Wouter Houthoofd

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.

Mononchoides composticola n. sp. (Nematoda: Diplogastridae) associated with composting processes: morphological, molecular and autecological characterisation

Mononchoides composticola n. sp. was isolated from compost and is described based on light and scanning electron microscopy, supplemented with SSU rDNA sequence data. It is characterised by the following features: a denticulate ridge in addition to the dorsal claw-like tooth, a small tooth-like swelling at the stegostom base, ca 26 longitudinal ridges on the female body, a uterine sac associated with two dumb-bell-shaped pouches, relatively small spicules (30-38 µm long), a simple gubernaculum shorter than half the spicule length, the genital subventral papillae (v6) consisting of three very small papillae, and a long filiform tail (female: 391-550 µm, 18-26 anal body diam.; male: 304-548 µm, 19-30 anal body diam.). Phylogenetic analyses placed the new species together with M. striatus, sister to Tylopharynx foetida. Since the use of nematodes as functional indicators often relies on the allocation of nematodes to feeding groups, experiments were performed to elucidate the feeding strategy of the new species. Both its ability to move actively to bacterial food sources and to prey on other compost nematodes were tested. Mononchoides composticola n. sp. actively moved towards the compost bacterium Achromobacter, a taxis that was temperature dependent, and also preyed on other nematodes. Predation was selective, with a higher predation rate on the relatively small and slow-moving Rhabditella sp. than on the considerably larger and more motile Rhabditis (Poikilolaimus) sp. Adults of M. composticola n. sp. have a dual feeding behaviour and can apparently alternate between bacterial and nematode prey

Lack of metabolic ageing in the long-lived flatworm Schmidtea polychroa

Freshwater planarians have a large totipotent stem cell population allowing high rates of cell renewal and morphological plasticity. It is often suggested that they are able to rejuvenate during fission, regeneration and starvation. These features, together with the rapidly expanding molecular toolset, make planarians such as Schmidtea polychroa and S. mediterranea interesting for ageing research. Yet, the basic demographic and physiological data are lacking or still based on fragmentary observations of one century ago. Here, we present the first longitudinal physiological study of the species S. polychroa. Survival, size and metabolic rate, measured by microcalorimetry, of a cohort of 28 individuals were followed over a period of three years. Sexual maturity was reached during the second month after which the worms continued growing up to 5 months. This initial growth phase was followed by alternating periods of synchronised growth and degrowth. Although mass-specific metabolic rates declined during the initial growth phase, no changes were found later in life. The absence of metabolic ageing may be explained by the very high rate of cell renewal during homeostasis and alternating phases of degrowth and growth during which tissues are renewed. Surprisingly, all deaths occurred in pairs of worms that were housed in the same culture recipient, suggesting that worms did not die from ageing. Taking into account the metabolic and demographic data, we suggest that S. polychroa shows negligible ageing. Detailed analyses of size and metabolic rate revealed a remarkable biphasic allometric scaling relation. During the initial growth phase (months 1-5) the allometric scaling exponent b was 0.86 while later in life, it increased to an unusually large value of 1.17, indicating that mass-specific metabolic rate increases with size in adult S. polychroa.

Analysis of the translationally controlled tumour protein in the nematodes Ostertagia ostertagi and Caenorhabditis elegans suggests a pivotal role in egg production.

The translationally controlled tumour protein (TCTP) is a conserved protein which has been described for a wide range of eukaryotic organisms including protozoa, yeasts, plants, nematodes and mammals. Several parasitic organisms have been shown to actively secrete TCTP during host infection as part of their immuno-evasive strategy. In this study, we have studied TCTP in Ostertagia ostertagi, a parasitic nematode of cattle, and in the free-living nematode Caenorhabditis elegans. An analysis of the transcription and expression patterns showed that TCTP was present in the eggs of both species. This localisation is consistent for some other Strongylida such as Teladorsagia circumcincta, Cooperia oncophora and Haemonchus contortus. TCTP was also detected at low levels in excretory-secretory material from adult O. ostertagi worms. The role of TCTP in nematode biology was also investigated by RNA interference in C. elegans. Knock-down of C. elegans tctp (tct-1) transcription reduced the numbers of eggs laid by the hermaphrodite in the F(0) and F(1) generations by 90% and 72%, respectively, indicating a pivotal role of TCTP in reproduction.

