Genes encoding proteins detected in virus-like particles with proteomics are shown with diagonal hatch marks on genes. Only one isoform of native F. arisanus genes is depicted for simplicity, and genes encoding proteins with unknown functions are indicated with locus names. Heavy black lines show junctions between nudivirus-like gene clusters and native F. arisanus genes verified by PCR and sequencing. Fig. 3. View largeDownload slide Genome architecture of endogenous virus elements in the Fopius arisanus genome. Regions of scaffolds containing more than one nudivirus-like gene are shown. Nudivirus-like genes and native F. arisanus genes are displayed in red and blue, respectively. Coding sequence exons and their transcriptional direction are indicated as arrows joined by lines (introns, if present), whereas untranslated regions are shown as boxes. Genes encoding proteins detected in virus-like particles with proteomics are shown with diagonal hatch marks on genes. Only one isoform of native F. arisanus genes is depicted for simplicity, and genes encoding proteins with unknown functions are indicated with locus names. Heavy black lines show junctions between nudivirus-like gene clusters and native F. arisanus genes verified by PCR and sequencing. Despite numerous attempts, we were not able to isolate DNA from purified VLPs. Combined with the lack of nucleic acid staining in virions, failure to isolate DNA suggested that FaENV VLPs do not package DNAs. In contrast, bracoviruses and ichnoviruses both package DNAs, which can be detected by sequencing DNAs isolated from virions or from whole ovaries (where DNAs are amplified) and mapping sequence reads to the wasp genome to look for areas of overrepresented coverage (Burke et al. 2015). Sequencing DNAs isolated from whole F. arisanus ovaries or from crude preparations of virus followed by mapping to the F. arisanus genome did not reveal regions of overrepresented coverage (even for nudivirus-like gene clusters), further verifying the lack of DNAs in FaENV (NCBI BioProject PRJNA420357). The lack of DNAs in VLPs indicates that unlike most other endogenous viruses of parasitoids, FaENV does not function as a gene delivery vehicle (of immune suppression or development-altering genes) and may function to promote parasitism of host insects via other mechanisms. The Function of FaENV within Wasps The function of FaENV in wasps can be explored via the presence or absence of key viral genes and their expression. Whereas functional characterization of nudivirus genes is mostly lacking, a wealth of information is available about the functions of homologous genes within the Baculoviridae (Rohrmann 2013). By extension, the functions of baculovirus genes can be used to predict the functions of FaENV genes. Based upon homology to baculovirus genes of known function, putative gene-product functions include DNA amplification and processing (helicase, lef-3), transcription (p47, lef-4, lef-5, lef-8, lef-9), structural nucleocapsid (vp39, 38K, ac146-like), and viral envelope proteins (p74, pif-1, pif-2, pif-3; multiple copies of pif-4; pif6, multiple copies of odv-e66; vp91, ac81-like, and ac92-like). An additional 30 nudivirus-like genes with unknown function were identified. We used proteomics to verify that VLPs are constructed of proteins encoded by nudivirus-like genes. Over half (28/55) of the nudivirus-like genes were detected from purified F. arisanus VLPs using proteomics, which included putative nucleocapsid and envelope proteins, the putative DNA-binding protein Lef-3, and 15 proteins encoded by nudivirus-like genes with unknown function (fig. 3, supplementary table S1, Supplementary Material online). These data confirm that genes of nudiviral origin in the wasp genome are producing the specific VLPs in F. arisanus ovaries. Universally conserved genes in nudiviruses and baculoviruses that are absent from FaENV include dnapol, vlf-1 and pif-5. The lack of the viral DNA polymerase gene dnapol is consistent with the lack of detectable DNAs packaged in VLPs (although the lack of a viral DNA polymerase does not prevent the packaging of host-amplified DNAs into virions produced by other endogenous viruses, Bézier et al. 2009; Volkoff et al. 2010; Burke et al. 2014; Pichon et al. 2015). In baculoviruses, Vlf-1 is a multi-functional protein involved in transcription, DNA processing, and packaging into virions (McLachlin and Miller 1994; Yang and