August Coomans

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.

Present status and future of nematode systematics

Since phylogenetic systematics became generally accepted and especially since informatics and molecular techniques for phylogenetic analysis were developed, systematics has undergone a conceptual and methodological revolution. Taxonomy, on the contrary, suffered a decline. Poor descriptions, too much routine work and low citation rates hampered it. As a result, the discipline became less attractive to young scientists. With only a small fraction of the biodiversity known, this situation will lead to serious problems in the future in all those fields of nematology depending on a correct identification of species. Phylogenetic analyses of nematodes have been mainly based on morphology, supplemented with developmental characters, but in recent years molecular methods have provided entirely new data sets. Phylogenetic estimates derived from independent data may provide new insights in character homologies through reciprocal illumination. Classifications of nematodes were often biased according to the expertise of the author and were only recently based on the principles of phylogenetic systematics. Recently, molecular and morphological data have been used to support a new overall classification with only (presumably) monophyletic taxa. In this classification plant, as well as animal, parasitic taxa are hierarchically downgraded in accordance with their phylogenetic history. Species occupy an important position in all aspects of biology, therefore the species concept matters. It determines, for example, the outcome of biodiversity assessments, distribution patterns, etc. However, several problems remain to be solved before a consensus will be reached about the choice of a concept. Future research in nematode systematics should comprise well-focused taxonomy based on a combination of classical and modern methods in a way that can raise the interest of young scientists as well as of funding agencies. It should be realised that, with the dwindling present taxonomic expertise, this is a very urgent matter.

Nematode systematics: past, present and future.

A brief overview is given of nematode taxonomy since its beginnings in the second half of the 19th century. Most of the work concerned α-taxonomy, but in the thirties and subsequently from the sixties several classifications have been proposed. Since the eighties phylogenetic systematics was introduced as the underlying theory. Due to the pioneering work of several famous nematode taxonomists in the first half of the 20th century an increased interest for the discipline arose. Very unfortunately, this interest decreased in more recent times to an alarming extent. The difficulty to correctly identify and to describe the rapidly increasing numbers of taxa has discouraged many young scientists and diverted them toward more appealing disciplines. Taxonomy provides nevertheless the basic knowledge for studies on biodiversity as well as for analyses of ecological and phylogenetic relationships. Molecular techniques have made systematics again exciting; when combined with the more traditional morphological approach, they will lead to new insights. Hopefully, these will arouse the interest of young scientists. Even then the future of nematode taxonomy will depend on the funding opportunities provided by national and international authorities. La systematique des nematodes est brievement revue depuis ses origines dans la seconde moitie du 19eme siecle. La plupart des travaux sont relatifs l’ α-taxinomie, mais dans les annees trente et ensuite dans les annees soixante, plusieurs classifications ont ete proposees. Depuis les annees 80, la systematique phylogenetique a ete introduite comme la theorie sous-jacente. Grâce au travail de pionnier de plusieurs taxinomistes celebres dans la premiere moitie du 20eme siecle, un interet accru est apparu pour la discipline. Tres malheureusement, cet interet a decru recemment jusqu’a un niveau alarmant. La difficulte d’identifier correctement et de decrire un nombre de taxons augmentant rapidement a decourage de nombreux jeunes scientifiques et les a pousse vers des disciplines plus attirantes. La taxinomie cependant apporte la connaissance de base tant pour des etudes de biodiversite que pour des etudes de relations ecologiques et phylogenetiques. Les techniques moleculaires ont rendu la systematique a nouveau attractive; lorsqu’elles seront combinees avec l’approche morphologique plus traditionnelle, elles conduiront a de nouveaux developpements. Heureusement, elles vont reveiller l’interet des jeunes chercheurs. Malgre cela, le futur de la taxinomie des nematodes dependra des opportunites de financement par les autorites nationales et internationales.

Embryonic cell lineage of the marine nematode Pellioditis marina.

