An ultrastructural and histochemical study of oogenesis in the trichostrongylid nematode Heligmosomoides polygyrus

Oogenesis in trichostrongylids has been examined for the first time in a light and electron microscopic investigation of Heligmosomoides polygyrus. The female reproductive tract is a single straight tube containing small oogonia (6 micron in diameter), which are arranged in a rosette pattern around a central rachis at the anterior end of the tract. Developing oocytes separate from the rachis and pass posteriorly in single file down the growth zone. Oocytes increase rapidly in volume due to the accumulation of cytoplasmic inclusion granules. These granules are of 3 types. Type 1 granules are amorphous and probably consist primarily of lipoprotein. Type 2 granules are large lipid inclusions and type 3 granules are electron-dense lipoprotein yolk bodies, which are probably used for energy reserves in the developing embryo. Histochemical studies show a more intense reaction for DNA in the nuclei of oogonia than in the nuclei of oocytes. There is a strong reaction for RNA in the nucleoli and in the cytoplasm of oogonia and oocytes. Ultrastructural studies indicate that this RNA is probably in the form of rRNA in the abundant ribosomes. Mature oocytes are cylindrical (60 X 70 micron), have a distinct nucleus with nuclear pores, and the cytoplasm is filled with inclusion granules and ribosomes but contains only small amounts of glycogen. Prior to fertilization the plasma membrane of oocytes acquires a flocculent coat. These oocytes contain 6 distinct bivalent chromosomes in diakinesis. Thus the major changes that occur in developing germ cells are 2-fold: nuclear changes that prepare the chromosomes for fertilization by initiating reduction division, and cytoplasmic changes that involve the synthesis and storage of inclusion granules.     

An ultrastructural study of septal development in hyphae of Polyporus biennis

A method was developed which allowed the ultrastructural study of septal formation in the basidiomycete Polyporus biennis. The method involved fixing and embedding single clamp connections. Clamp connections with septa at desired developmental stages were located by light microscopy. The septum grew by the incorporation of vesicles of wall material precursors. The rim of the developing septum was drawn centripetally inwards by a contracting collar of microfilaments within a darkly staining matrix. The inflation of the central pore swelling was governed by realigned microfilaments. The parenthesomes were formed, starting at the apex, by the utilization of the microfilament/matrix material lying along the septum. On completion of the parenthesomes a transient striated structure, governed by microfilaments, was formed in the pore channel and the areas enclosed by the parenthesomes. The maturation of the septum involved the laying down of ER along the septum and the occlusion of each end of the pore channel.     

Microsatellite markers for the human nematode parasite Ascaris lumbricoides: development and assessment of utility

We describe 35 microsatellite markers from the human parasitic nematode Ascaris lumbricoides. We found 7 sex-linked markers and demonstrate that 26 autosomal loci can be scored reliably. These markers have high genetic variability and provide the tools to address multiple questions concerning the epidemiology, fine-scale genetic structure, host specificity, and mating systems of this parasite.     

Population dynamics of pinewood nematode and the endoparasitic fungus Esteya vermicola: interactions under experimental conditions

Esteya vermicola (Ophiostomataceae), an endoparasitic fungus, exhibits great potential as a biological control agent against pinewood nematodes (PWNs). The present study reports the interaction between PWNs and E. vermicola at different spore concentrations, number of PWNs and the time of culture. The addition of PWNs enhanced the sporulation of E. vermicola after 10 days of culture. The 5-day-old cultures of E. vermicola prior to addition of PWNs increased the highest amount of sporulation than that of 10- or 15-day-old cultures. The PWNs were completely killed by E. vermicola in the pine tree powder culture medium at the concentrations of 10⁷ and 10⁸ colony-forming-units (CFU) per ml. The interaction of the PWNs and E. vermicola was that PWNs provide nutrition to E. vermicola, however, the PWNs can also feed on mycelium of E. vermicola. The effect of E. vermicola on control of PWNs was determined by the population size, time of pest infection and the duration of co-infection.     