Miller 1998; Vanarsdall et al. 2006). The lack of vlf-1 in FaENV is also consistent with the absence of DNA in virions and could play a role in extreme nucleocapsid length and the lack of closed ends, similar to a vlf-1 knockout of Autographa californica nucleopolyhedrosis virus (AcMNPV; Li et al. 2005; Vanarsdall et al. 2006). The retention of what appear to be intact helicase and lef-3 genes is unexpected given empty FaENV VLPs. These genes may have uncharacterized alternative functions or may be nonfunctional and likely to be eventually lost from the wasp genome. The pif-5 gene encodes an envelope protein and an oral infectivity factor in AcMNPV (Braunagel et al. 1996). Given that FaENV is injected into host insects during oviposition, the absence of pif-5 is of unknown functional significance. In summary, the FaENV gene set is reflective of a virus that has undergone modifications that still enable the production of virions but prevent packaging of DNAs and consequently replication outside of wasp hosts. Investigation of the infectivity of VLPs inoculated onto host cells and the role of FaENV in parasitism is currently in progress. FaENV Expression is Suggestive of Ongoing Adaptation in Viral Gene Regulation Co-option and rearrangement of viral genes in the wasp genome enables the adaptation of regulatory sequences to control FaENV replication and mitigate potential pathogenic effects upon the wasp. In baculoviruses, virus replication proceeds through a series of cascading transcriptional stages (Rohrmann 2013). After viral DNAs have entered into cell nuclei, the first viral genes to be expressed (immediate early genes) are transcribed by the host RNA polymerase (Fuchs et al. 1983). The genes p47, lef-4, lef-8, and lef-9 are “early” genes and encode the subunits required to produce the viral RNA polymerase holoenzyme that selectively transcribes “late” genes (Rohrmann 1986). We hypothesized that viral particle assembly may be constrained by wasps by controlling the initial stages of transcription and limiting the downstream transcriptional cascade to calyx cells where VLPs are observed. This has occurred for nudivirus-derived genes that have been present in the genomes of bracovirus-producing wasps for ∼100 My; early genes are expressed specifically in calyx cells and not in other ovary tissues or in males (Bézier et al. 2009). To test this hypothesis, nudivirus-like gene expression was measured in several tissue types and life stages. First, data from four previously published transcriptome samples (adult female, adult male, pupae, and larvae) were re-analyzed to incorporate the properly annotated nudivirus-like gene set (supplementary fig. S7, table S1, Supplementary Material online). Most nudivirus-like genes were expressed in adult females, whereas expression in adult males, pupae and larvae was detected at low levels for <15 genes in each life stage (Reads Per Kilobase of exon model Mapped [RPKM] ≤ 3, supplementary fig. S7, table S4, Supplementary Material online). Newly generated data from 3-day old adult ovaries demonstrated that all but one of the nudivirus-like genes are expressed in ovaries, including the most highly expressed gene vp39 encoding the putative major nucleocapsid protein. Quantitative PCR (qPCR) of representative viral genes was used to assess specificity of expression in wasp tissues and throughout development (fig. 4). Our choices included the early genes lef-8 and lef-9, and the late genes vp39 and pif-1 (a putative envelope gene, Rohrmann 2013). lef-9 was expressed as expected with transcription detected solely in female ovaries where VLPs are produced. In contrast, lef-8 was expressed in reproductive tissues in both males and females; transcripts were detected in ovaries, testes, and Hagen’s glands (ectodermally derived scent glands, Buckingham and Sharkey 1988; Williams et al. 1988), whereas very little expression was observed in venom glands of females (Quicke et al. 1997) or nonreproductive tissues in either sex (fig. 4A). These data show that for lef-8, the early stages of viral gene transcription are not ovary-specific but occur in male reproductive tissues as well. lef-8 translation products produced in male tissues are unlikely to be functional because other early gene products are not present to produce the viral RNA polymerase holoenzyme. While late genes were highly expressed in ovaries, expression was negligible in testes and Hagen’s