Chromosome-Wide Evolution and Sex Determination in the Three-Sexed Nematode Auanema rhodensis.

Trioecy, a mating system in which males, females and hermaphrodites co-exist, is a useful system to investigate the origin and maintenance of alternative mating strategies. In the trioecious nematode Auanema rhodensis, males have one X chromosome (XO), whereas females and hermaphrodites have two (XX). The female vs. hermaphrodite sex determination mechanisms have remained elusive. In this study, RNA-seq analyses show a 20% difference between the L2 hermaphrodite and female gene expression profiles. RNAi experiments targeting the DM (doublesex/mab-3) domain transcription factor dmd-10/11 suggest that the hermaphrodite sexual fate requires the upregulation of this gene. The genetic linkage map (GLM) shows that there is chromosome-wide heterozygosity for the X chromosome in F2 hermaphrodite-derived lines originated from crosses between two parental inbred strains. These results confirm the lack of recombination of the X chromosome in hermaphrodites, as previously reported. We also describe conserved chromosome elements (Nigon elements), which have been mostly maintained throughout the evolution of Rhabditina nematodes. The seven-chromosome karyotype of A. rhodensis, instead of the typical six found in other rhabditine species, derives from fusion/rearrangements events involving three Nigon elements. The A. rhodensis X chromosome is the smallest and most polymorphic with the least proportion of conserved genes. This may reflect its atypical mode of father-to-son transmission and its lack of recombination in hermaphrodites and males. In conclusion, this study provides a framework for studying the evolution of chromosomes in rhabditine nematodes, as well as possible mechanisms for the sex determination in a three-sexed species.

A Diffuse Interface Framework for Modeling the Evolution of Multi-cell Aggregates as a Soft Packing Problem Driven by the Growth and Division of Cells.

We present a model for cell growth, division and packing under soft constraints that arise from the deformability of the cells as well as of a membrane that encloses them. Our treatment falls within the framework of diffuse interface methods, under which each cell is represented by a scalar phase field and the zero level set of the phase field represents the cell membrane. One crucial element in the treatment is the definition of a free energy density function that penalizes cell overlap, thus giving rise to a simple model of cell-cell contact. In order to properly represent cell packing and the associated free energy, we include a simplified representation of the anisotropic mechanical response of the underlying cytoskeleton and cell membrane through penalization of the cell shape change. Numerical examples demonstrate the evolution of multi-cell clusters and of the total free energy of the clusters as a consequence of growth, division and packing.

Two independent sulfation processes regulate mouth-form plasticity in the nematode Pristionchus pacificus.

Sulfation of biomolecules, like phosphorylation, is one of the most fundamental and ubiquitous biochemical modifications with important functions during detoxification. This process is reversible, involving two enzyme classes: a sulfotransferase, which adds a sulfo group to a substrate; and a sulfatase that removes the sulfo group. However, unlike phosphorylation, the role of sulfation in organismal development is poorly understood. In this study, we find that two independent sulfation events regulate the development of mouth morphology in the nematode Pristionchus pacificus. This nematode has the ability to form two alternative mouth morphologies depending on environmental cues, an example of phenotypic plasticity. We found that, in addition to a previously described sulfatase, a sulfotransferase is involved in regulating the mouth-form dimorphism in P. pacificus However, it is unlikely that both of these sulfation-associated enzymes act upon the same substrates, as they are expressed in different cell types. Furthermore, animals mutant in genes encoding both enzymes show condition-dependent epistatic interactions. Thus, our study highlights the role of sulfation-associated enzymes in phenotypic plasticity of mouth structures in Pristionchus.

Cloning and expression analysis of the DEAD-box/RNA helicase Oslaf-1 in Ovomermis sinensis.