An ultrastructural study of early endosperm development and synergid changes in unfertilized cotton ovules

Excised, unfertilized cotton (Gossypium hirsutum L.) ovules were cultured for 1–5 days postanthesis and embryo-sac development was studied with the electron microscope. In some ovules the two polar nuclei fuse and the diploid endosperm nucleus goes through a limited number of free nuclear divisions after 2–3 days in culture. Each nucleus has two nucleoli, in contrast to nuclei of fertilized triploid endosperm which have three nucleoli. Precocious cell walls form between the endosperm nuclei on the 3rd day in culture. The morphology of the plastids, mitochondria, rough endoplasmic reticulum (RER), dictyosomes and microbodies, and the amount of starch and lipid in the diploid cellular endosperm are similar to those of the central cell. A few large helical polysomes appear close to plastids and mitochondria. After 2 days in culture, one of the two synergids in the unfertilized cultured ovules shows degenerative changes which in fertilized ovules are associated with the presence of the pollen tube, i.e., increase in electron density, collapse of vacuoles, irregular darkening and thickening of mitochondrial and plastid membranes, disappearance of the plasmalemma and the membranes of the plasmalemma and the membranes of the RER. The second synergid remains unchanged in appearance. The egg cell does not shrink or divide or show structural changes characteristic of the cotton zygote. Embryo-sac development is arrested on the 4th and 5th days in culture. The nucellus continues growth and at 14 days crushes the degenerate embryo sac.     

The interaction of phagocytes and the large-sized parasite Cryptococcus neoformans: Cytochemical and ultrastructural study

Summary The yeast Cryptococcus neoformans may develop under certain conditions a large polysaccharide capsule 50–100 M in diameter and therefore cannot be phagocytosed by either polymorphonuclear cells (PMN's) or mononuclear phagocytes (MN's). The cellular defense mechanism — in various animals — against the yeast is composed by formation of ringlike structure of PMN's or MN's cells which surround the C. neoformans. Ring structures develop either in vivo or in vitro in tissue culture; destruction of the yeast occurs within 36–72 hours.Several hydrolases, such as acid phosphatase, -glucuronidase and non-specific esterase were found to be released from the phagocytic cells into the enclosed yeast. Considerable reduction of NBT used as a marker for oxidative activity was observed in MN rings at contact regions of the MN cells and the yeast. Electron microscopic studies indicate that the phagocytic cells in the ring structure have many pseudopodes penetrating into the polysaccharide capsule of the yeast. Disintegration of the capsule was observed as well as phagocytosis of its material. A possible analogy between normal phagocytosis of small-sized bodies and the ring structure obtained when large bodies are involved is discussed.     

Mitochondrial genome of the nematode endoparasitic fungus Hirsutella vermicola reveals a high level of synteny in the family Ophiocordycipitaceae

Ophiocordycipitaceae is a diverse fungal family comprising multiple ecologically, economically, medicinally, and culturally important fungal species; however, only four species of the family have available mitochondrial genomes (mitogenomes). In this study, the complete mitogenome of the nematode endoparasitic fungus Hirsutella vermicola in Ophiocordycipitaceae was sequenced, and a comparative mitogenomic analysis of Ophiocordycipitaceae was performed. We found that the 53,793-bp circular mitogenome of H. vermicola, except for standard fungal mitochondrial genes, harbors seven introns acquired possibly through lateral transfer from other fungi and three free-standing open reading frames (ORFs) coding for hypothetical proteins. Phylogenetic analysis based on concatenated mitochondrial protein sequences confirmed its placement in Ophiocordycipitaceae. Comparison on five mitogenomes of Ophiocordycipitaceae revealed great variation on their sizes, from 35.2 kb in Tolypocladium ophioglossoides to 157.5 kb in Ophiocordyceps sinensis, mainly due to variable numbers of introns (from 7 to 54) as well as variable lengths of intergenic regions. The five mitogenomes, however, are highly syntenic to each other in terms of gene order, the presence of an intronic ORF encoding ribosomal protein S3 within rnl, and the nad2/nad3 joining pattern. Our study is the first report of the mitogenome of H. vermicola and has facilitated the understanding of mitogenome evolution of Ophiocordycipitaceae.

