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Janina Schenk, Karin Hohberg, Johannes Helder, Kai Ristau and Walter Traunspurger

Reliable and well-developed DNA barcode databases are indispensable for the identification of microscopic life. However, effectiveness of molecular barcoding in identifying terrestrial specimens, and nematodes in particular, has received little attention. In this study, ca 600 ribosomal large subunit DNA fragments (D3-D5 region) were successfully amplified for 79 limnic and terrestrial nematode species sampled at 147 locations across Germany. Distinctive DNA motifs in the LSU region were identified in 80% of all species examined. For 13 supposedly single morphospecies, 2-7 LSU barcode groups were detected with a wide range of intraspecific variations (0.09-7.9%). This region seems to be more suitable for the assessment of limno-terrestrial nematode diversity than the frequently used mitochondrial gene COI, as amplification success of the latter fragment is low for several nematode species. Our reference database for nematodes may serve as a starting point for applied and fundamental studies for these ubiquitous, ecologically highly relevant, organisms.

Kourosh Azizi, Ali Eskandari, Akbar Karegar, Reza Ghaderi, Sven van den Elsen, Martijn Holterman and Johannes Helder

The genus Pratylenchoides has recently been transferred from the family Pratylenchidae to Merliniidae. To investigate further the relationship between these ‘Pratylenchus-like’ species (residing in the subfamily Pratylenchoidinae) and the subfamily Merliniinae, more than 500 soil samples were collected from various natural and agronomic habitats in the northern and north-western provinces of Iran. In this study, paratypes or populations of 22 species of Pratylenchoides, including the Iranian populations of P. alkani, P. crenicauda, P. erzurumensis, P. laticauda, P. nevadensis, P. ritteri and an undescribed species, were studied. Intra- and interspecies variation of the following characters were investigated: position of the pharyngeal gland nuclei, shape of female and male head, striation of female tail terminus, number of lateral lines at mid-body and in phasmid region for females, presence of intestinal fasciculi, and shape of sperm. Combining morphological and molecular data prompted us to propose two clusters of related Pratylenchoides species. One cluster includes P. crenicauda, P. variabilis and P. erzurumensis, whereas the second cluster consists of P. alkani, P. nevadensis and P. ritteri. Our data point to a sister positioning of P. magnicauda vis-à-vis all Pratylenchoides species included in this research. Analyses of SSU rDNA (for family and subfamily relationships) and partial LSU rDNA sequences (for intrageneric relationships) data revealed: i) the distal and nested positioning of all Pratylenchoidinae within the Merliniidae; ii) the single transition from ectoparasitism to migratory endoparasitism within the family Merliniidae corresponds with the current subfamily partitioning; and iii) support for the monophyletic nature of the genus Pratylenchoides.

Mark Phillips, Vivian Blok, Linda Cardle, Johannes Helder, Geert Smant, John Jones, Herman Popeijus and Erin Bakker


Expressed sequence tag (EST) projects offer a rapid route to the discovery of novel genes. Genes expressed in a wide range of parasitic nematodes of medical or veterinary importance have been investigated using EST analysis but these techniques have not yet been applied to plant parasitic nematodes. We describe a small scale EST project using cDNA libraries made from the two species of potato cyst nematode, Globodera rostochiensis and G. pallida, and assess the utility of this approach to identify mRNAs encoding abundantly expressed secreted proteins and other proteins present in the nematode at the onset of parasitism. Approximately 1000 sequences were obtained from G. rostochiensis and 100 from G. pallida. A variety of genes was characterised and approximately 11% of the cDNAs sequenced were apparently PCN specific. Secreted proteins identified included a novel PCN homologue of chorismate mutase, a cDNA recently cloned from the gland cells of Meloidogyne javanica. The results obtained justify a much larger scale application of this technology to these parasites. Utilisation de L'Expressed Sequence Tag pour l'analyse de gènes s'exprimant chez les juvéniles de deuxième stade des nématodes à kyste de la pomme de terre Globodera rostochiensis et G. pallida - l'Expressed Sequence Tag (EST) ouvre une voie rapide vers la découverte de nouveaux gènes. Des gènes s'exprimant chez un large éventail de nématodes parasites d'importance médicale ou vétérinaire ont été étudiés par analyse EST alors que cette technique n'a pas encore été appliquée aux nématodes phytoparasites. Nous décrivons ici un projet EST à petite échelle utilisant les banques d'ADNc constituées à partir de deux espèces de nématodes à kyste de la pomme de terre (PCN), Globodera rostochiensis et G. pallida et nous évaluons l'utilité de cette approche pour identifier les ARNs codant les protéines abondamment sécrétées - et les autres protéines - présentes chez les nématodes lors de l'attaque parasitaire. Mille séquences environ ont été obtenues chez G. rostochiensis et 100 chez G. pallida. Des gènes variés ont été caractérisés et environ 11% des ADNc séquencés sont apparemment spécifiques des PCN. Parmi les protéines sécrétées identifiées figurent un nouvel homologue PCN de la chorismate mutase ainsi qu'un ADNc récemment cloné à partir de cellules glandulaires de Meloidogyne javanica. Les résultats ainsi obtenus justifient une utilisation à plus grande échelle de l'EST pour l'étude de ces parasites.

