Wnt gene loss in flatworms.

Wnt genes encode secreted glycoproteins that act in cell-cell signalling to regulate a wide array of developmental processes, ranging from cellular differentiation to axial patterning. Discovery that canonical Wnt/ß-catenin signalling is responsible for regulating head/tail specification in planarian regeneration has recently highlighted their importance in flatworm (phylum Platyhelminthes) development, but examination of their roles in the complex development of the diverse parasitic groups has yet to be conducted. Here, we characterise Wnt genes in the model tapeworm Hymenolepis microstoma and mine genomic resources of free-living and parasitic species for the presence of Wnts and downstream signalling components. We identify orthologs through a combination of BLAST and phylogenetic analyses, showing that flatworms have a highly reduced and dispersed complement that includes orthologs of only five subfamilies (Wnt1, Wnt2, Wnt4, Wnt5 and Wnt11) and fewer paralogs in parasitic flatworms (5-6) than in planarians (9). All major signalling components are identified, including antagonists and receptors, and key binding domains are intact, indicating that the canonical (Wnt/ß-catenin) and non-canonical (planar cell polarity and Wnt/Ca(2+)) pathways are functional. RNA-Seq data show expression of all Hymenolepis Wnts and most downstream components in adults and larvae with the notable exceptions of wnt1, expressed only in adults, and wnt2 expressed only in larvae. The distribution of Wnt subfamilies in animals corroborates the idea that the last common ancestor of the Cnidaria and Bilateria possessed all contemporary Wnts and highlights the extent of gene loss in flatworms.

The embryonic development of the bodywall and nervous system of the cestode flatworm Hymenolepis diminuta.

Cestodes (tapeworms) are a derived, parasitic clade of the phylum Platyhelminthes (flatworms). The cestode body wall represents an adaptation to its endoparasitic lifestyle. The epidermis forms a non-ciliated syncytium, and both muscular and nervous system are reduced. Morphological differences between cestodes and free-living flatworms become apparent already during early embryogenesis. Cestodes have a complex life cycle that begins with an infectious larva, called the oncosphere. In regard to cell number, cestode oncospheres are among the simplest multicellular organisms, containing in the order of 50-100 cells. As part of our continuing effort to analyze embryonic development in flatworms, we describe here the staining pattern obtained with acTub in embryos and larvae of the cestode Hymenolepis diminuta and, briefly, the monogenean Neoheterocotyle rhinobatidis. In addition, we labeled the embryonic musculature of Hymenolepis with phalloidin. In Hymenolepis embryos, two different cell types that we interpret as neurons and epidermal gland cells express acTub. There exist only two neurons that develop close to the midline at the anterior pole of the embryo. The axons of these two neurons project posteriorly into the center of the oncosphere, where they innervate the complex of muscles that is attached to the hooklets. In addition to neurons, acTub labels a small and invariant set of epidermal gland cells that develop at superficial positions, anteriorly adjacent to the neurons, in the dorsal midline, and around the posteriorly located hooklets. During late stages of embryogenesis they spread and form a complete covering of the embryo. We discuss these data in the broader context of platyhelminth embryology.

Predictability of morphological gradients in the tapeworm, Hymenolepis diminuta.

The strobila of an adult tapeworm represents a continual gradient of developmental stages from immature to gravid proglottids. The purpose of this study was to determine if organogenesis (as measured by the developmental gradient) in tapeworms within a single host and among different hosts occurred at the same rates. Rats were infected with Hymenolepis diminuta and the tapeworms were recovered 20 days post-infection. The total number of proglottids in each worm was determined, and five 'benchmarks' of organogenesis were quantified. The data demonstrated that organogenesis in worms from a single host occurred at a relatively constant rate, but that rates in tapeworms from different hosts were different.

Effects of ox bile on the growth and organogenesis of Hymenolepis microstoma in vitro.

Hymenolepis microstoma has been cultured in vitro from 4 to 11 days of age using Eagle's medium (BME) supplemented with horse serum, lamb's liver extract and ox gall (dehydrated bile) in five different concentrations (0.025%, 0.1%, 0.2%, 0.5% and 1% all in BME) including a control without the added ox gall. The results of these experiments demonstrated that growth is related inversely to the concentration of ox gall in the media. However, the number of proglottids containing fertilized ova in the "lateral sacs" becomes higher in worms grown in the medium containing 0.025% ox bile than in those grown in the control medium. This number increases with the bile concentration up to 0.2%. It is concluded that bile stimulates oogenesis and in higher concentrations retards the growth of worms.

Hymenolepis nana: the fine structure of the embryonic envelopes.

The fine structure of the envelopes surrounding hatched and unhatched oncospheres of Hymenolepis nana has been investigated by transmission and scanning electron microscopy (SEM), together with light microscope histochemical observations of JB-4 embedded material. The oncosphere is surrounded by 3 layers--the capsule, the outer envelope and the inner envelope, the latter giving rise to the embryophore and the 'oncospheral membrane'. An additional layer--the polar filament layer--lies between the 'oncospheral membrane' and the oncosphere. Shell material is deposited on the capsule as a thin layer. It is secreted by the outer envelope, which degenerates once shell formation is complete. The uterus may also contribute to shell formation. The embryophore forms a thin incomplete and peripheral layer within the inner envelope. In the basal region of this envelope, partial development of an 'oncospheral membrane' takes place, but it does not become detached as a separate layer. The polar filaments, which are characteristic of the oncosphere of H. nana, are derived from the epithelial covering of the oncosphere itself, which delaminates to form a separate polar filament layer. The filaments arise from knob-like projections at opposite poles of this layer. The design of the embryonic envelopes in H. nana show a number of modifications from the basic cyclophyllidean pattern, and these can be related to the demands of its 'direct' life-cycle.

A comparative study of the two alternative larval forms of Hymenolepis nana, the dwarf tapeworm, with special reference to the process of excystment.

Studies of the cysticercoids of Hymenolepis nana from insects and from mouse villi revealed important differences in cyst structure and function. The insect form resists low pH unless treated with bile salts which render the cyst permeable and reduce infectivity to mice. Bile salts are not essential for scolex activation. Activation is inhibited by pH 2.5 and under and by 1% succinic acid up to pH 4.0. The importance of scolex immobility and energy conservation in relation to cyst impermeability is discussed. The villus cysticercoid has no special insulating layer. It is vulnerable to low pH and cannot infect mice orally. Bile salts are without effect and excystment occurs unaided by external agents. The structural differences between the two forms revealed by the electron microscope may be attributed to changes in the relative rates of development of the various tissues.

The fine structure of the cysticercoid of Hymenolepis diminuta. III. The scolex.

Transmission electron microscopy of the scolex of the 8-day-old Hymenolepis diminuta cysticercoid demonstrates its resemblance to the scolex of the adult. A syncytial tegument composed of external and internal layers is connected by cytoplasmic extensions. Fully developed microtriches are present. Furthermore, a basement membrane, muscle layers, and medullary region containing flame cells, nerve tissue, and other cell bodies are observed. Of particular interest is the presence of discrete sensory endings whose function is discussed.