The D. immitis
genome sequence described here is only the second to be determined for an onchocercid nematode, despite the social and economic importance of these parasites. Three genomes were cosequenced: the mitochondrial (at ~4000-fold read coverage of the 13.6-kb genome; this had been determined previously; ref. 17
); the genome of the Wolbachia
symbiont wDi (at ~1000-fold coverage of the 0.9-Mb genome); and the nuclear genome (at ~150-fold coverage of the estimated 95-Mb genome). We used high-throughput, short-read Illumina technology, stringent quality filtering and optimized assembly methods to derive genomes of good draft quality (73
). After redundancy reduction, the span of the nuclear assembly was 84.2 Mb, slightly smaller than the 88.3 Mb assembled for B. malayi
). Overall, although the number of scaffolds was approximately equivalent, the contiguity of the D. immitis
genome assembly was lower than that of B. malayi
, because of the availability of long-range scaffolding information for the latter species. The predicted nuclear gene set was much smaller than that of C. elegans
, but of a size similar to that of B. malayi
. The two onchocercid nematodes also have a lower proportion of species-unique proteins. These two differences may be a feature of the Onchocercidae, because the unpublished L. loa
genome has only 15,444 predicted proteins (Filarial Worms Sequencing Project, Broad Institute of Harvard and MIT; http://www.broadinstitute.org/
). Another possibility is that the richer analytic environment for C. elegans
in particular has permitted the identification of many unique genes using biological evidence (such as transcript information). We will continue to develop and improve the assembly and annotation of D. immitis
and wDi as additional tools and biological resources become available.
Two peculiarities of the assembled D. immitis
genome are striking: the lack of genetic diversity and the lack of active transposable elements. The lack of diversity was convenient, in that it allowed us to pool data obtained from two different D. immitis
isolates, one from Pavia, Italy, and the other from Athens, Georgia, USA. Polymorphisms called from the independent sequencing of the two isolates yielded a per-nucleotide diversity of 0.04%. Both sequenced isolates fall within the single eastern United States population defined by microsatellite analyses (36
). The hypovariability may be a result of the recent admixture of European and American heartworm populations through movement of domestic animals or arise from the very recent introduction of heartworm into the New World by Europeans (74
). The first report of dirofilariasis in the United States dates from only 1847, as opposed to a 1626 observation from Italy. The lack of genetic diversity in the nuclear genome will make identification of mutations conferring drug resistance much easier. The lack of DNA transposons and active retrotransposons in D. immitis
is a strong negative result, because active elements are easy to identify (they are present in multiple, highly similar copies). We identified only fragmented and functionally inactivated segments of Pao-type retrotransposons, similar to those found in and probably still active in B. malayi
. To our knowledge, this is the first metazoan genome devoid of active transposable elements. The presence of putatively active Pao elements in B. malayi
suggests that their loss was an evolutionary recent event in D. immitis
The Wolbachia wDi genome, with 823 predicted proteins, complements the D. immitis nuclear genome in that it encodes enzymes for anabolic pathways that are missing in the latter, e.g., biosynthesis of heme, purine, or pyrimidines (). In contrast to wBm, wDi also carries the genes for folate synthesis, suggesting that folate too might be supplied by the endosymbiont. However, essential metabolites could also be taken up from the mammalian or insect host, and so it remains to be shown whether such metabolites are actually delivered from wDi to D. immitis. Analysis of orthology between wBm and wDi revealed that both organisms possess many unique genes (approximately one-third of the total gene complement of each genome). The representation of genes in the different COG categories was similar for wBm and wDi, suggesting that most gene losses occurred before the split of the two lineages or that there have been no biases in gene losses/acquisition after the evolutionary separation. Analysis of protein distances revealed that proteins involved in cell wall/membrane biogenesis (COG category M) displayed more variation between the two organisms compared with the other functional categories. It is reasonable to conclude that the interface between the symbiotic bacterium and the host environment is a place where evolutionary rates are elevated, either as part of an arms race underpinning conflict between the two genomes or as a feature of the dynamic exploitation of the interface in adaptation of the symbiosis. In any case, the endosymbiont, being essential for proliferation of D. immitis, represents a target for control of the heartworm. Screening the predicted wDi proteome returned expected antibiotic drug targets such as Fts and Sec proteins, but also the products of the Mur operon required for peptidoglycan synthesis.
Many of the anthelmintics used in human medicine were originally developed for the veterinary sector. We pursued two approaches to identify potential drug targets in D. immitis
: top-down, starting from the known anthelmintic targets of C. elegans
(), and bottom-up, narrowing down the predicted D. immitis
proteome to a list of essential, unique, and druggable targets (). Although the majority of the current anthelmintics activate their target (thereby interfering with synaptic signal transduction), the aim of the second approach was to identify inhibitable targets. The criteria applied—presence of an essential ortholog in C. elegans
, absence of any significantly similar protein in human or dog, and absence of paralogs in D. immitis
—admittedly missed many of the known anthelmintic targets, e.g.
, proteins that are not conserved in C. elegans
or that possess a mammalian ortholog. The aim of the approach was to maximize the specificity of in silico
target prediction at the cost of low sensitivity. Our goal was to end up with a manageable, rather than complete, list of unique D. immitis
proteins that are likely to be essential and druggable. Some of the candidates identified are worth further investigation, based on their presumed role in signal transduction, e.g.
, the nematode-specific G protein-coupled receptors or hedgehog proteins (). Others have already been validated as drug targets in other systems: sterol-C-24-methyltransferase (EC 220.127.116.11) is a target of sinefungin, chitin synthase (EC 18.104.22.168) is the target of the insecticide lufenuron, and the mannosyltransferase bre-3 is required for interaction of Bacillus thuringiensis
toxin with intestinal cells (52
). The discovery of new D. immitis
drug targets would be timely because resistance to macrocyclic lactones has recently been reported from the southern United States (75
Filarial nematodes modulate the immune systems of their hosts in complex ways that result in an apparently intact immune system that ignores a large parasite residing, sometimes for decades, in tissues or the bloodstream. They may also require intact immune systems to develop properly (76
). Often immune responses result in a pathologic condition for the host in addition to parasite clearance, and Wolbachia
may exacerbate these responses (12
). We identified a wide range of putative immunomodulatory molecules and, in addition, highlight two D. immitis
products that may deflect or distract the host immune response: one similar to SOCS5 and the other similar to IL-18. The host-encoded versions of both of these molecules have been implicated in antifilarial immune responses. Strategies for development of a vaccine against filariases depend on delivering the correct antigens to the right arm of the immune system, avoiding induction of dangerous responses, and deflecting or stopping immune suppression by the parasite. We identified homologs of all the current roster of filarial vaccine candidates in our genome, and these can now be moved rapidly into testing in the dog heartworm model. In addition, we defined a large number of potentially secreted D. immitis
proteins that may contribute to the host-parasite interaction and also be accessible to the host immune system.
Onchocercid parasites share not only a fascinating biology involving immune evasion, arthropod vectors, and Wolbachia endosymbionts but also a pressing need for new drugs, improved diagnostic methods, and, ideally, vaccines. We hope that the genome sequence of the heartworm presented here will contribute to an increased understanding of its biology and to new leads for control.