glands. Consistent with expression data, no VLPs were found in testes visualized with transmission electron microscopy. Expression of nudivirus-like genes was also measured throughout ovary development in female wasp abdomens (fig. 4B). Expression of lef-8 and lef-9 was first upregulated in brown-eyed pupae, whereas vp39 and pif-1 expression was significantly upregulated in newly emerged adults. This is consistent with the transcription of early viral RNA polymerase genes in developing ovaries prior to the predicted structural late genes, which are putatively transcribed by the nudivirus-like RNA polymerase holoenzyme. Fig. 4. View largeDownload slide Mean transcript abundance ± standard error of selected nudivirus-like genes from Fopius arisanus as determined by qPCR. Transcript abundance is presented for (A) adult tissue types from 3-day old wasps (from three independently collected tissue samples) or (B) from female abdomens of increasing maturity throughout pupal development (as indicated by pupal eye color) until adulthood (from five independently collected tissue samples). Summary statistics for the differences between means (ANOVA) are listed and significant differences (Tukey’s HSD) are indicated by letters. Fig. 4. View largeDownload slide Mean transcript abundance ± standard error of selected nudivirus-like genes from Fopius arisanus as determined by qPCR. Transcript abundance is presented for (A) adult tissue types from 3-day old wasps (from three independently collected tissue samples) or (B) from female abdomens of increasing maturity throughout pupal development (as indicated by pupal eye color) until adulthood (from five independently collected tissue samples). Summary statistics for the differences between means (ANOVA) are listed and significant differences (Tukey’s HSD) are indicated by letters. We next examined FaENV gene structure and correlation with timing of gene expression in calyx cells. Strikingly, three of the four FaENV genes that contain introns are putative early genes. lef-4, lef-5, lef-9 and GbNV gp78-like each contain an intron, ranging in size from 77 bp to 17 kb (fig. 3). To date, introns have not been observed in RNA polymerase genes from nudiviruses or baculoviruses, suggesting that introns in FaENV genes have been gained since introduction of the viral genome into the wasp genome. The presence of introns in early genes and their advance expression suggests these genes cannot be transcribed by the viral RNA polymerase they encode. Instead, they may be recognized and transcribed by the host RNA polymerase in reproductive tissues, allowing the host to control viral gene expression and constrain replication to calyx cells. Together, our hypothesis that viral particle assembly may be constrained by wasps is supported by 1) the presence of introns in key early genes, 2) the absence of expression of lef-9, vp39 and pif-1 outside of ovaries, and 3) the lack of VLPs in tissues outside of calyx cells. However, the expression of lef-8 in several tissue types is not what was predicted and differs from the expression of early genes in more ancient parasitoid wasp-viral associations derived from nudiviruses. The expression of lef-8 in male testes and Hagen’s glands suggests lef-8 transcription is controlled by a developmental pathway active in both female and male reproductive tissues. As each nudivirus-like gene has independent cis-regulatory elements and as early gene products must be produced together to comprise the viral RNA polymerase holoenzyme, VLP assembly appears to be limited to ovaries by expression of at least one of the remaining early genes (i.e. lef-9) in this tissue only. Thus, FaENV gene expression patterns are suggestive of ongoing adaptation of viral gene expression to optimally produce VLPs in wasp ovaries for injection into hosts. Conclusions The discovery of FaENV and related endogenous viruses in the genus Fopius is significant because it represents a recent virus acquisition and suggests that the repeated evolution of these associations could be major drivers of parasitoid biodiversity. Our data are suggestive of the relatively common and recurrent introduction of nudivirus DNAs into parasitoid genomes. Nudivirus endogenization into wasp genomes could relate to the ability of some nudivirus taxa to form latent infections via integration into host genomes (Strand and Burke 2014; Drezen et al. 2017). This recently derived example