View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Virology 363 (2007) 26–35 www.elsevier.com/locate/yviro Shared and species-specific features among ichnovirus genomes Kohjiro Tanaka a,1,2, Renée Lapointe b,2, Walter E. Barney a, Andrea M. Makkay c, Don Stoltz c, ⁎ ⁎ Michel Cusson b, , Bruce A. Webb a, a Department of Entomology, University of Kentucky, Lexington, KY 40503, USA b Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du PEPS, PO Box 10380 Ste-Foy stn, Quebec City, QC, Canada G1V 4C7 c Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 Received 3 August 2006; returned to author for revision 13 September 2006; accepted 13 November 2006 Available online 15 February 2007 Abstract During egg-laying, some endoparasitic wasps transmit a polydnavirus to their caterpillar host, causing physiological disturbances that benefit the wasp larva. Members of the two recognized polydnavirus taxa, ichnovirus (IV) and bracovirus (BV), have large, segmented, dsDNA genomes containing virulence genes expanded into families. A recent comparison of IV and BV genomes revealed taxon-specific features, but the IV database consisted primarily of the genome sequence of a single species, the Campoletis sonorensis IV (CsIV). Here we describe analyses of two additional IV genomes, the Hyposoter fugitivus IV (HfIV) and the Tranosema rostrale IV (TrIV), which we compare to the sequence previously reported for CsIV. The three IV genomes share several features including a low coding density, a strong A+T bias, similar estimated aggregate genome sizes (∼250 kb) and the presence of nested genome segments. In addition, all three IV genomes contain members of six conserved gene families: repeat element, cysteine motif, viral innexin, viral ankyrin, N-family, and a newly defined putative family, the polar-residue-rich proteins. The three genomes, however, differ in their degree of segmentation, in within-family gene frequency and in the presence, in TrIV, of a unique gene family (TrV). These interspecific variations may reflect differences in parasite/host biology, including virus-induced pathologies in the latter. © 2007 Published by Elsevier Inc. Keywords: Ichnovirus; Polydnavirus; Genome evolution Introduction generation through the wasp germ line (Stoltz, 1990). Importantly, replication and virion assembly are restricted to The two recognized polydnavirus (PDV) taxa, ichnovirus the calyx cells, which are located at the junction of the ovarioles (IV) and bracovirus (BV), share an obligate mutualistic and the lateral oviduct. The PDV genome consists of multiple association with endoparasitic wasps of the families Ichneu- circular double stranded DNA segments, ranging in size from 2 monidae and Braconidae, respectively. A unique trait of PDVs to 42 kb (Kroemer and Webb, 2004). The number of genome is that their genome is integrated into the chromosomal DNA of segments and their size distribution vary from one PDV species the wasp carrier and is thus vertically transmitted to the next to another. The estimated size of characterized PDV genomes ranges from 187 to 567 kb (Dupuy et al., 2006; Espagne et al., 2004; Webb, 1998; Webb et al., 2006). ⁎ Corresponding authors. During oviposition, PDV virions are injected into the E-mail addresses: [email protected] (K. Tanaka), lepidopteran host hemocoel, along with wasp eggs, venom [email protected] (R. Lapointe), [email protected] (W.E. Barney), and ovarian proteins (Webb, 1998). PDV infection causes [email protected] (A.M. Makkay), [email protected] (D. Stoltz), physiological alterations in the parasitized larva, including an [email protected] (M. Cusson), [email protected] (B.A. Webb). arrest or delay in development and/or a suppression of the 1 Present address: Institute for Biological Resources and Functions, National Institute of Advanced Industrial Sciences and Technology (AIST), Tsukuba, immune response that would otherwise lead to encapsulation Ibaraki 305-8566, Japan. and death of wasp eggs by host hemocytes. PDV genes are 2 These authors contributed equally to the work. classified in three groups according to the host specificity of 0042-6822/$ - see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.virol.2006.11.034 K. Tanaka et al. / Virology 363 (2007) 26–35 27 their expression (Webb, 1998). Genes expressed exclusively in For both HfIV and TrIV, we constructed genomic libraries the wasp or in the lepidopteran host are designated as class I and that we used for whole genome shotgun sequencing. In parallel, class II genes, respectively, whereas class III genes are we used the EZ∷TN transposon system (Epicentre) to purify expressed in both hosts. Given that affected wasps are individual genome segments, which were then submitted for asymptomatic, class II genes have received the greatest sequencing so as to facilitate sequence assembly and sequence attention; indeed most of the proteins they encode are believed assignment to genome segments. Our results indicate