Parascaris Univalens Research Articles

Problems of Bad Contrast in Conventional Microscopy Solution and Speckle Elimination with a Laser Fourier Holographic Microscope

The problem of bad contrast in conventional microscopy is well-known and was solved in part by colouring the samples. It is shown theoretically that a laser Fourier holographic microscope produces images undisturbed by speckle-noise. A laser holographic microscope (LHM) is investigated experimentally. The instrument uses visible radiation of λ = 0.514 µm, Mach – Zehnder scheme optical setup, and CCD detector of the hologram. Images are reconstructed digitally. The standard slide of Parascaris Univalens Iarva (ascaris) is studied without any drying as for electron microscope. Comparison of the pictures of the same ascaris cell, observed by the LHM and high-quality Nikon conventional optical microscope with immersion oil and green filter indicates dramatically different contrast. The ultrahigh contrast of the LHM gives much more micromorphological information.

Parascaris univalens--a victim of large-scale misidentification?

The equine ascarid parasite Parascaris equorum is well known as a ubiquitous parasite infecting foals. A sibling species, Parascaris univalens, was first described over 130 years ago, but very little attention has been given to its existence and possible implications for anthelmintic resistance, clinical disease, or host age spectrum. P. univalens only possesses one germ line chromosome pair as opposed to two for P. equorum, but the two species are otherwise considered morphologically identical. For the present study, live worms obtained from the University of Kentucky parasitology horse herd were dissected and identified using karyotyping techniques. With no exception, all specimens (n = 30) were identified to be P. univalens. Further, the karyotyping technique was adapted to ascarid eggs derived from fecal samples and carried out on samples collected from 25 Thoroughbred foals from three farms in Central Kentucky. P. equorum was not identified among these, whereas P. univalens was found in 17 samples, with the remaining being inconclusive. The mitochondrial genome was sequenced, assembled, and annotated from one male worm identified as P. univalens, and comparison with available sequence reads labeled as P. equorum revealed only 0.16% nucleotide differences. However, it is unlikely that the sequences available in public databases have been unequivocally identified to species level by karyotyping. Taken together, these data suggest that P. univalens is likely the main species now observed in equines and that perhaps the designation Parascaris spp. should be used unless cytological characterization has confirmed the species.

Chromatin diminution leads to rapid evolutionary changes in the organization of the germ line genomes of the parasitic nematodes A. suum and P. univalens.

Chromatin diminution in the parasitic nematodes Ascaris suum and Parascaris univalens represents a rather complex molecular phenomenon that includes chromosomal breakage, DNA degradation and new telomere formation. At a given elimination site, DNA breakage and new telomere addition does not take place at a single chromosomal locus but at many different places within a several kilobase long chromosomal region, referred to as chromosomal breakage region (CBR). Here we describe the cloning and the characterisation of seven CBRs from A. suum and P. univalens and we show that the process has been conserved between the two species. A detailed sequence comparison provides evidence that the sequences of the CBRs and their flanking regions are not directly important for the specification of the elimination sites. Six out of the seven CBRs are conserved between the two nematode species, suggesting that they have already existed in a common ancestor. We present a hypothesis stating that the elimination process ensures the maintenance of a functional somatic genome and concomitantly allows extremely rapid and profound changes in the germ line genome, thereby allowing the development of new germ line specific functions and thus providing a selective advantage for the chromatin eliminating nematodes during further evolution.

Chromatin diminution in the parasitic nematodes Ascaris suum and Parascaris univalens

Chromatin diminution in Parascaris univalens and Ascaris suum undoubtedly represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. It is a complex mechanism involving chromosomal breakage, new telomere addition and DNA degradation, and occurs in all presomatic cells. The process is rather specific with respect to its developmental timing and the chromosomal regions that are eliminated. The functional significance of chromatin diminution still remains an enigma. The fact, however, that single-copy, protein-coding genes are contained in the eliminated DNA demonstrates that in P. univalens and A. suum, there is a qualitative difference between germ-line and somatic genomes, and suggests that chromatin diminution may be used as a “throw-away” approach to gene regulation. We present a hypothesis as to how, during evolution, a partial genome duplication might have been linked to the process of chromatin diminution, in order to provide a selective advantage to parasitic DNA-eliminating nematodes.

A pentamer-repeat-containing DNA sequence in Texas bluebonnet (Lupinus texensis Hook.)