Ovomermis sinensis is a potentially-valuable nematode for controlling insect pests. The parasitic stage of the nematode absorbs nutrients in its host's hemolymph to maintain its growth development and then kills the host when it emerges. At present, little known about its reproductive development, particularly the responsible molecular mechanism. More detailed research on the genes of reproductive development will not only help us understand the mechanisms underlying sex differentiation in the nematode, but would also be valuable for successfully cultivating them in vitro and using them for biocontrol. In this study, we used the homology cloning method to clone the full-length cDNA of a DEAD-box family gene (Oslaf-1) from O. sinensis. Then, using qRT-PCR technology to detect the expression pattern of the Oslaf-1 gene at different development stages and tissues, the gene was found to be highly expressed in the post-parasitic stage (P < 0.01) and ovarian (P < 0.05) of O. sinensis. Western blot analysis showed the same result that the gene is associated with gonadal development and function, but is not gonad-specific. In situ hybridization further demonstrated that the gene is widely expressed in early embryos and is mainly distributed in the gonadal area. However, the signal was mainly concentrated in the reproductive primordia in pre-parasitic juveniles. RNA interference (RNAi) studies revealed that the sex ratio of O. sinensis soaked in dsRNA of Oslaf-1 was not statistically different than the gfp dsRNA treated groups. Our results suggest that Oslaf-1 may play a vital role in the reproductive systems of the nematode. In addition, we speculate that the Oslaf-1 gene plays an important role during embryonic development and that it occurs and develops in the gonads of pre-parasitic juveniles of O. sinensis.

Evolution of mitotic spindle behavior during the first asymmetric embryonic division of nematodes.

Asymmetric cell division is essential to generate cellular diversity. In many animal cells, the cleavage plane lies perpendicular to the mitotic spindle, and it is the spindle positioning that dictates the size of the daughter cells. Although some properties of spindle positioning are conserved between distantly related model species and different cell types, little is known of the evolutionary robustness of the mechanisms underlying this event. We recorded the first embryonic division of 42 species of nematodes closely related to Caenorhabditis elegans, which is an excellent model system to study the biophysical properties of asymmetric spindle positioning. Our recordings, corresponding to 128 strains from 27 Caenorhabditis and 15 non-Caenorhabditis species (accessible at http://www.ens-lyon.fr/LBMC/NematodeCell/videos/), constitute a powerful collection of subcellular phenotypes to study the evolution of various cellular processes across species. In the present work, we analyzed our collection to the study of asymmetric spindle positioning. Although all the strains underwent an asymmetric first cell division, they exhibited large intra- and inter-species variations in the degree of cell asymmetry and in several parameters controlling spindle movement, including spindle oscillation, elongation, and displacement. Notably, these parameters changed frequently during evolution with no apparent directionality in the species phylogeny, with the exception of spindle transverse oscillations, which were an evolutionary innovation at the base of the Caenorhabditis genus. These changes were also unrelated to evolutionary variations in embryo size. Importantly, spindle elongation, displacement, and oscillation each evolved independently. This finding contrasts starkly with expectations based on C. elegans studies and reveals previously unrecognized evolutionary changes in spindle mechanics. Collectively, these data demonstrate that, while the essential process of asymmetric cell division has been conserved over the course of nematode evolution, the underlying spindle movement parameters can combine in various ways. Like other developmental processes, asymmetric cell division is subject to system drift.

An asymmetric attraction model for the diversity and robustness of cell arrangement in nematodes.

During early embryogenesis in animals, cells are arranged into a species-specific pattern in a robust manner. Diverse cell arrangement patterns are observed, even among close relatives. In the present study, we evaluated the mechanisms by which the diversity and robustness of cell arrangements are achieved in developing embryos. We successfully reproduced various patterns of cell arrangements observed in various nematode species in Caenorhabditis elegans embryos by altering the eggshell shapes. The findings suggest that the observed diversity of cell arrangements can be explained by differences in the eggshell shape. Additionally, we found that the cell arrangement was robust against eggshell deformation. Computational modeling revealed that, in addition to repulsive forces, attractive forces are sufficient to achieve such robustness. The present model is also capable of simulating the effect of changing cell division orientation. Genetic perturbation experiments demonstrated that attractive forces derived from cell adhesion are necessary for the robustness. The proposed model accounts for both diversity and robustness of cell arrangements, and contributes to our understanding of how the diversity and robustness of cell arrangements are achieved in developing embryos.

Mechanisms of animal diapause: recent developments from nematodes, crustaceans, insects, and fish.

