Marjolein Couvreur

A New Class of Ubiquitin Extension Proteins Secreted by the Dorsal Pharyngeal Gland in Plant Parasitic Cyst Nematodes

By performing cDNA AFLP on pre- and early parasitic juveniles, we identified genes encoding a novel type of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in the cyst nematode Heterodera schachtii. The proteins consist of three domains, a signal peptide for secretion, a mono-ubiquitin domain, and a short C-terminal positively charged domain. A gfp-fusion of this protein is targeted to the nucleolus in tobacco BY-2 cells. We hypothesize that the C-terminal peptide might have a regulatory function during syncytium formation in plant roots.

The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification

BackgroundGlobin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles.ResultsIn the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection.ConclusionOur results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying selection subsequently dampened further evolution and facilitated fixation of the duplicated genes in the genome.

A new preparation method to study fresh plant structures with X-ray computed tomography

Since the development of X‐ray computed tomography as a medical diagnostic tool, it was adapted and extended for many scientific applications, including plant structure research. As for many biological studies, sample preparation is of major importance to obtain good‐quality images. Therefore, we present a new preparation method for fresh material which includes critical point drying and heavy metal staining. This technique enhances the contrast of fresh tissues, prevents artefacts such as tissue compression, and requires no embedding.

High-pressure freezing and freeze substitution of gravid Caenorhabditis elegans (Nematoda: Rhabditida) for transmission electron microscopy

Because chemical fixatives have a profound negative influence on tissue morphology and antigenicity, alternative fixation methods must be applied for some purposes. In this work we used high-pressure freezing (HPF) followed by freeze substitution to maximally preserve antigenicity and morphology. We developed a pipette method for bringing living Caenorhabditis elegans nematodes into the HPF recipient. Using cellulose tubes, it is possible to select individual nematodes for fixation. We were able to HPF complete adults and preserve the morphology in an enhanced fashion compared to chemically fixed tissue. Cellular organelles, especially mitochondria, were much better preserved. Uterine embryos protected by the intact eggshell were excellently preserved without the need for elaborate techniques. Antigenicity with MH27 and ICB4 antisera was tested. With the MH27 serum, an adequate, reproducible, specific binding pattern with chemically fixed tissue could only be achieved using purified antibodies, whereas with highpressure freezing, unpurified MH27 antisera was effective. For ICB4 antisera, a reproducible specific binding pattern was achieved at a concentration of primary antiserum 1000 × lower than that for chemically fixed tissue.

Soft dentin results in unique flexible teeth in scraping catfishes.

Teeth are generally used for actions in which they experience mainly compressive forces acting toward the base. The ordered tooth enamel(oid) and dentin structures contribute to the high compressive strength but also to the minor shear and tensile strengths. Some vertebrates, however, use their teeth for scraping, with teeth experiencing forces directed mostly normal to their long axis. Some scraping suckermouth catfishes (Loricariidae) even appear to have flexible teeth, which have not been found in any other vertebrate taxon. Considering the mineralized nature of tooth tissues, the notion of flexible teeth seems paradoxical. We studied teeth of five species, testing and measuring tooth flexibility, and investigating tooth (micro)structure using transmission electron microscopy, staining, computed tomography scanning, and scanning electron microscopy–energy-dispersive spectrometry. We quantified the extreme bending capacity of single teeth (up to 180°) and show that reorganizations of the tooth (micro)structure and extreme hypomineralization of the dentin are adaptations preventing breaking by allowing flexibility. Tooth shape and internal structure appear to be optimized for bending in one direction, which is expected to occur frequently when feeding (scraping) under natural conditions. Not all loricariid catfishes possess flexible teeth, with the trait potentially having evolved more than once. Flexible teeth surely rank among the most extreme evolutionary novelties in known mineralized biological materials and might yield a better understanding of the processes of dentin formation and (hypo)mineralization in vertebrates, including humans.