of symbiogenesis in some species of Fopius wasps allows us to impose a rapid timeline for changes between FaENV and its pathogenic ancestors, including genomic rearrangement, loss of packaged DNA and key DNA processing proteins, and host control of viral gene expression with the gain of introns in early genes. The repeated evolution of viral genome rearrangement and the loss of the DNA polymerase gene in all endogenous nudivirus-like lineages in parasitoid wasps (bracoviruses, VcVLPs, and FaENV) and some ichnoviruses is indicative of their importance in establishing persistent associations with wasps (Bézier et al. 2009; Volkoff et al. 2010; Burke and Strand 2012a; Burke et al. 2014; Pichon et al. 2015). Prior to the discovery of FaENV, the timeline of these events was unknown because bracoviruses (and possibly ichnoviruses) represent ancient associations in which initial events have been obscured by sequence changes over time. VcVLP represents an ichnovirus-for-nudivirus replacement event of unknown age, although the pseudogenization of potentially “unnecessary” nudivirus-like genes in the V. canescens genome suggests this replacement could also be quite recent (Pichon et al. 2015; Leobold et al. 2018). It is unclear whether the dispersed genome architecture of bracoviruses and ichnoviruses was due to a single viral integration event followed by re-arrangement, or multiple integration events into different locations in the wasp ancestor’s genome followed by differential gene loss (Burke and Strand 2012b). From FaENV, we now know that changes in genome architecture can derive from a single integration event and that rearrangement can occur very rapidly. Adaptation towards calyx cell specific viral gene expression as found for bracoviruses seems to be a process that is ongoing in F. arisanus that lags behind changes in viral genome architecture. The lack of DNAs packaged in FaENV virions prevents any conclusions about the source and original timing of DNAs packaged into bracoviruses and ichnoviruses. A final common feature of independently derived endogenous viruses is the restriction of viral particle assembly to wasp calyx cells. The function of these cells in wasp species that do not produce VLPs is currently unknown. The characteristics of calyx cells that make them particularly accommodating for the production of massive titers of virus are interesting from a developmental standpoint that should be given some attention in future research. Materials and Methods Laboratory Colonies and Rearing Conditions Unlike most opiine parasitoid wasp species that parasitize fly host larvae, F. arisanus is primarily an egg parasitoid that undergoes development in the host’s larva and pupa and emerges from its host at the pupal stage. Adult wasp samples were derived from USDA-ARS Pacific Basin Agricultural Research Center insectary line of F. arisanus, maintained on B. dorsalis as its host species. Fopius arisanus parasitoids were reared on B. dorsalis in artificial diet as previously described (Bautista et al. 1999; Manoukis et al. 2011). Viral Gene Annotation Viral genes within the F. arisanus genome were identified using homology to genes in other viral genomes. All Open Reading Frames (ORFs) >33 amino acids in size were found in the F. arisanus genome using getORF from the EMBOSS package v. 6.6.0.0 (Rice et al. 2000). F. arisanus ORFs were searched against a database of all currently known viral proteins available from NCBI downloaded on July 19, 2017 (https://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi? taxid=10239). All ORFs that had a hit to the viral database with an e-value of at least 0.001 were selected and searched against the nonredundant (nr) database available from NCBI on August 8, 2017. ORFs with best hits to genes in other Hymenoptera were removed, whereas ORFs with hits to viral genes were retained and manually curated further. The annotations for all viral genes were further checked and refined using the JBrowse instance hosted at https://apollo.nal.usda.gov/fopari/jbrowse (Skinner et al. 2009). Previously generated transcriptome data from adult females, adult males, and larvae and pupae of mixed sex and levels of maturity displayed as bigwig coverage plots in JBrowse were used to annotate boundaries of transcribed but untranslated regions and any introns that were present in viral genes. Gene models are curated at the USDA Ag Data Commons under accession