Natsumi Kanzaki

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Evaluating high‐throughput sequencing as a method for metagenomic analysis of nematode diversity

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time‐consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400‐bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.

A PCR primer set for determination of phylogenetic relationships of Bursaphelenchus species within the xylophilus group

The phylogenetic relationships of Bursaphelenchus conicaudatus with B. abruptus, B. fraudulentus, B. mucronatus and B. xylophilus species were analysed based on the DNA base sequences of 18S, 5.8S, ITS1 and ITS2 of rDNA and the partial code of mitochondrial cytochrome oxidase subunit I (COI) gene. The monophyly of B. abruptus and the other species in the xylophilus group was not supported, and B. abruptus could be excluded from the xylophilus group. The other species, i.e ., B. conicaudatus, B. fraudulentus, B. mucronatus and B. xylophilus were assumed to be monophyletic. The ancestor of these four species is assumed to have originated in the eastern part of the Eurasian continent as a free-living nematode inhabiting broad-leaved trees. First, B. conicaudatus branched from the ancestor in eastern Asia. Then B. fraudulentus and B. mucronatus separated from the ancestor. B. mucronatus changed its host from broad-leaved trees to conifers and spread throughout the coniferous forest over the Eurasian continent and North America. B. xylophilus might originate from a population of B. mucronatus remaining in North America after B. mucronatus had diversified.

A Rapid and Precise Diagnostic Method for Detecting the Pinewood Nematode Bursaphelenchus xylophilus by Loop-Mediated Isothermal Amplification

ABSTRACT Bursaphelenchus xylophilus is the causal agent of pine wilt disease, which is a major forest disease in Japan, Korea, China, Taiwan, and Portugal. A diagnostic method which is rapid, precise, and simple could greatly help the proper management of this disease. Here, we present a novel detection method using loop-mediated isothermal amplification (LAMP) targeting the internal transcribed spacer region of ribosomal DNA of the nematode. Specificity of the primers and LAMP was confirmed using DNA from various nematode species related to B. xylophilus. Our experimental results suggest that LAMP can detect B. xylophilus faster and with higher sensitivity than the traditional diagnostic method. Moreover, because it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions.

Tropical nematode diversity: vertical stratification of nematode communities in a Costa Rican humid lowland rainforest

Comparisons of nematode communities among ecosystems have indicated that, unlike many organisms, nematode communities have less diversity in the tropics than in temperate ecosystems. There are, however, few studies of tropical nematode diversity on which to base conclusions of global patterns of diversity. This study reports an attempt to estimate nematode diversity in the lowland tropical rainforest of La Selva Biological Research Station in Costa Rica. We suggest one reason that previous estimates of tropical nematode diversity were low is because habitats above the mineral soil are seldom sampled. As much as 62% of the overall genetic diversity, measured by an 18S ribosomal barcode, existed in litter and understorey habitats and not in soil. A maximum‐likelihood tree of barcodes from 360 individual nematodes indicated most major terrestrial nematode lineages were represented in the samples. Estimated ‘species’ richness ranged from 464 to 502 within the four 40 × 40 m plots. Directed sampling of insects and their associated nematodes produced a second set of barcodes that were not recovered by habitat sampling, yet may constitute a major class of tropical nematode diversity. While the generation of novel nematode barcodes proved relatively easy, their identity remains obscure due to deficiencies in existing taxonomic databases. Specimens of Criconematina, a monophyletic group of soil‐dwelling plant‐parasitic nematodes were examined in detail to assess the steps necessary for associating barcodes with nominal species. Our results highlight the difficulties associated with studying poorly understood organisms in an understudied ecosystem using a destructive (i.e. barcode) sampling method.

Description of Three Pristionchus Species (Nematoda: Diplogastridae) from Japan that Form a Cryptic Species Complex with the Model Organism P. pacificus

Three new species of Pristionchus (P. exspectatus, P. arcanus, and P. japonicus) are described from Japan. They are morphologically similar, with P. exspectatus and P. arcanus being almost indistinguishable from the model organism P. pacificus. Reproductive isolation, namely the inability to produce interfertile F1 hybrids, separates all species pairs in the species complex. Additionally, all three new species are distinguished from P. pacificus Sommer, Carta, Kim, and Sternberg, 1996 by having a gonochoristic instead of hermaphroditic mode of reproduction. In addition to its reproductive isolation, P. japonicus is distinct from other Pristionchus species by its arrangement of genital papillae. All species in the complex are separated from each other by molecular sequence divergence, as indicated by analysis of 27 nuclear protein-coding genes and unique sequences of the small subunit ribosomal RNA gene. The identification of a species complex that includes P. pacificus is invaluable for studies of population genetics, speciation, and macroevolution, particularly the evolution of hermaphroditism in the genus.