Anthelmintic tolerance in free-living and facultative parasitic isolates of Halicephalobus (Panagrolaimidae)

Studies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.

Stem Cells Propagate Their DNA by Random Segregation in the Flatworm Macrostomum lignano

Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts) are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized ‘Label-Retaining Cells’ (LRCs). Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells.We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU− double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.

Different roads to form the same gut in nematodes

SUMMARY The morphogenesis of a gut from the endoderm has been well studied among the animal kingdom and is also well described in the nematode Caenorhabditis elegans. But are there other ways to build a nematode intestine? Sulston et al. (1983) described a different intestinal cell lineage in the species Panagrellus redivivus and Turbatrix aceti that includes two programmed cell deaths. However, no details are known about the three‐dimensional (3D) configuration and the role of the cell deaths. Here, we describe the intestinal morphogenesis of P. redivivus and five other nematode species by means of four‐dimensional microscopy, which gives us a 3D representation of gut formation at the cellular level. The morphological pathway of gut formation is highly conserved among these distantly related species. However, we found the P. redivivus pattern in another related species Halicephalobus gingivalis. In this pattern, the intestinal precursors migrate inward in concert with the mesoderm precursors. Based on the observations, we propose a hypothesis that could explain the differences. The positions of the mesoderm precursors create a possible spatial constraint, by which the establishment of bilateral symmetry in the intestine is delayed. This symmetry is corrected by cell migrations; other cells are eliminated and compensated by supplementary cell divisions. This pattern leads to the same result as in the other nematodes: a bilateral symmetrical intestine with nine rings. This illustrates how conserved body plans can be achieved by different developmental mechanisms.

The embryonic cell lineage of the nematode Halicephalobus gingivalis (Nematoda: Cephalobina: Panagrolaimoidea)

This paper describes the nearly complete embryonic cell lineage of the terrestrial nematode, Halicephalobus gingivalis, up to somatic muscle contraction, resulting in the formation of 536 cells, of which 24 undergo programmed cell death. Halicephalobus gingivalis has a 94% lineage homology with both Caenorhabditis elegans and Pellioditis marina, and a fate homology of only 86% and 78%, respectively. Although H. gingivalis belongs to a different superfamily than C. elegans and P. marina, its cell lineage is remarkably consistent with them. Variations in the fate distribution of cells among the different species were only observed at the end of the cell lineage. The data presented here show that the polyclonal cell specification is much more widespread in clades 9 and 10 and is not a highly derived trait that is specifically linked to the fast development of the model organism C. elegans.

The early embryonic development of the satellite organism Pristionchus pacificus: differences and similarities with Caenorhabditis elegans

As a comparative counterpart for the model organism Caenorhabditis elegans , the nematode Pristionchus pacificus was established as a satellite organism to study developmental processes. However, these studies mainly focused on post-embryonic development and little is known about the early embryonic development. Using 4D microscopy we reconstructed the early embryonic cell lineage of 12 individuals of P. pacificus . By analysing several parameters of early development, including the division sequence, the spatial arrangement of blastomeres, the cell cycle patterns of the AB lineage and cell-cell contacts in different cell stages of the embryo, it was shown that the early embryonic development is nearly identical to C. elegans . Known cell-cell contacts necessary for induction of blastomere fates in C. elegans are also present in P. pacificus . Thus, the spatio-temporal conditions that would allow possible homologous inductions are present. However, at least one model for blastomere specification seems not to apply to P. pacificus since the third division in the AB lineage differs from that of C. elegans . Furthermore, naturally occurring variability of early development was demonstrated, which is clearly permitted since there seems to be no influence on further development into an adult worm.

Measurement of S-phase duration of adult stem cells in the flatworm Macrostomum lignano by double replication labelling and quantitative colocalization analysis

Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo. These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated Ts (S‐phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano. A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity‐based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object‐based colocalization approach was devised to score the relative number of double‐positive cells. Using this approach, Ts (S‐phase duration) in the main population of neoblasts was ∼13 h. During early regeneration, no significant change in Ts was observed.