A 654-bp EcoRI restriction fragment called Lup4 was shotgun cloned from Texas bluebonnet (Lupinus texensis Hook.) nuclear genomic DNA that had been extracted from plant leaves. Its sequence was determined on both strands. Southern blot analysis of EcoRI-digested Texas bluebonnet DNA revealed clone hybridization throughout the approximately 1.0 to > 23 kb size range; the darkest hybridization was in the high molecular weight range (approximately 6 to > 23 kb). Using dot blot analysis, the copy number of the 654-bp clone was estimated to be approximately 4.75 x 10(4) copies per haploid genome, and thus this repetitive sequence comprises 3.5-4.1% of the nuclear genome. Sequence analyses showed that this clone contains multiple repeats: mainly the ATTGG pentamer, but also derivative pentamers (e.g., GTTGG, TTTGG, ATAGG, and GGATT). Examination of DNA base occurrence revealed a strand bias in base composition. Data-base searches using the algorithm FASTA revealed no alignment with existing plant data base sequences, but showed sequence identity with several telomeric and satellite DNA sequences. The two highest-scoring alignments were with another pentamer-containing sequence from a telomeric region of the nematode Parascaris univalens.

PUMA1: a novel protein that associates with the centrosomes, spindle and centromeres in the nematode Parascaris

We have identified a 227 kDa spindle- and centromere-associated protein in Parascaris, designated PUMA1 (Parascaris univalens mitotic apparatus), using a monoclonal antibody (mAb403) generated against Parascaris embryonic extracts. PUMA1 distribution was studied by immunofluorescence microscopy in mitotic and meiotic Parascaris cells, where centromere organization differs greatly. In mitosis, PUMA1 associates throughout cell division with the centrosomes and kinetochore-microtubules, and it concentrates at the continuous centromere region of the holocentric chromosomes. PUMA1 also localizes to the spindle mid-zone region during anaphase and at the midbody during telophase. In meiosis, PUMA1 associates with the centrosomes and with the discrete centromeric regions lacking kinetochore structures. The analysis of colchicine-treated embryos indicated that the association of PUMA1 with the centromeric region depends on microtubule integrity. mAb403 also recognizes spindle components in Drosophila. A series of overlapping cDNAs encoding the gene were isolated from a Parascaris embryonic expression library. Analysis of the nucleotide sequence identified an open reading frame capable of encoding a protein of 227 kDa. Analysis of the protein sequence indicated that PUMA1 is predicted to be a coiled-coil protein containing a large central alpha-helical domain flanked by nonhelical terminal domains. The structural features and cellular distribution of PUMA1 suggest that it may play a role in the organization of the spindle apparatus and in its interaction with the centromere in Parascaris.

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens.

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A3/ distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are located in the middle of the euchromatic central regions. This location implies that no rDNA sequences are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

Chromatin diminution is strictly correlated to somatic cell behavior in early development of the nematode Parascaris univalens.

We have studied the relationship between the occurrence of chromatin diminution and the developmental behavior of somatic blastomeres in early development of the nematode Parascaris univalens. A cytological and immunocytochemical analysis of chromatin diminution was performed in P. univalens embryos exposed to 'vegetalizing' (LiCl) and 'animalizing' (NaSCN) substances during early developmental stages. We have also analyzed chromatin diminution in embryos displaying only symmetrical 'somatic-like' divisions due to a brief cytochalasin B treatment at the pronuclear stage. The results show that LiCl treatment induces chromatin diminution in P0-P4 pregerminal blastomeres while NaSCN treatment prevents it. Pregerminal cells undergoing chromatin diminution in 'vegetalized' embryos behaved like somatic cells with respect to division and cleavage patterns. NaSCN treatment results in undiminuted polynucleated embryos that are not capable of cleavage. In cytochalasin B-pulsed embryos, chromatin diminution occurs in all blastomeres. From our results we conclude that chromatin diminution and somatic cell behavior are not separable and constitute strictly correlated events in Parascaris. Moreover, the results indicate that the segregation of the cytoplasmic factors involved in chromatin diminution in early Parascaris development are microfilament-mediated. Here, we also report the formation of a latter pregerminal cell precursor (P5) not susceptible to LiCl-induced chromatin diminution.

Kinetochores and chromatin diminution in early embryos of Parascaris univalens.

