Free-living Nematode Panagrellus Silusiae Research Articles

Genomic arrangement of repeated PS700 elements in the nematode Panagrellus silusiae

When genomic DNA from the free-living nematode Panagrellus silusiae is digested with the restriction endonuclease BamHI and separated by electrophoresis, a band in the 700 base pair size range is evident after ethidium bromide staining. One of the 0.7-kilobase fragments (PS700-1) was characterized and found to be a member of a moderately repetitive DNA family (T. Warren and J.J. Pasternak. 1988. Nucleic Acids Res. 16: 10,833-10,847). In the current study, DNA sequence analyses of three independently isolated copies of the PS700 DNA family showed the same nucleotide sequence and greater than 98% similarity to PS700-1. Four EMBL-4 bacteriophage clones were isolated from a Panagrellus genomic DNA library with PS700-1 as the probe and were analyzed by restriction endonuclease site mapping and Southern blot DNA hybridization. These clones contain 31 copies of the PS700 DNA family. In each case, the units are arranged in head-to-tail arrays. One of the EMBL-4 clones contains copies of a novel variant of the PS700 elements. The maintenance of both nucleotide sequence and restriction endonuclease restriction site homogeneity among members of the dispersed PS700 DNA family may denote a functional role for these sequences.

Ascaris lumbricoides: Zonal localization of immunoreactive collagen in the cuticle

Monoclonal antibodies that were raised against cuticular components from the free-living nematode Panagrellus silusiae were found to react with a cuticular collagenous domain from Ascaris lumbricoides. One of these monoclonal antibodies was used to localize the collagenous epitope within sectioned Ascaris cuticles. By indirect immunofluorescence, accessible binding sites were observed in the basal zone of the cuticle. Immunological staining occurred in the innermost lamella of the basal zone, i.e., basal lamella, in which the fibrillar palisade gave a strong response. The three layers of the spiral fiber system of the basal zone exhibited a distinctive immunofluorescence pattern. In each of these layers, irregular shaped blocks, often quadrangular, were immunostained; whereas, adjacent blocks were immunonegative. Immunostaining was, for the most part, absent from the cortical and medial zones of the cuticle as well as from other tissues within the worm.

Toxicity of Seven Heavy Metals On Panagrell Us Silusiae: the Efficacy of the Free-Living Nematode as an in Vivo Toxicological Bioassay

The efficacy of the free-living nematode Panagrellus silusiae as a toxicological assay organism was studied using nickel, cadmium, lead, chromium, mercury, copper and zinc as test substances. Acute lethal toxicity values (LC50 values) were obtained for each heavy metal and Panagrellus was found to be highly resistant. When the LC50 values are ordered and correlated, there was no difference between rankings for juvenile (J2) and adult worms. Juvenile worms tend to be slightly more sensitive than adults but this trend was not significant. Testing of the chronic effects of the seven heavy metals revealed that (1) copper, chromium and cadmium effectively blocked growth at all stages of development, (2) the highest soluble concentrations of lead and nickel were without effect on growth of the worms, (3) high concentrations of zinc (>500 mg/l) partially blocked growth and (4) mercury was either lethal (20 mg/l) or ineffective in blocking growth (10 mg/l). When compared with the toxicological data that have been obtained for a number of other organisms, Panagrellus may have some fundamental shortcomings as a model in vivo bioassay organism if the present results with heavy metals pertain to other kinds of toxic substances.

Isolation and characterization of the cuticle from the free-living nematode Panagrellus silusiae

The cuticle (0.15 to 0.5 μm thick) of the microscopic free-living nematode Panagrellus silusiae was isolated intact by incubating worms with 1% sodium dodecyl sulfate at 37°C overnight. After shearing and further treatment with detergent, electron microscopy revealed that the cuticular pieces were free of contaminating material and retained their characteristic in situ ultrastructure. From amino acid determinations, the cuticle is collagen-like with high levels of glycine (≈31 residue %), proline (≈20 residue %) and alanine (≈21 residue %) although the hydroxyproline (2.6 residue %) content is low. Half-cystine (≈1 residue %) is present in purified cuticles. Treatment with 8 M guanidine hydrochloride-2% β-mercaptoethanol can solubilize more than 85% of the cuticular preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized cuticles from juvenile, adult and old dead worms revealed, at least, 18 discrete components. Estimated molecular weights ranged from about 26 000 (peak 1) to 250 000 (peak 18).