Life cycle delays are beneficial for opportunistic species encountering suboptimal environments. Many animals display a programmed arrest of development (diapause) at some stage(s) of their development, and the diapause state may or may not be associated with some degree of metabolic depression. In this review, we will evaluate current advancements in our understanding of the mechanisms responsible for the remarkable phenotype, as well as environmental cues that signal entry and termination of the state. The developmental stage at which diapause occurs dictates and constrains the mechanisms governing diapause. Considerable progress has been made in clarifying proximal mechanisms of metabolic arrest and the signaling pathways like insulin/Foxo that control gene expression patterns. Overlapping themes are also seen in mechanisms that control cell cycle arrest. Evidence is emerging for epigenetic contributions to diapause regulation via small RNAs in nematodes, crustaceans, insects, and fish. Knockdown of circadian clock genes in selected insect species supports the importance of clock genes in the photoperiodic response that cues diapause. A large suite of chaperone-like proteins, expressed during diapause, protects biological structures during long periods of energy-limited stasis. More information is needed to paint a complete picture of how environmental cues are coupled to the signal transduction that initiates the complex diapause phenotype, as well as molecular explanations for how the state is terminated. Excellent examples of molecular memory in post-dauer animals have been documented in Caenorhabditis elegans It is clear that a single suite of mechanisms does not regulate diapause across all species and developmental stages.

Nuclear receptors in nematode development: Natural experiments made by a phylum.

The development of complex multicellular organisms is dependent on regulatory decisions that are necessary for the establishment of specific differentiation and metabolic cellular states. Nuclear receptors (NRs) form a large family of transcription factors that play critical roles in the regulation of development and metabolism of Metazoa. Based on their DNA binding and ligand binding domains, NRs are divided into eight NR subfamilies from which representatives of six subfamilies are present in both deuterostomes and protostomes indicating their early evolutionary origin. In some nematode species, especially in Caenorhabditis, the family of NRs expanded to a large number of genes strikingly exceeding the number of NR genes in vertebrates or insects. Nematode NRs, including the multiplied Caenorhabditis genes, show clear relation to vertebrate and insect homologues belonging to six of the eight main NR subfamilies. This review summarizes advances in research of nematode NRs and their developmental functions. Nematode NRs can reveal evolutionarily conserved mechanisms that regulate specific developmental and metabolic processes as well as new regulatory adaptations. They represent the results of a large number of natural experiments with structural and functional potential of NRs for the evolution of the phylum. The conserved and divergent character of nematode NRs adds a new dimension to our understanding of the general biology of regulation by NRs. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Differences in life-histories refute ecological equivalence of cryptic species and provide clues to the origin of bathyal Halomonhystera (Nematoda).

The discovery of morphologically very similar but genetically distinct species complicates a proper understanding of the link between biodiversity and ecosystem functioning. Cryptic species have been frequently observed to co-occur and are thus expected to be ecological equivalent. The marine nematode Halomonhystera disjuncta contains five cryptic species (GD1-5) that co-occur in the Westerschelde estuary. In this study, we investigated the effect of three abiotic factors (salinity, temperature and sulphide) on life-history traits of three cryptic H. disjuncta species (GD1-3). Our results show that temperature had the most profound influence on all life-cycle parameters compared to a smaller effect of salinity. Life-history traits of closely related cryptic species were differentially affected by temperature, salinity and presence of sulphides which shows that cryptic H. disjuncta species are not ecologically equivalent. Our results further revealed that GD1 had the highest tolerance to a combination of sulphides, high salinities and low temperatures. The close phylogenetic position of GD1 to Halomonhystera hermesi, the dominant species in sulphidic sediments of the Håkon Mosby mud volcano (Barent Sea, 1280 m depth), indicates that both species share a recent common ancestor. Differential life-history responses to environmental changes among cryptic species may have crucial consequences for our perception on ecosystem functioning and coexistence of cryptic species.

A host beetle pheromone regulates development and behavior in the nematode Pristionchus pacificus.