doi:10.15482/USDA.ADC/1417880, and are visible as a track on JBrowse hosted at the i5k Workspace at https://apollo.nal.usda.gov/fopari. Verification of junctions between nudivirus-like and native F. arisanus genes was performed with long-range PCR and gene-specific primers (supplementary SI Appendix, table S1, Supplementary Material online). DNA was extracted from a single adult male with the DNeasy Blood and Tissue extraction kit (QIAGEN). PCR was performed using the KAPA LongRange HotStart PCR kit in 25 µl reactions. The final concentrations for the PCR master mix were: 1x Kapa LongRange Buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.675 U Kapa LongRange HotStart DNA polymerase, and primers at 0.7 µM. 20 ng of F. arisanus DNA was added to each reaction, and the cycling conditions were as follows: initial denaturation for 3 min at 95°C, followed by 35 cycles of 95°C for 25 s, 55°C for 15 s, and 68°C for 5.5 min. A final extension was performed for 10 min at 68°C. Standard PCR with cDNA generated from 3-day old wasp ovaries was used to validate intron predictions for lef-4, lef-5, and lef-9. Extraction of total RNA was performed using the QIAGEN RNeasy kit following the standard kit protocol with a 15 min on-column DNase treatment and elution in 30 µl of RNAse-free H2O. RNA concentration was measured using a Nanodrop Spectrophotometer and cDNA was synthesized from 100 ng of total RNA (normalized across samples). First-strand cDNA synthesis was performed using Invitrogen reagents including the Superscript III enzyme and oligo(dT) primers as outlined by the manufacturer (Invitrogen). Detecting FaENV in Relatives of F. arisanus Wasp samples were identified by Robert Wharton (supplementary table S2, Supplementary Material online). DNA was isolated from wasp specimens using the QIAGEN DNeasy Blood and Tissue Kit insect protocol B without sample homogenization and with overnight proteinase K incubation. Vouchers are stored at Texas A&M University. Fopius ceratitivorus sequences were derived from insects maintained in culture in Hilo, HI, originally derived from the MOSCAMED strain maintained in Petapa, Guatemala. Degenerate primers targeting lef-8 in alphanudiviruses were designed to PCR screen samples for the presence of an endogenous nudivirus (supplementary table S5, Supplementary Material online). Prior to PCR, sample DNAs were diluted to ∼10 ng/µl. The quality of the DNA samples was verified using PCR with the Lep1 primer set to amplify a region of the cytochrome C oxidase I gene (COI, supplementary table S5, Supplementary Material online). Lep1 PCR was performed in 10uL reactions, with the following final concentrations for the master mix: 1x HotMaster Taq buffer, 1 mM dNTPs, 0.4 U Quantabio HotMaster Taq DNA Polymerase, and primers at 0.8 µM. Cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 20 s, 55 °C for 30 s, and 65 °C for 1.5 min. Final extension was performed at 65 °C for 7 min. Lef-8 PCRs with degenerate primers were performed in 15 µl reactions. Final concentrations for the lef-8 PCR master mix were: 1× HotMaster Taq buffer, 1 mM dNTPs, 0.4 U Quantabio HotMaster Taq DNA Polymerase, and primers at 0.8 µM. Cycling conditions were as follows: initial denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 20 s, 51 °C for 30 s, and 65 °C for 1.5 min. Final extension was performed at 65 °C for 7 min. PCR products were cloned with the StrataClone PCR cloning kit and a consensus was calculated with sequences generated from 2 to 4 clones per sample. Proteomic Analysis of F. arisanus VLPs Calyx fluid was released from 50 dissected F. arisanus ovaries by gently puncturing the calyx region, allowing VLPs to diffuse into a PBS solution. Then, the supernatant was spun down at 20,000 × g for 30 min and washed once with PBS to collect VLPs as described previously (Burke et al. 2013). The virus pellet was resuspended in Proprep buffer containing 1× Laemelli loading dye and 5% beta-mercaptoethanol, sonicated, and incubated at 90 °C for 10 min before electrophoresis on a Mini PROTEAN TGX 4–20% polyacrylamide gel at 100 V for 30 min (Bio-Rad). The entire lane was cut into four pieces to separate proteins by size. In-gel trypsin digestion was performed for each gel slice, followed by proteomic analysis by an Orbitrap Elite mass spectrometer, coupled to an Easy-nLC II Liquid Chromatography (LC) instrument (Thermo Fisher Scientific) as previously described (Burke et al. 