Isolation and translation in vitro of poly(A)+ RNA from the free-living nematode Panagrellus silusiae

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)-RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)- RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.

Characterization of the genome of the free-living nematode Panagrellus silusiae: absence of short period interspersion

The complexity of the DNA of the free-living nematode Panagrellus silusiae has been examined. Reassociation kinetics of pressure-sheared fragments (approximately 290 nucleotides) in 0.18 M Na+ at 60 degrees C showed the presence of foldback, repetitive, and unique DNA sequence elements. The three classes comprise 9.3%, 26.1%, and 61.3% of the total DNA, respectively. The mean length of the foldback duplex DNA after digestion with S1 nuclease is about 185 nucleotides. There are about 1.8 x10(4) inverted repeats per genome. Sequence arrangement was deduced from (1) renaturation kinetic profiles of long and short fragments on hydroxylapatite; (2) the pattern of renaturation of tracer DNA, labeled in vitro with 125I, of various sizes after incubation with excess short fragments; and (3) thermal denaturation behavior of DNA that had been reassociated to various C0t values. It was found that DNA fragments of the repetitive fraction that are, at least, 2000 nucleotides in length are virtually free of unique sequences. Moreover, it is estimated that the repeated segments in this species could extend for 10,000 nucleotide pairs. Thus, Panagrellus DNA lacks the pattern of extensive short period interspersion that is typified by the DNA of Xenopus.

In vitro translation of nematode cuticular collagens

Phenanthroline treatment of growing cultures of the free-living nematode Panagrellus silusiae was used to lower the degree of hydroxylation of nascent collagen chains at the polysomal level. Under these conditions, the bound pentasome-hexasome fraction provided substrate for prolyl hydroxylase. When this polysomal fraction was subsequently tested in a cell-free wheat germ system, collagenase-susceptible translation products were observed after sodium dodecyl sulfate-acrylamide gel electrophoresis. The electrophoretic mobilities of each of these four major collagen products were similar to four collagens that are isolated from intact cuticles. In addition, purified polysomal RNA that adhered to unmodified cellulose directed the synthesis of four pepsin-resistant polypeptides that had molecular weights that coincided with four pepsin-resistant collagens that can be purified from the cuticle of this species. Thus, the polysomal site of the messenger RNAs for the cuticular collagens of P. silusiae was located. Although precursor forms of the cuticular collagens were not produced in the cell-free system, the question whether additional amino acid segments occur on the primary translational products of the cuticular collagens in vivo remains open.

Partial purification and characterization of prolyl hydroxylase from the free-living nematode Panagrellus silusiae

Prolyl hydroxylase was isolated from the free-living nematode Panagrellus silusiae by ammonium sulfate precipitation and calcium phosphate gel ion exchange from Triton X-100 treated homogenates. The enzyme preparation was highly purified but not to homogeneity as judged by agarose gel filtration and sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. Gel filtration indicated that the molecular weight of the enzyme approximated 285 000. Analysis by SDS–acrylamide electrophoresis revealed subunits of ~67 000. Complete enzymic activity required α-ketoglutarate, Fe2+, ascorbate, catalase, atmospheric oxygen, and dithiothreitol. This activity was dramatically inhibited by α, α-dipyridyl, phenanthroline, and polyproline II. The purified enzyme preparation synthesized 50 to 100 μg hydroxyproline per milligram chick protocollagen per hour at 30 °C with an estimated Km for the substrate of 80 μg. Thus, a purified prolyl hydroxylase from a simple invertebrate possesses many of the properties of the prolyl hydroxylases of various vertebrates.