Rapid diversification associated with a macroevolutionary pulse of developmental plasticity

Developmental plasticity has been proposed to facilitate phenotypic diversification in plants and animals, but the macroevolutionary potential of plastic traits remains to be objectively tested. We studied the evolution of feeding structures in a group of 90 nematodes, including Caenorhabditis elegans, some species of which have evolved a mouthpart polyphenism, moveable teeth, and predatory feeding. Comparative analyses of shape and form, using geometric morphometrics, and of structural complexity revealed a rapid process of diversification associated with developmental plasticity. First, dimorphism was associated with a sharp increase in complexity and elevated evolutionary rates, represented by a radiation of feeding-forms with structural novelties. Second, the subsequent assimilation of a single phenotype coincided with a decrease in mouthpart complexity but an even stronger increase in evolutionary rates. Our results suggest that a macroevolutionary ‘pulse’ of plasticity promotes novelties and, even after the secondary fixation of phenotypes, permits sustained rapid exploration of morphospace. DOI: http://dx.doi.org/10.7554/eLife.05463.001

A nested PCR-based method for detecting the pine wood nematode, Bursaphelenchus xylophilus , from pine wood

Several methods were examined for extracting DNA from the woody tissues of Japanese black pine, including the general CTAB method which proved to be the most successful. To detect the pine wood nematode, the causal agent of pine wilt disease (PWD) in pine wood tissues, PWN-specific PCR was done using a DNA mixture obtained from 80 mg of pine wood tissues and a single pine wood nematode as the DNA template. The results showed that nested PCR could amplify the ITS region specific to the PWN, even though the amount of nematode DNA was extremely small. Also, the PWN could be detected in diseased tissues of several Pinus spp. which had been inoculated with nematodes. The method described here can also be used as a simple and quick detection method for a wide range of wood-inhabiting pathogens.

Pseudaphelenchus yukiae n. gen., n. sp. (Tylenchina: Aphelenchoididae) associated with Cylindrotermes macrognathus (Termitidae: Termitinae) in La Selva, Costa Rica

A species of aphelenchoidid nematode was isolated from a subterranean termite, Cylindrotermes macrognathus, during a survey of termite-associated nematodes in a conserved forest in La Selva, Costa Rica. The nematode was morphologically intermediate between the families Aphelenchidae and Aphelenchoididae, i.e., the nematode had a true bursa supported by bursal limb-like genital papillae but lacked a clear pharyngeal isthmus. The molecular phylogenetic status of the new nematode among tylenchid, cephalobid, panagrolaimid, aphelenchid and aphelenchoidid genera was analysed based on ca 1.2 kb of SSU ribosomal DNA sequence and the inferred position was basal to the family Aphelenchoididae. It was clearly not part of the clade containing the genus Aphelenchus (=Aphelenchidae). This nematode is described herein as Pseudaphelenchus yukiae n. gen., n. sp., and the family definition of Aphelenchoididae is emended to include the unique morphological characters of this new genus. The molecular phylogenetic analysis supported the paraphyly of the three Aphelenchoidinae genera Aphelenchoides, Laimaphelenchus and Schistonchus and the monophyly of Ektaphelenchinae, Seinura (Seinurinae) and Noctuidonema (Acugutturinae). However, many more representatives are needed to resolve the family-genus level phylogeny of Aphelenchoididae.

Phylogeography of Coptotermes gestroi and Coptotermes formosanus (Isoptera: Rhinotermitidae) in Taiwan.

ABSTRACT The Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), is primarily reported from subtropical and warm temperate regions, whereas Coptotermes gestroi (Wasmann) is reported from many areas of the tropics. Taiwan is one of a few areas where the distributions of the two species overlap. By analyzing partial mitochondrial sequences of COII, 12S rRNA, and 16S rRNA, we found that most Taiwanese C. formosanus populations were closely related to Japanese and some Chinese populations and that Taiwanese C. gestroi populations were most closely related to those from the Philippines and Hawaii rather than populations from Thailand, Malaysia, Singapore, and Indonesia. The intraspecific variation of C. formosanus was 0.7–0.8% for three genes among seven Taiwanese populations, whereas all Taiwanese C. gestroi populations had identical sequences. The results support that Taiwan may be a center of origin for C. formosanus, but a recent introduction site for C. gestroi.