Tiasei Kikuchi, Peter J.A. Cock, Johannes Helder and John T. Jones

While the majority of Aphelenchoides species are fungivorous, some species are plant parasites that have retained the ability to feed on fungi. Aphelenchoides besseyi is an important and widespread pathogen that causes ‘white tip’ disease on rice. This migratory endoparasitic nematode makes a significant contribution to the estimated $US 16 billion worth of damage caused by nematodes to rice crops. Here we describe a small-scale analysis of the transcriptome of A. besseyi. After sequencing, QC and assembly, approximately 5000 contigs were analysed. Bioinformatic analysis allowed 375 secreted proteins to be identified, including orthologues of proteins known to be secreted by other nematodes. One contig could encode an A. besseyi orthologue of a GHF45 cellulase, similar to those present in Bursaphelenchus xylophilus. No transcripts similar to GHF5 cellulases were present in this dataset.

Katarzyna Rybarczyk-Mydłowska, Hanny van Megen, Sven van den Elsen, Paul Mooyman, Gerrit Karssen, Jaap Bakker and Johannes Helder

In 2000 Siddiqi formulated a hypothesis stating that root-knot nematodes (Meloidogyne spp.) constitute a branch arising from yet another important group of plant parasites, the migratory Pratylenchidae. This hypothesis was solely based on morphological characteristics. Ribosomal DNA (rDNA) sequence analysis supports this hypothesis in its broad sense, but the more precise question about the identity of a migratory Pratylenchidae representative being closest to the most basal Meloidogyne species could not be addressed due to a lack of backbone resolution (Holterman et al., 2009). Here we present an extended small subunit rDNA sequence analysis and a data set of partial RNA polymerase II sequences from Pratylenchidae and basal Meloidogynidae. Our data point at members of the genus Pratylenchus as being closest to the common ancestor of the root-knot nematodes, but it was not possible unequivocally to identify a candidate lesion nematode species. Pratylenchus is a species-rich genus (ca 70 valid species), and we suggest that the species closest to the most basal root-knot nematode should be sought outside of the group of relatively well-characterised, agronomically relevant, species.

Jaap Bakker, Agnieszka Doroszuk, Andre van der Wurff, Martijn Holterman, Tom Bongers, Hanny van Megen, Johannes Helder and Gerard Korthals

Sven van den Elsen, Oleksandr Holovachov, Gerrit Karssen, Hanny van Megen, Johannes Helder, Tom Bongers, Jaap Bakker, Martijn Holterman and Paul Mooyman


As a result of the scarcity of informative morphological and anatomical characters, nematode systematics have always been volatile. Differences in the appreciation of these characters have resulted in numerous classifications and this greatly confuses scientific communication. An advantage of the use of molecular data is that it allows for an enormous expansion of the number of characters. Here we present a phylogenetic tree based on 1215 small subunit ribosomal DNA sequences (ca 1700 bp each) covering a wide range of nematode taxa. Of the 19 nematode orders mentioned by De Ley et al. (2006) 15 are represented here. Compared with Holterman et al. (2006) the number of taxa analysed has been tripled. This did not result in major changes in the clade subdivision of the phylum, although a decrease in the number of well supported nodes was observed. Especially at the family level and below we observed a considerable congruence between morphology and ribosomal DNA-based nematode systematics and, in case of discrepancies, morphological or anatomical support could be found for the alternative grouping in most instances. The extensiveness of convergent evolution is one of the most striking phenomena observed in the phylogenetic tree presented here – it is hard to find a morphological, ecological or biological characteristic that has not arisen at least twice during nematode evolution. Convergent evolution appears to be an important additional explanation for the seemingly persistent volatility of nematode systematics.

Myriam Claeys, Nurul Dwi Handayani, Vladimir V. Yushin, Prabowo Lestari, Antarjo Dikin, Johannes Helder and Wim Bert


Ultrastructural analysis of the early development of nematodes is hampered by the impermeability of the eggshell to most commonly used fixatives. High-pressure freezing (HPF), a physical cryo-fixation method, facilitates a fast rate of fixation, and by using this method the issue of the uneven delivery of fixative is circumvented. Although HPF results in a superior preservation of the fine structure, the equipment costs impede a wider application of this method. Self-pressurised rapid freezing (SPRF) is an alternative low-cost cryo-fixation method, and its usefulness was evaluated in an ultrastructural study of the eggshell and the cuticle of unhatched second-stage juveniles (J2) of Globodera rostochiensis and Heterodera schachtii. A comparison with conventional (chemical) fixation demonstrates that SPRF fixation results in a remarkably well-preserved ultrastructure of the entire egg including both the eggshell and the cellular details of the unhatched J2. Therefore, SPRF fixation is proposed as an affordable, relatively easy-to-use and time-efficient technique to study the ultrastructure of unhatched J2 and eggs of nematodes.