2013). Proteins were identified by searching against a database of all annotated protein-coding sequences from F. arisanus, including correctly annotated nudivirus-like genes using the Mascot v2.3 algorithm (Matrix Science Inc.). Data were visualized with ProteomeDiscoverer v1.3 (Thermo Fisher Scientific). Peptides with scores greater than the identity score (P < 0.05) were considered significant matches. Only ORFs that were matched by at least two unique peptide spectra were considered positive identifications. Microscopy Ovaries were dissected from adult wasps in a plastic petri dish in a droplet of PBS. Light images of representative ovaries were generated on a Leica MZLFIII dissecting stereomicroscope with a Leica DFC420 digital camera. For Transmission Electron Microscopy (TEM), ovaries from adult females were dissected and fixed in 3% glutaraldehyde in phosphate buffer at 4 °C. Tissues were postfixed in 1% osmium tetroxide, and block stained in 0.5% uranyl acetate prior to being embedded. Tissues were dehydrated in graded ethanol solutions, followed by infiltration in graded propylene oxide and Epon/Araldite. Tissues were sectioned with an ultramicrotome and sections were placed on grids and stained with Reynald’s lead citrate and uranyl acetate before examination with TEM. Negative staining was performed by dissecting ovaries from 30 adult female wasps and gently releasing virions into 50 µl of PBS, adsorbing virions onto a Formvar coated grid and staining with 3% phosphotungstic acid. Deep Sequencing to Verify the Absence of DNA in VLPs In the wasp species M. demolitor, deep sequencing of circularized DNAs encapsidated in virions or DNAs amplified in wasp ovaries followed by read mapping to the wasp genome clearly indicated which sequences were packaged or amplified by differences in read coverage (relative to low, background levels of coverage across the wasp genome; Burke et al. 2014, 2015). DNA was isolated and sequenced from whole F. arisanus ovaries or from a crude virus preparation to verify that regions of extremely high sequence coverage, and thus packaging of DNAs in to virions, do not occur in this wasp species. Ovaries were dissected from nine individual three day-old adult wasps, and most ovariole tissue was removed with opthalmology scissors. A crude virus preparation was obtained by explanting ovaries from 50 three day-old adult wasps, and gently pulling the calyx regions apart to let virus diffuse into a 50 µl drop of phosphate buffered saline (PBS). PBS was added to both samples to a total volume of 500 µl, and the calyx regions of ovaries were homogenized. 250 mg of proteinase K (Roche) and 2% (wt/vol) Sarkosyl were added, followed by incubation at 62 °C for 1 h. After phenol–chloroform extraction and addition of 25 µg of glycogen, DNA was precipitated with 0.3 M sodium acetate (pH 5.2) and isopropanol. The DNA pellets were then resuspended in 50 µl of water. Small insert libraries were prepared using the Illumina TruSeq DNA kit with standard conditions. These two libraries were sequenced (PE150) on an Illumina MiSeq or NextSeq instrument at the University of Georgia Genomics and Bioinformatics Core. This generated 51.6 million reads (ovaries) or 9.4 million reads (virus preparation) that were filtered to retain reads with a minimum Phred quality score equivalent of 30 for >90% of the nucleotides in a read to a total of 25.6 million (ovaries) or 4.7 million (virus preparation) reads using the FASTX toolkit (http://hannonlab.cshl.edu). Mapping of sequence reads to the genome was performed with the Burrows–Wheeler Aligner algorithm bwa-mem (version 0.7.15; Li and Durbin 2009) resulted in 83.5% (ovaries) and 91.5% (virus preparation) of the reads mapping to the F. arisanus genome. Manual inspection of read alignments was performed using Tablet (Milne et al. 2013). Transcriptomic Analysis of Gene Expression and qPCR To quantify expression of viral genes in the F. arisanus genome, we used existing Illumina transcriptome data in conjunction with newly generated sequence data. Existing sequence reads (PE100) came from the four transcriptome samples described above (Calla et al. 2015), NCBI BioSamples SRS691550, SRS691551, SRS69153, and SRS691554; BioProject PRJNA259570). To generate an ovary transcriptome, total RNA was extracted from ovaries of ten 3-day old adult female wasps followed by standard stranded RNA library construction and Illumina NextSeq PE150 