     

Effect of temperature on infection, development and reproduction of the parasitic nematode Thripinema nicklewoodi in Frankliniella occidentalis

The parasitic nematodeThripinema nicklewoodi Siddiqi (Tylenchida:Allantonematidae) is currently underinvestigation for use in inoculative releasestrategies against western flower thrips (WFT),Frankliniella occidentalis Pergande(Thysanoptera: Thripidae) infesting greenhousefloricultural crops. The aim was to determinewhether temperatures within greenhouses wouldpermit the establishment of T.nicklewoodi. The abilities of T.nicklewoodi to infect, develop and reproducein WFT were assessed under a range of constantand fluctuating temperatures in the laboratory.At constant temperatures, T. nicklewoodiinfected WFT over the range of 1–30 °C,although the temperature-related infectionprofile followed an asymmetric distributionaround an optimum 20 °C (80%infection). The lower and upper thresholds forT. nicklewoodi in vivo development andreproduction were higher than for infection, at10 °C and 35 °C, respectively.Climate data recorded over 1999–2000 in acommercial greenhouse (Texas) revealed atemperature range of 15 °C to31 °C from early March through mid June,when WFT were most abundant. While low(nighttime) greenhouse temperatures areconducive for T. nicklewoodi, upperdaytime temperatures are close to the upperthreshold for infection and may reducereproductive output. However, fluctuatingtemperature bioassays in the laboratorydemonstrated that T. nicklewoodimaintained separately at the upper thresholdtemperatures for infection (30 °C) anddevelopment (35 °C) readily infected anddeveloped in WFT when they were allowedintermittent (10 h daily) exposure to apermissive temperature in the range10–20 °C. Drawing on the results, thediurnal temperature-fluctuations of variousgreenhouses growing ornamentals would permitthe establishment of T. nicklewoodi.     

Rhabditis terricola: An opportunistic nematode parasite of earthworm cocoons

During a study on the breeding rate of the earthworms Lumbricus rubellus and Eisenia foetida, nematodes identified as Rhabditis terricola were found consistently in large numbers in earthworm cocoons that failed to hatch after an adequate incubation period. This nematode species, which was previously known as a saprophyte, was found to invade earthworm cocoons and reproduce within, causing extensive productivity losses in earthworm cultures. In this study, R. terricola was effectively eradicated from earthworm cultures by rinsing the earthworms in tap water and transferring them repeatedly to sterile bedding.     

An ultrastructural study of osmiophilia in the human rectum

Synopsis Rectal biopsies from subjects with a normal rectum and from patients with various forms of inflammatory bowel disease were studied by the prolonged osmication technique. No consistent ultrastructural differences were observed between these groups, but there were striking differences between individual epithelial cells in the same biopsy and between the epithelium and the cells of the lamina propria. The Golgi apparatus was demonstrated occasionally in the epithelium, often in endothelium. Endoplasmic reticulum, perinuclear cisterna and mitochondria were variably outlined. In plasma cells, there were striking differences in osmiophilia.The underlying mechanism of the different staining patterns is not clear. The findings do not appear to help in the differential diagnosis of inflammatory bowel disease nor to shed any new light on their underlying pathogenic mechanisms.     

Plant parasitic nematode proteins and the host parasite interaction.

This review focuses on the proteins and secretions of sedentary plant parasitic nematodes potentially important for plant-nematode interactions. These nematodes are well equipped for parasitism of plants. Having acquired the ability to manipulate fundamental aspects of plant biology, they are able to hijack host-cell development to make their feeding site. They feed exclusively from feeding sites as they complete their life cycle, satisfying their nutritional demands for development and reproduction. Biochemical and genomic approaches have been used successfully to identify a number of nematode parasitism genes. So far, 65 204 expressed sequence tags (ESTs) have been generated for six Meloidogyne species and sequencing projects, currently in progress, will underpin genomic comparisons of Meloidogyne spp. with sequences of other pathogens and generate genechip microarrays to undertake profiling studies of up- and down-regulated genes during the infection process. RNA interference provides a molecular genetic tool to study gene function in parasitism. These methods should provide new data to help our understanding of how parasitic nematodes infect their hosts, leading to the identification of novel pathogenicity genes.     

An ultrastructural and histochemical study of the development of secondary palate in Japanese quail, Coturnix coturnix japonica

Embryonic development of the secondary palate of the Japanese quail, Coturnix coturnix japonica, was studied. The palatal shelves appeared on day 5 of incubation in a horizontal direction over the dorsal surface of the tongue. Subsequently, unlike those in mammals, the opposing palatal shelves do not fuse, and a physiological cleft persists between them. In comparison with the chick, where the palatal shelves do not fuse and the medial edge epithelium (MEE) becomes orthokeratinized stratified squamous type, the quail MEE differentiates into a parakeratinized stratified squamous type. The basal cells of the quail MEE differentiated from cuboidal to columnar and showed reorganization of their cytoplasmic organelles. In contrast to both the chick and mammalian MEE, quail MEE showed very little ruthenium red (RR) binding at the plasma membrane of both the basal and superficial cells. Initial development in a horizontal direction, lack of fusion, and an absence of programmed cell death in MEE of developing quail palate distinguished it from the mammalian palatogenesis. Also, although the morphogenesis of palate in quail and chick was similar, the pattern of cytodifferentiation in their MEE was different, which may be attributed to their ontogenesis.     