Nematodes and insects are the two most speciose animal phyla and nematode-insect associations encompass widespread biological interactions. To dissect the chemical signals and the genes mediating this association, we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus. We found that while the beetle pheromone is attractive to P. pacificus adults, the pheromone arrests embryo development, paralyzes J2 larva, and inhibits exit of dauer larvae. To uncover the mechanism that regulates insect pheromone sensitivity, a newly identified mutant, Ppa-obi-1, is used to reveal the molecular links between altered attraction towards the beetle pheromone, as well as hypersensitivity to its paralyzing effects. Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types, including the amphid neuron sheath and excretory cells. Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny.

Bioinformatic analysis of nematode migration-associated genes identifies novel vertebrate neural crest markers.

Neural crest cells are highly motile, yet a limited number of genes governing neural crest migration have been identified by conventional studies. To test the hypothesis that cell migration genes are likely to be conserved over large evolutionary distances and from diverse tissues, we searched for vertebrate homologs of genes important for migration of various cell types in the invertebrate nematode and examined their expression during vertebrate neural crest cell migration. Our systematic analysis utilized a combination of comparative genomic scanning, functional pathway analysis and gene expression profiling to uncover previously unidentified genes expressed by premigratory, emigrating and/or migrating neural crest cells. The results demonstrate that similar gene sets are expressed in migratory cell types across distant animals and different germ layers. Bioinformatics analysis of these factors revealed relationships between these genes within signaling pathways that may be important during neural crest cell migration.

Developmental variations among Panagrolaimid nematodes indicate developmental system drift within a small taxonomic unit.

Comparative studies of nematode embryogenesis among different clades revealed considerable variations. However, to what extent developmental differences exist between closely related species has mostly remained nebulous. Here, we explore the correlation between phylogenetic neighborhood and developmental variation in a restricted and morphologically particularly uniform taxonomic group (Panagrolaimidae) to determine to what extent (1) morphological and developmental characters go along with molecular data and thus can serve as diagnostic tools for the definition of kinship and (2) developmental system drift (DSD; modifications of developmental patterns without corresponding morphological changes) can be found within a small taxonomic unit. Our molecular approaches firmly support subdivision of Panagrolaimid nematodes into two monophyletic groups. These can be discriminated by distinct peculiarities in early embryonic cell lineages and a mirror-image expression pattern of the gene skn-1. This suggests major changes in the logic of cell specification and the action of DSD in the studied representatives of the two neighboring nematode taxa.

Nematicidal activity of microbial pigment from Serratia marcescens.

Ineffectiveness of available nematicides and the high damage caused by plant-parasitic nematodes result in the urgent need to find some natural remedy for their control. Bioactivity of the pigment extracted from Serratia marcescens was screened for controlling nematodes at their juvenile stage. Test pigment was found effective against juvenile stages of Radopholus similis and Meloidogyne javanica at low concentrations (LC50 values, 83 and 79 µg/mL, respectively) as compared with positive control of copper sulphate (LC50 values, 380 and 280 µg/mL, respectively). The pigment also exhibited inhibition on nematode egg-hatching ability. Characterisation of extracted pigment with TLC, FTIR, HPLC, HPTLC and spectroscopic analysis confirmed the presence of prodigiosin as a bioactive metabolite. Considering the sensory mechanism of pathogen recognition by nematodes, the use of microbial secondary metabolites can be effective for nematode control rather than using whole organism.

Nematode model systems in evolution and development.

The free-living nematode Caenorhabditis elegans is one of the most important model organisms in all areas of modern biology. Using the knowledge about C. elegans as a baseline, nematodes are now intensively studied in evolution and development. Evolutionary developmental biology or for short, 'evo-devo' has been developed as a new research discipline during the last two decades to investigate how changes in developmental processes and mechanisms result in the modification of morphological structures and phenotypic novelty. In this article, we review the concepts that make nematode evo-devo a successful approach to evolutionary biology. We introduce selected model systems for nematode evo-devo and provide a detailed discussion of four selected case studies. The most striking finding of nematode evo-devo is the magnitude of developmental variation in the context of a conserved body plan. Detailed investigation of early embryogenesis, gonad formation, vulva development, and sex determination revealed that molecular mechanisms evolve rapidly, often in the context of a conserved body plan. These studies highlight the importance of developmental systems drift and neutrality in evolution.