Cytophotometric analysis of basic nuclear proteins during gametogenesis in the free-living nematode Panagrellus silusiae.

The cytochemical staining characteristics of basic nuclear proteins during gametogenesis in both sexes of the free-living nematode Panagrellus silusiae were monitored by absorption microspectrophotometry. During spermatogenesis, the levels of both of the nuclear-bound Sakaguchi and 1-fluoro-2, 4-dinitrobenzene (FDNB) reagents per Feulgen-DNA content increase prior to meiosis and subsequently drop off during spermiogenesis. During oogenesis, the amount of Sakaguchi-protein to DNA ratio rises slightly whereas the FDNB-protein to DNA ratio undergoes a meagre decline. Since the oocyte of Panagrellus accumulates DNA in excess of a 4C DNA equivalent, the relative constancy of the ratio of basic nuclear protein content to DNA amount during oogenesis signifies that these proteins are being continously produced during oocyte maturation. After treatment with alkaline fast green (pH 8.1) the cytoplasmic and nuclear regions of the gonocytes of both sexes yield such an intense reaction that microspectrophotometric measurements could not be taken. The basis of the cytoplasmic staining reaction to the fast green (pH 8.1) dye was not determined. The rapid, highly deterministic programme of embryogenesis in Panagrellus may require a substantial stockpiling of basic nuclear proteins during oogenesis.

Relative ribosomal RNA cistron multiplicity in oocytes and postembryonic stages of the eutelic nematode Panagrellus silusiae.

The number of ribosomal RNA cistrons has been measured in the total DNA extracted from L2 juvenile and adult stages of the free-living nematode Panagrellus silusiae. Saturation hybridization studies with homologous rRNA indicate that both stages have about 275 ribosomal genes per haploid equivalent. Using homologous 125I-labelled rRNA for in situ hybridization, the mean number of silver grains per DNA content for oocyte, hypodermis and gut nuclei was similar. The mean DNA contents of maturing oocyte, hypodermis and gut nuclei are about 20C, 2C, and 10C respectively. We conclude that rDNA amplication alone is insufficient to account for the variation in DNA content of oocytes and that postembryonic development in this eutelic organism occurs without a significant differential increase in the number of ribosomal cistrons per worm.

Programmed synthesis of collagen during postembryonic development of the nematode Panagrellus silusiae

The relative rate of collagen synthesis in the free-living nematode Panagrellus silusiae during postembryonic development was found to be discontinuous by measuring either the incorporation of tritium into material extracted as collagen or the amount of collagen-bound tritiated proline and hydroxyproline after 2-hr incubations of whole worms with [ 3H]proline. A peak of collagen production preceded each of the three molts that were examined. Moreover, protocollagen prolyl hydroxylase activity during each intermolt period paralleled the pattern of collagen synthesis. On the other hand, a triphasic pattern was not observed when noncollagenous proteins were labeled with either [ 3H]tryptophan or [ 3H]leucine. In addition, the level of soluble radioactive proline that accumulates in whole organisms after 2-hr incubation periods did not fluctuate appreciably during postembryonic development. The mean ratio of hydroxy-proline to proline in a number of collagen samples extracted at various times during the maturation phase was 0.113 ± 0.040. Pulse and chase experiments with [ 3H]proline indicated that most of the collagen synthesized during a peak period is lost after the second ecdysis following the labeling interval. In contrast, a considerable proportion of the collagen synthesized during nonpeak periods is retained throughout the postembryonic period. It is postulated that the modulated pattern of collagen biosynthesis in Panagrellus reflects, for the most part, a quantitative regulation of the production of cuticular collagen during postembryonic development.