that HfIV to be responsible for the pathological effects observed in the and TrIV share several genome properties and that each lepidopteran host and, therefore, hold some potential for the contains members of the gene families found in CsIV, including development of new pest-control strategies (Kroemer and a newly described group of related putative genes; however, we Webb, 2004). also observed some disparities in genome composition, which The genomes of the Campoletis sonorensis ichnovirus may reflect differences in the types of disturbances the viruses (CsIV), Microplitis demolitor bracovirus (MdBV) and Cotesia cause in their respective hosts. congregata bracovirus (CcBV) were recently sequenced and compared with respect to their organization and gene content Results (Espagne et al., 2004; Webb et al., 2006). They were observed to share various attributes, including segmentation, a lack of General features of the HfIV and TrIV genomes genes required for DNA replication and strong gene expansion into families. On the other hand, the comparison between CsIV The HfIV genome and MdBV indicated that these ichnovirus and bracovirus We identified and sequenced 56 unique HfIV genome genomes contain few shared gene families; however, a segments, 11 of which can yield smaller, “daughter” or “nested” preliminary comparison of available sequence data from several segments, which are generated by intramolecular recombination PDV species suggested that the gene families identified so far of larger genome segments (Kroemer and Webb, 2004). This are well conserved within the IV and BV taxa (Webb et al., degree of genome segmentation is higher than that assessed for 2006). either TrIV or CsIV (Fig. 1A), making HfIV the most highly In the present article, we report on the genome sequences of segmented IV genome characterized to date. Its genome two campoplegine IVs, the Hyposoter fugitivus ichnovirus segments range in size between 2.6 and 8.9 kb, with a median (HfIV) and the Tranosema rostrale ichnovirus (TrIV). In size of 4.0 kb (Fig. 1B), and its aggregate genome size is undertaking this work, we wished to determine whether the estimated at 246 kb (Fig. 1C). The HfIV genome has a G+C general features described for the CsIV genome are shared by content of 43.1%, a value comparable to that determined for other selected IV representatives, with the aim of achieving a TrIV and CsIV (Fig. 1D). Overall, coding sequences represent level of intrataxonomic comparison similar to that which is 30% of its genome (Fig. 1E), although coding density is higher currently possible for the BV group. Earlier work has shown among larger genome segments (>5 kb: 38.9%) than among that at least one gene family, the rep family, is shared by three smaller ones (<5 kb: 26.8%; see Fig. 2). IV species (Volkoff et al., 2002). In addition, we wanted to assess the feasibility of sequencing an entire IV genome (TrIV The TrIV genome in this case) using material obtained from a wild wasp We completed the sequencing of 20 TrIV genome segments, population because the documented occurrence of polymor- which included genome segment F whose sequence had been phism in PDV genomes (Stoltz and Xu, 1990) was expected to reported earlier and shown to contain members of the rep gene make genome assembly more difficult. To conduct work on family (Volkoff et al., 2002). These 20 genome segments make TrIV, we have had to rely on yearly field collections of up 130,961 bp, to which we must add seven partially (∼60%) parasitized host larvae inasmuch as maintenance of a T. rostrale sequenced and annotated genome segments (Fig. 1A) as well as colony in the laboratory has so far proven impossible (Cusson five ORF-containing sequences that could not be assigned to et al., 1998a). specific genome segments. Thus, although the sequenced In selecting the viruses of H. fugitivus and T. rostrale, which portions of the TrIV genome add up to 176,243 bp, we estimate are parasitoids of the forest tent caterpillar Malacosoma disstria at ∼200 kb the aggregate size of the 27 genome segments (Stoltz et al., 1986) and the spruce budworm Choristoneura characterized here, a value obtained by computing the full sizes fumiferana (Cusson et al., 1998a), respectively, we also wanted of the partially sequenced genome segments, which were to determine whether differences in pathological effects are assessed by gel electrophoresis of the associated EZ∷TN clones reflected in viral gene composition. Unlike other IVs charac- (data not shown). Many additional small contigs, totaling terized to date, TrIV has been shown to have little or no apparent ∼75 kb (most of which contained no detectable ORFs), have impact on the host cellular immune response, although it not yet been assigned to any genome segment, but several of induces a strong inhibition of caterpillar metamorphosis these are expected to partially fill the ∼25 kb gaps of the seven (Cusson et al., 1998b; Doucet and Cusson, 1996a, 1996b).