Life history of a free-living marine nematode Daptonema normandicum reared in laboratory.

Life history of a free-living meiobenthic nematode Daptonema normandicum (DeMan, 1890) was studied in the laboratory. Live specimens were primarily collected from the sewage outlet site near the mouth of the Mandovi estuary, Goa This species was the most dominant (> 67%) among the meiobenthic nematodes. Vertically, nematode abundance was highest at the surface sediment and correlated with the organic carbon and sediment chlorophyll-a. Considering their dominance in the meiofauna, attempts were made to rear D. normandicum in laboratory. Salinity of the culture medium was maintained at 14 to 17 PSU (same as the collection site). All the culture experiments were conducted in semisolid nutrient agar media at 27 +/- 2 degrees C temperature for 12 hr dark: 12 hr light conditions. The food consists primarily of an unidentified bacterium and mixed algae, but diatom and ciliates were also observed in culture. Females produced first batch of eggs at the age of 23 days. Gravid female normally carry 8-10 eggs. Embryonic development is completed in -72 hr and entire life cycle (egg to adult) was completed in 22-24 days. Average size of juveniles at the hatching was 0.189 mm. Young individuals attains a maximum size of 1.23 mm (male) and 1.04 mm (female) in -21-23 days. Growth, in terms of length was augmented upto 23rd day and ceased thereafter. The daily growth increment for the first 5 days was 0.01-0.04 mm which increased upto 0.05-0.08 mm d(-1) during the maturation (10-18 days). Male : female ratio was 1:2. In this laboratory study, we provided information on the embryonic development, the life cycle and ecology Our results demonstrated that D. normandicum can be reared successfully under the controlled conditions, suggesting possible use of this species in toxicological and aquaculture studies. The culture method described is very handy and can be applicable for rearing other meiobenthic species particularly the nematodes with comparable feeding habits.

Pristionchus pacificus daf-16 is essential for dauer formation but dispensable for mouth form dimorphism.

The nematode Pristionchus pacificus shows two forms of phenotypic plasticity: dauer formation and dimorphism of mouth form morphologies. It can therefore serve as a model for studying the evolutionary mechanisms that underlie phenotypic plasticity. Formation of dauer larvae is observed in many other species and constitutes one of the most crucial survival strategies in nematodes, whereas the mouth form dimorphism is an evolutionary novelty observed only in P. pacificus and related nematodes. We have previously shown that the same environmental cues and steroid signaling control both dauer formation and mouth form dimorphism. Here, we examine by mutational analysis and whole-genome sequencing the function of P. pacificus (Ppa) daf-16, which encodes a forkhead transcription factor; in C. elegans, daf-16 is the target of insulin signaling and plays important roles in dauer formation. We found that mutations in Ppa-daf-16 cause strong dauer formation-defective phenotypes, suggesting that Ppa-daf-16 represents one of the evolutionarily conserved regulators of dauer formation. Upon strong dauer induction with lophenol, Ppa-daf-16 individuals formed arrested larvae that partially resemble wild-type dauer larvae, indicating that Ppa-daf-16 is also required for dauer morphogenesis. By contrast, regulation of mouth form dimorphism was unaffected by Ppa-daf-16 mutations and mutant animals responded normally to environmental cues. Our results suggest that mechanisms for dauer formation and mouth form regulation overlap partially, but not completely, and one of two key transcriptional regulators of the dauer regulatory network was either independently co-opted for, or subsequently lost by, the mouth form regulatory network.

Cellular and muscular growth patterns during sipunculan development.

Sipuncula is a lophotrochozoan taxon with annelid affinities, albeit lacking segmentation of the adult body. Here, we present data on cell proliferation and myogenesis during development of three sipunculan species, Phascolosoma agassizii, Thysanocardia nigra, and Themiste pyroides. The first anlagen of the circular body wall muscles appear simultaneously and not subsequently as in the annelids. At the same time, the rudiments of four longitudinal retractor muscles appear. This supports the notion that four introvert retractors were part of the ancestral sipunculan bodyplan. The longitudinal muscle fibers form a pattern of densely arranged fibers around the retractor muscles, indicating that the latter evolved from modified longitudinal body wall muscles. For a short time interval, the distribution of S-phase mitotic cells shows a metameric pattern in the developing ventral nerve cord during the pelagosphera stage. This pattern disappears close to metamorphic competence. Our findings are congruent with data on sipunculan neurogenesis, as well as with recent molecular analyses that place Sipuncula within Annelida, and thus strongly support a segmental ancestry of Sipuncula.