sequencing at the Georgia Genomics and Bioinformatics Core at the University of Georgia. Raw reads were filtered by quality as described above. Data were reduced by 15–26% to 3.9–5.3 Gb per sample. Quality filtered reads were mapped to the F. arisanus genome using HISAT2 v. 2.1.0, sorted with samtools v. 1.3.1 and read Fragments Per Kilobase of exon model Mapped (FPKM) was calculated using StringTie v. 1.2.0 (Li et al. 2009; Pertea et al. 2016). The reference GFF3 file used was modified to include all previously annotated F. arisanus genes along with newer manually curated gene models for nudivirus-like genes. We used qPCR to detect differences in expression of target genes in specific tissues and across development. Tissues were dissected from ovaries, venom glands, testes and Hagen’s glands (10 individuals/sample), male and female pelt tissue (3 individuals/sample), or abdomens from female pupae (1 individual/sample), and cDNAs were generated as described above. An absolute standard curve was generated via PCR amplification of the corresponding cDNA for each gene of interest using specific primers with methods similar to Burke and Strand (2012a) (supplementary table S5, Supplementary Material online). Each product was cloned into pSC-A-amp/kan, and after propagating and isolating each plasmid from minipreps, its identity was confirmed by sequencing. Standard curves were generated followed by determination of copy numbers from serially diluted amounts (102–107 copies) of each plasmid standard. qPCR was performed on a Rotor-Gene Q using the SYBR Green QuantiTect PCR Kit with 1 µM primers and 1 µl of undiluted cDNA per 10 µl reaction (Qiagen). After 15 min of denaturation at 95 °C, a three-step amplification cycle with 95 °C for 15 s denaturation, 60 °C for 30 s of annealing and 72 °C for 30 s for extension was used for 45 cycles. Melting curve analyses were performed to ensure that amplified products were specific for the gene of interest. Three (for tissues) or five (for developmental stages) independently acquired biological replicates were analyzed for each gene, with each sample technically replicated four times. Phylogenetic Analyses Amino acid sequences from representative taxa previously used in a similar analysis within the Baculoviridae, Nudiviridae, Hytrosaviridae, parasitoid wasps and the planthopper N. lugens were collected for 19 core genes conserved in baculoviruses and nudiviruses (Bézier et al. 2015). Two other core genes were excluded because they were not consistently single-copy (pif-5) or had uncertain alignment (p6.9). The sequences used for alignment were obtained from species listed in supplementary table S3, Supplementary Material online. Sequences were aligned using the Mafft linsi algorithm (Katoh and Standley 2013) and were curated with GBlocks using default settings with modifications (the minimum number of sequences for a flanking position = 50% + 1, maximum number of contiguous nonconserved positions = 10, minimum length of a block = 5, allowed gap positions = all; Talavera and Castresana 2007). A maximum likelihood phylogeny was derived for individual gene alignments and a concatenated alignment using RAxML with default parameters and 1,000 rapid bootstrap replicates (Stamatakis 2014). Acknowledgments We thank Jeff Takano and Stephanie Gayle for their assistance in rearing colonies and collecting wasp samples for this study. We would also like to thank Monica Poelchau and the i5k project, Mary Ard at the UGA electron microscopy lab for sample preparation and microscope operation, Jena Johnson for wasp photography, and Robert Wharton, Sonja Scheffer, and Matthew Lewis for contributing Fopius DNA samples. This work was supported by the US Department of Agriculture—Agricultural Research Service (to S.M.G.), US National Science Foundation (DEB-1622986 to G.R.B.), the USDA National Institute of Food and Agriculture Hatch project (1013423 to G.R.B.) and the University of Georgia's Research Foundation, Office for the Vice President for Research, and Agricultural Experiment Station. 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TI - Rapid Viral Symbiogenesis via Changes in Parasitoid Wasp Genome Architecture
JF - Molecular Biology and Evolution
DO - 10.1093/molbev/msy148
DA - 2018-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/rapid-viral-symbiogenesis-via-changes-in-parasitoid-wasp-genome-iXpIk5ueaz
SP - 2463
VL - 35
IS - 10
DP - DeepDyve
ER -