Properties and synthesis of ribosomal RNA in the free-living nematode Panagrellus silusiae

The nominal S values of ribosomes and ribosomal subunits extracted from the free-living nematode Panagrellus silusiae are similar to those of other eukaryotic organisms. Upon heating (70°C, 15 min) a 5.5-S RNA is released from the larger ribosomal RNA (27 S; 1.40 · 10 6 daltons). Unlike other protostomes, however, the 27-S rRNA does not dissociate in an orderly manner to yield 18-S components. The electrophoretic mobility of the small ribosomal RNA species (18 S; 0.72 · 10 6 daltons) is unaltered by heat treatment. The base composition of both Panagrellus rRNA species has a 51% G+C content. Labelling worms with either [ Me- 3H]methionine or [ 14C]uridine at various stages of postembryonic development and subsequent analysis of the extracted RNA revealed eight radioactive fractions, two of which were the mature ribosomal RNAs. From both the time sequence of appearance of the eight RNA species and the relative degree of methylation of the various RNA fractions two maturation pathways for ribosomal RNA processing were deduced. A 37-S (3.6 · 10 6 daltons) RNA is the precursor to both 27-S and 18-S RNA. During processing about two-fifths of the original transcriptional unit is lost. In this organism the “transcribed spacer” regions may be methylated although alternate interpretations have not been precluded.

DNA accumulation during oogenesis in the free-living nematode Panagrellus silusiae

DNA contents of nuclei at different stages of gonadogenesis, gametogenesis and embryogenesis in the free-living nematode Panagrellus silusiae were measured by Feulgen microspectrophotometry. Somatic nuclei of gonadal tissue in both males and females have DNA values that range from 2C to 4C. Relative DNA values identified meiotic processes during spermatogenesis. In the adult female, developing oocytes amass an amount of nuclear DNA in excess of the expected 4C equivalent. The chromatin of these nuclei is diffuse with varyingsized clumps of Feulgen-positive material. Throughout oogenesis maturing oocytes remain uninucleolate. — The extra oocyte DNA is distributed proportionately at each of the first four cleavage divisions; thereafter, its presence is not resolvable. — A standardized microphotometric comparison indicated that the amount of DNA in the unit genome of P. silusiae is about 0.1 pg. The oviduct oocytes accumulate about 3.6 pg DNA in excess of a 4C equivalent. The kind of DNA that builds up during oogenesis in this organism is not known.

Nematode Morphogenesis: Localization of Controlling Regions by Laser Microbeam Surgery

Laser microbeam studies reveal that postembryonic development of the free-living nematode Panagrellus silusiae is under the control of specific regions. Growth is regulated by the hindgut, and ecdysis by nerve cell bodies situated anterior to the nerve ring, and gonad development is under the control of the nerve ring. This latter event is presumably neuronally mediated, while the other events are under hormonal control.

Regulation of nematode behavior by physical means

Application of microwave radiation, laser microbeam irradiation, and short pulses of electric current stimulates changes in normal swimming behavior of the free-living nematode Panagrellus silusiae. The altered behavior, termed activated behavior, involves increased movement of the ends of the nematode, rapid changes in orientation, and an increase in the proportion of the swimming time that the nematode is curved such that the ends are pointing in the same direction. Laser microbeam studies indicate that the receptor for mating attraction is situated on the spicule. Panagrellvs silusiae shows mating attraction with two strains of P. redivivus, but the P. redivivus strains only attract P. silusiae or the other P. redivivus strain. Experiments with pulses of electric current indicate that the nematodes can be conditioned and that this conditioning requires RNA and protein synthesis.

Effects of cyclohexamide and hydroxyurea on mating behavior and its development in the free-living nematode Panagrellus silusiae (de Man 1913) Goodey, 1945

Females of the free-living dioecious nematode Panagrellus silusiae that have undergone postembryonic development in hydroxyurea do not attract males, and treated males do not respond to normal females. Nematodes placed in hydroxyurea as adults have normal gonads and retain the sexual attraction system. Hydroxyurea has no effect upon copulatory behavior of adult animals. Treatment of adult females with actidione stops production of attractant, although production resumes within 24 h after removal of the females from actidione. Actidione treatment of males has no effect upon the response to attractant by males or on copulation. It is proposed that sexual attraction depends on complete development of the reproductive system, but that copulation does not.