Conservation of MAP kinase activity and MSP genes in parthenogenetic nematodes.

BACKGROUND: MAP (mitogen-activated protein) kinase activation is a prerequisite for oocyte maturation, ovulation and fertilisation in many animals. In the hermaphroditic nematode Caenorhabditis elegans, an MSP (major sperm protein) dependent pathway is utilised for MAP kinase activation and successive oocyte maturation with extracellular MSP released from sperm acting as activator. How oocyte-to-embryo transition is triggered in parthenogenetic nematode species that lack sperm, is not known. RESULTS: We investigated two key elements of oocyte-to-embryo transition, MSP expression and MAP kinase signaling, in two parthenogenetic nematodes and their close hermaphroditic relatives. While activated MAP kinase is present in all analysed nematodes irrespective of the reproductive mode, MSP expression differs. In contrast to hermaphroditic or bisexual species, we do not find MSP expression at the protein level in parthenogenetic nematodes. However, genomic sequence analysis indicates that functional MSP genes are present in several parthenogenetic species. CONCLUSIONS: We present three alternative interpretations to explain our findings. (1) MSP has lost its function as a trigger of MAP kinase activation and is not expressed in parthenogenetic nematodes. Activation of the MAP kinase pathway is achieved by another, unknown mechanism. Functional MSP genes are required for occasionally emerging males found in some parthenogenetic species. (2) Because of long-term disadvantages, parthenogenesis is of recent origin. MSP genes remained intact during this short interval although they are useless. As in the first scenario, an unknown mechanism is responsible for MAP kinase activation. (3) The molecular machinery regulating oocyte-to-embryo transition in parthenogenetic nematodes is conserved with respect to C. elegans, thus requiring intact MSP genes. However, MSP expression has been shifted to non-sperm cells and is reduced below the detection limits, but is still sufficient to trigger MAP kinase activation and embryogenesis.

Experimental studies on the development of Contracaecum rudolphii (Nematoda: Anisakidae) in copepod and fish paratenic hosts.

The larval development of the nematode Contracaecum rudolphii (Rudolphi, 1819), a common parasite of the proventriculus of cormorants, was experimentally studied. Within the eggs cultivated in freshwater under laboratory temperatures of 20-22 degrees C, the developing larva undergoes two moults on days 4-5, attaining the third larval stage. Most of the ensheathed third-stage larvae, 291-457 microm long, hatch spontaneously from egg shells on days 5-6. Experiments have indicated that hatched ensheated third-stage larvae and those still inside egg capsules are already infective to copepods and fishes, which both can be considered paratenic (meta-paratenic) hosts. Five copepod species, Acanthocyclops vernalis, Cyclops strenuus, Ectocyclops phaleratus, Eucyclops serrulatus and Megacyclops viridis, the isopod Asellus aquaticus and small carps Cyprinus carpio were infected by feeding them these larvae. In addition, 9 fish species, Alburnoides bipunctatus, Anguilla anguilla, Barbatula barbatula, Cyprinus carpio, Gobio gobio, Perca fluviatilis, Phoxinus phoxinus, Poecilia reticulata and Tinca tinca, were successfully infected by feeding them copepods previously infected with C. rudolphii third-stage larvae. In fishes, larvae from copepods penetrate through the intestinal wall to the body cavity, where, in a few weeks, they become encapsulated; the larvae substantially grow in fish, attaining the body length up to 4.87 mm. In carp fry, the nematode third-stage larvae survived for about 15 months (up to 18 months in fish infected directly, i.e., without copepods). One small cormorant (Phalacrocorax carbo sinensis) was successfully infected by feeding it with copepods harbouring C. rudolphii third-stage larvae.