Embryonic origins of hull cells in the flatworm Macrostomum lignano through cell lineage analysis: developmental and phylogenetic implications

The development of macrostomid flatworms is of interest for evolutionary developmental biology research because these taxa combine characteristics of the canonical spiral cleavage pattern with significant deviations from this pattern. One such deviation is the formation of hull cells, which surround the remaining embryonic primordium during early development. Using live observations with a 4D microscope system, histology, and 3D reconstructions, we analyzed the ontogeny of these hull cells in the macrostomid model organism Macrostomum lignano. Our cell lineage analysis allowed us to find the precursors of the hull cells in this species. We discuss the relation between macrostomid development and the development of other spiralians and the question of whether hull cells are homologous within rhabditophoran flatworms.

The pitfalls of molecular species identification : a case study within the genus Pratylenchus (Nematoda: Pratylenchidae)

Comprehensive morphological and molecular analyses revealed that published ITS sequences of the economically important plant-parasitic nematode Pratylenchus goodeyi are actually sequences from distantly free-living bacterivorous ‘cephalobids’. We demonstrated that this incorrect labelling resulted in a cascade of erroneous interpretations, as shown by the reports of ‘ P. goodeyi ’ on banana in China and on cotton in India. This clearly illustrates the risk of mislabelled sequences in public databases. Other mislabelled Pratylenchus cases are discussed to illustrate that this is not an isolated case. Herein, P. lentis n. syn. is considered a junior synonym of P. pratensis while P. flakkensis was for the first time linked to DNA sequences using topotype material. As taxonomic expertise is decreasing and sequence-based identification is growing rapidly, the highlighted problem may yet increase and a strong link between morphology and DNA sequences will be of crucial importance in order to prevent, or at least minimise, sequence-based misidentifications.

mtCOI successfully diagnoses the four main plant-parasitic Aphelenchoides species (Nematoda: Aphelenchoididae) and supports a multiple origin of plant-parasitism in this paraphyletic genus

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides, revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.

Deceptive morphological variation in Hirschmanniella mucronata (Nematoda: Pratylenchidae) and a polytomous key to the genus

Hirschmanniella mucronata populations isolated from two Cambodian provinces were characterised using morphological, morphometric and molecular criteria. Examination of 1024 specimens from 60 different paddy fields revealed high intraspecific variation in morphology and morphometrics, especially in tail terminus shape and stylet length. Sequence results confirmed that morphologically divergent individuals represent a single species, suggesting that neglecting morphological variation has led to an overestimation of Hirschmanniella diversity in former studies. Phylogenetic analysis of the SSU, D2-D3, LSU and ITS1-5.8S-ITS2 regions revealed three concordant clades, H. mucronata having a sister relationship with H. kwazuna and H. loofi. Plotting the diagnostic features, including tail terminus shape, stylet length and lip region morphology on the phylogenetic framework, revealed that none of them supported the clades and represented convergent features. All three molecular markers were able to discriminate all Hirschmanniella species, but the D2-D3 region was the easiest, fastest and most successful region to be amplified. Species delimitation and the diagnostic features of Hirschmanniella were re-evaluated. Hirschmanniella abnormalis and H. exacta are considered to be junior synonyms of H. oryzae and H. mannai a species inquirenda. Finally, a list of valid species with indication of synonyms and a polytomous key are provided.

Steinernema kandii n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from northern Benin

Two nematode isolates from the genus Steinernema were collected in northern Benin. Morphological, morphometric, molecular and cross-hybridisation studies placed these nematodes into a new species, Steinernema kandii n. sp., within the bicornutum-group. Phylogenetic analyses based on both ITS and D2-D3 regions of 28S rDNA revealed that S. kandii n. sp. is different from all known Steinernema species and sister to S. abbasi (97.3-97.6% ITS nucleotide similarity) and S. bifurcatum (98.3-98.4% D2-D3 similarity). Steinernema kandii n. sp. can be separated from other members of the bicornutum-group by the greater infective juvenile (IJ) max. body diam. of 35 (27-48) μm (type isolate). It differs from S. abbasi by the greater IJ body length 707 (632-833) μm (type isolate), EP distance 55 (52-60) μm (type isolate), spicule length 67 (57-75) μm (type isolate) and the occurrence of one pair of genital papillae at the cloacal aperture.