Cytoplasmic organelles present during spermatogenesis in the free-living nematode Panagrellus silusiae

By electron microscopy it was found that two kinds of cytoplasmic organelles occur during spermatogenesis in the free-living nematode Panagrellus silusiae. One type of organelle is called a vesicular body (v-body) and lines the complete internal cell surface of the spherical and aflagellate sperm. V-bodies (about 0.35 μm × 0.6 μm) are formed in the primary spermatocytes and after meiosis they migrate to the surface of the spermatid. In the primary spermatocytes, before the appearance of v-bodies, many double membrane - bound, ovoid crystalline bodies (c-bodies) are formed. These bodies (about 0.5 μm × 0.7 μm) subsequently degenerate and no trace of their fibrillar material is observed in the spermatozoon.The mature sperm is devoid of microtubules, centrioles, Golgi material, and acrosomal-like structures; however, numerous mitochondria lie beneath the surface vesicles and in the central region of the mature sperm the chromatin occurs as discrete condensed clumps.

Number and DNA content of nuclei in the free-living nematode Panagrellus silusiae at each stage during postembryonic development.

Nuclei from the four major tissues of the nematode Panagrellus silusiae were enumerated and examined using Feulgen microspectrophotometry at each stage during postembryonic development. The number of nuclei in the hypodermis, nerve, and intestine remains fairly constant during maturation, but there is a slight increase (∼57%) in the number of muscle nuclei. Thus, this organism is not stringently eutelic. The total number of somatic nuclei is about 600. DNA values of hypodermis and nerve nuclei were unimodal and adult nuclei had 2C amounts of DNA. The DNA distribution of muscle nuclei reflects the pattern expected for a tissue in which a portion of the nuclei are undergoing DNA synthesis. Intestinal nuclei accumulated DNA in the absence of nuclear division and in the adult the nuclei fall into discrete DNA classes which correspond to a geometric series of the 2C value. It is concluded that chromatin diminution does not occur in this species. In addition, the relationship in the different tissues of nuclear DNA content to nuclear volume and cell size is discussed.

Nematode morphogenesis: fine structure of the molting cycles in Panagrellus silusiae (de Man 1913) Goodey 1945

The mode of formation of a new cuticle in the free-living nematode Panagrellus silusiae is similar at each of the three postpartum molts. Molting begins with the appearance of filaments adjacent to the hypodermis. The new cuticle accumulates material and lacks organization during the early stage of the molt. The edge of the new cuticle adjacent to the old cuticle is composed of an amorphous row of particles and a filamentous region abuts the hypodermis. Eventually, a complete cuticle is produced beneath the preexisting one.The shedding of the old cuticle varies in different molts. In the larval molts (L2 to L3 and L3 to L4) and the molt to the adult female the cuticle is discarded piecemeal. During the molt to the adult male the old cuticle splits and is shed as a single piece.Before completion of ecdysis in the final female molt, the new cuticle folds extensively. This folding does not occur during any other molt.Resorption of the old cuticle is never observed. There is no microscopic evidence of protein synthesis in the interchordal hypodermis during the molting process.

Nematode morphogenesis: fine structure of the cuticle of each stage of the nematode, Panagrellus silusiae (de Man 1913) Goodey 1945.

The fine structure of the cuticle of each of the five stages of the free-living nematode Panagrellus silusiae (de Man 1913) Goodey 1945 has been examined. The cuticle plan is made up of a few regions: (1) an outer region (~350 Å) which consists of three layers: a thin outer layer, an electron transparent layer, and a thin but diffuse inner layer; (2) an inner region which has virtually no resolvable substructure other than a striated layer. This layer is not usually seen in the early stages of development but is readily apparent in the L4 and adult stages. The pattern of the cuticle is the same for each stage.The lateral fields—alae—reveal a definite differentiation during the life cycle; in the larval stages they are mere bulges but in the adults they take on a distinct four-lobed shape. The cuticle lining the pharynx consists of two regions, and there is never a striated band present nor does the cuticle increase in width during nematode growth.The interchordal hypodermis is devoid of endoplasmic reticulum, Golgi material, and mitochondria, but in some regions glycogen is found.