We describe the complete embryonic cell lineage of the marine nematode Pellioditis marina (Rhabditidae) up to somatic muscle contraction, resulting in the formation of 638 cells, of which 67 undergo programmed cell death. In comparison with Caenorhabditis elegans, the overall lineage homology is 95.5%; fate homology, however, is only 76.4%. The majority of the differences in fate homology concern nervous, epidermal, and pharyngeal tissues. Gut and, remarkably, somatic muscle is highly conserved in number and position. Partial lineage data from the slower developing Halicephalobus sp. (Panagrolaimidae) reveal a lineage largely, but not exclusively, built up of monoclonal sublineage blocs with identical fates, unlike the polyclonal fate distribution in C. elegans and P. marina. The fate distribution pattern in a cell lineage could be a compromise between minimizing the number of specification events by monoclonal specification and minimizing the need for migrations by forming the cells close at their final position. The latter could contribute to a faster embryonic development. These results reveal that there is more than one way to build a nematode.

Use of the Verrucomicrobia-Specific Probe EUB338-III and Fluorescent In Situ Hybridization for Detection of “Candidatus Xiphinematobacter” Cells in Nematode Hosts

ABSTRACT Fluorescent in situ hybridization with a 16S rRNA probe specific for Verrucomicrobia was used to (i) confirm the division-level identity of and (ii) study the behavior of the obligate intracellular verrucomicrobium “Candidatus Xiphinematobacter” in its nematode hosts. Endosymbionts in the egg move to the pole where the gut primordium arises; hence, they populate the intestinal epithelia of juvenile worms. During the hosts molt to adult female, the endosymbionts concentrate around the developing ovaries to occupy the ovarian wall. Some bacteria are enclosed in the ripening oocytes for vertical transmission. Verrucomicrobia in males stay outside the testes because the tiny spermatozoids are not suitable for transmission of cytoplasmic bacteria.

An SXP/RAL-2 protein produced by the subventral pharyngeal glands in the plant parasitic root-knot nematode Meloidogyne incognita

Meloidogyne incognita is a major parasite of numerous plant families, including many crop species. Upon infection of the plant root, it induces several multinucleate giant cells by the injection of pharyngeal gland secretions into the root cells. In order to obtain a better understanding of the nematode-plant interaction, characterization of the pharyngeal gland secretions is a necessity. By differential display, a nematode gene was identified that encodes a new member of the SXP/RAL-2 protein family. The gene is specifically expressed in the subventral pharyngeal glands and the protein is most likely secreted.

Observations On Spear-Formation in Xiphinema

The process of spear-formation in Xiphinema could be traced by studying larvae of different stages fixed while moulting. It was found that growth of the new odontostyl started during the early stages of moulting and ended before the old cuticle was entirely shed. The phenomena described below are supposed not to be restricted to the species involved and even not to the genus Xiphinema, e.g. the observations of Siddiqi (1962). Special reference is made to the terminology of different parts of the spear.

Embryonic lineage evolution in nematodes

Because of the high number of species and its ancient roots in evolution, the phylum Nematoda is well suited for comparative embryonic study. Using 4D microscopy we have reconstructed the embryonic lineages of several nematodes. This allows us to identify changing developmental strategies in the phylum Nematoda. Generally there has been a shift in the phylum from a non-determined, non-strict development to a faster, highly determined embryonic development. En raison du nombre eleve d’especes et de ses racines anciennes dans l’evolution, le phylum Nematoda est bien approprie a des etudes d’embryologie comparee. A l’aide d’un microscope 4D, les lignages embryonnaires de plusieurs nematodes ont ete reconstruits. Cela nous a permis d’identifier les modifications de strategie developpementales dans le phylum. Generalement, il y a eu un changement dans le phylum depuis un developpement non determine et non precis jusqu’a un developpement embryonnaire tres rapide et hautement determine.

The demanian system, traumatic insemination and reproductive strategy in Oncholaimus oxyuris Ditlevsen (Nematoda, Oncholaimina)

Observations on living specimens confirm the hypothesis formulated by Chabaud et al. about traumatic insemination in Oncholaimus oxyuris. A detailed description is given of the copulation, the formation of copulation pores, interstitial channels and the injection of glandular material and sperm. The terminal structures of the demanian system of O. oxyuris are not pre‐existing structures, but are built by the combined action of males, ejaculatory and female substances. The injected sperm is temporarily stored around the uvette in the main duct; from there they reach the uterus through the ductus uterinus. With each copulation, a new copulation pore and interstitial channel are formed. After withdrawal of the spicules a ‘wound plug’ seals the pore. When copulations follow at short intervals, sperm of the previous insemination are evacuated to the intestine through the osmosium. The hypothesis is formulated that subsequent copulations result in the fertilization of subsequent egg masses by different males.

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.