An ultrastructural study of sporidium formation during infection of a rhabditid nematode by large gun cells of Haptoglossa heteromorpha

Recently fired gun cells of Haptoglossa heteromorpha, an aplanosporic nematode parasite, were examined ultrastructurally. The everted tubes of the fired cells had penetrated the cuticle of a nematode, and infective sporidia were developing inside the host body. The nematode cuticle was penetrated by the narrow, walled part of the tube below the needle chamber. The lower unwalled part of the tube tail formed the sporidium. The developing sporidium had a multilayered fibrous outer coating and the plasma membrane was separated from the wall in places. Sporidia contained biphasic membrane-bound vesicles that had been generated by the Golgi dictyosome during gun cell development. Immediately following gun cell firing, the nuclear envelope of the sporidium nucleus was not apparent, and the sporidium nucleus contained clusters of electron-dense particles concentrated in the nucleolar region. We compare the structures and organelles found in the mature gun cell with those in the fired cell and attempt to identify the membranous layers around the sporidium.     

Morphogenesis of the hydrogenosome: An ultrastructural study

Summary— The morphogenesis of hydrogenosomes in several trichomonad species (Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas gallinae, Tritrichomonas augusta and Monocercomonas sp) was investigated by transmission electron microscopy of thin sections and freeze-fracture replicas of whole cells or the isolated organelle. Close proximity, and even continuity, between endoplasmic reticulum and hydrogenosomes was observed. Structures were seen connecting hydrogenosomes to each other and to cytoplasmic structures. Morphological evidence is presented showing that in all the trichomonads here studied, hydrogenosomes, like mitochondria, may divide by two distinct processes: segmentation and partition. In the segmentation process, the hydrogenosome grows, becoming enlongated with the appearance of a constriction in the central portion. Microfibrillar structures appear to help the furrowing process, ending with a total fission of the organelle. In the partition process, the division begins by an invagination of the inner hydrogenosome membrane, forming a transversal septum, separating the organelle matrix into two compartments. We suggest that myelin-like structures seen either in close contact with or in the vicinity of the hydrogenosomes may be a source of membrane lipids for hydrogenosome growth.     

An ultrastructural study of the development of the dermal iridophores and structural pigmentation in Poecilia reticulata (peters)

Three general stages of iridophore development were found in Poecilia reticulata that correspond to the development of structural pigmentation. The first stage was prevalent in fish embryos about to hatch to young fish 4 months old. Dermal cells containing elements of endoplasmic reticulum and a Golgi apparatus developed into iridophores. The endoplasmic reticulum early in iridophore development became a few sparse cisternae, and the Golgi apparatus elaborated long rectangular vacuoles with two membranes. From 5 to 15 vacuoles were arranged in parallel stacks in each developing iridophore. Crystals of guanine were deposited within the inner compartment of each vacuole. At this stage of development, the young fish had only a few dermal iridophores next to the lateral muscle. Fish 4 to 6 months old had a more advanced type of iridophore development including several layers of iridophore cells in the dermis. The innermost iridophores near the muscle had many mature crystal-containing vacuoles (iridosomes). Each cell had upt to three stacks of 10–20 iridosomes with their long axis oriented at a slight oblique angle to the surface of the fish. The outer layers of iridophores resembled the immature developing cells found in very young fish. The third developmental stage was found in sexually functional adults. All dermal iridophores contained 2–3 groups of 10–20 mature iridosomes. In mature iridophores, the Golgi apparatus was not found in the cytoplasm. The thickness of the guanine crystals (70 nm) and cytoplasmic intervals (90 nm) results in a constructive interference reflection of 496 nm (blue-green). This iridescence increased concomitantly with the increase in iridophore cells in the dermis and the maturation of their iridosomes.     

An ultrastructural study of the human micropolycystic ovary

The basement membrane of follicles in the micropolycystic ovaries of infertile women was thickened compared to that in the ovaries of normal women or those with typical polycystic ovaries.