In Parascaris the mitotic chromosomes of gonial germline cells are holocentric and possess a continuous kinetochore along their entire length. By contrast, in meiotic cells, the centromeric activity is restricted to the heterochromatic tips where direct insertion of spindle microtubules into chromatin without any kinetochore plate is seen. In the presomatic cells of early embryos, which undergo heterochromatin elimination, only euchromatin shows kinetic activity. After developing a technique to separate the very resistant egg shell from the embryos, we studied the cell divisions during early embryogenesis by immunochemical and EM approaches. The results reported here show that in presomatic cells microtubules bind only the euchromatin where a continuous kinetochore plate is present. We also report observations suggesting that the binding of the long kinetochores to the mitotic spindle initiates to a limited number of sites and extends along the entire length, during chromosome condensation. The existence of different centromere stages in different cell types, rends Parascaris chromosomes a very good model to study centromere organization.

The highly variable pentameric repeats of the AT-rich germline limited DNA in Parascaris univalens are the telomeric repeats of somatic chromosomes

We have characterized the organization of the germline limited DNA of P. univalens by means of sequence analysis. The repeat unit of this satellite DNA is the pentanucleotide 5'TTGCA, although there is a high degree of sequence variation. Repeat variants are not arranged in tandem but in a disperse, nonrandom manner. In the somatic genome which arises from the germline genome through extensive genomic rearrangement early in development, copies of these pentamers represent the telomeric repeats, indicated by their sensitivity to Bal 31 and their presence in a somatic endlibrary. Unlike telomeric sequences from other species the P. univalens telomeres do not display consecutive guanines and no strand bias for that base, recently suggested as universal features of eukaryotic telomeres. Investigation of fragments that carry pentameric repeats along with sequences of different type identifies a 5 bp consensus sequence at the junction point. We suggest a model in which pentameric repeats originate via amplification by a terminal transferase (telomerase) in both the germline and the somatic genome.

Centromere organization in meiotic chromosomes of Parascaris univalens.

The chromosomes of Parascaris univalens possess a continuous centromeric structure spanning their entire length in gonial cells. A cytological and ultrastructural analysis of P. univalens meiotic chromosomes was performed. The results show that during meiosis the holocentric germline chromosomes of male P. univalens undergo restriction of kinetic activity to the heterochromatic terminal regions. These regions lack kinetochore structures and interact directly with spindle microtubules.

Cytological analysis of chromosomes in the two species Parascaris univalens and P. equorum

Chromosome organization and the phenomenon of “chromatin diminution” in the two species Parascaris univalens (2n=2) and P. equorum (2n=4) were cytologically analysed by a variety of staining techniques (Quinacrine, Hoechst 33258, Chromomycin A3 and C-banding). The results show that: (1) the chromosomes of the two species differ markedly in both the location and the type of heterochromatin they contain; (2) in both species there is a strong chromosome polymorphism which, however, ranges within a basic species-specific phenotype; (3) the heterochromatin can be eliminated in presomatic cells during early embryogenesis at two different stages and in both cases the consequence of this process is the generation of somatic cells with a 2n=60 karyotype. Moreover, evidence suggesting the sterility of hybrids between the two species is provided.

GENETIC VARIATION OF ASCARIDOID WORMS WITH DIFFERENT LIFE CYCLES.

The hypothesis that genetic variation of populations is positively related to the degree of environmental variation they experience was tested by several authors (e.g., Bryant, 1974a, 1974b; Hedrick et al., 1976; Valentine, 1976; Steiner, 1977; Nevo, 1976, 1978; Mitter and Futuyma, 1979; Nevo et al., 1984). These studies yielded contradictory results, partly because of the experimental design and/or the organisms compared. For example, some animal groups are physiologically more buffered than others against environmental variation, and this can affect their levels of genetic variability (Selander and Kaufman, 1973). The effect of environmental variation might be detectable only when the organisms studied are closely related and ecologically similar. In the present study, the genetic structure of several Ascaridoid worms is compared by means of multilocus electrophoresis. Evidence is given that species whose life cycle is carried out on a single homeothermic host show lower genetic variability (by a factor of about 1/3) than do those needing both poikilothermic and homeothermic hosts. Natural selection is suggested to be the major factor promoting and maintaining the different patterns of genetic variability found in these parasites. The 17 species studied belong to the superfamily Ascaridoidea; nine of them (i.e., Ascaris lumbricoides, A. suum, Parascaris univalens, P. equorum, Neoascaris vitulorum, Toxocara canis, T. cati, Toxascaris leonina, Baylisascaris transfuga) are single-host species, while eight (i.e., Anisakis simplex A, A. simplex B, A. physeteris, Phocascaris cystophorae, Contracaecum osculatum A, C. osculatum B, C. rudolphii A, C. rudolphii B) are multiple-host species. The former parasitize terrestrial mammals, whereas the latter live as larvae on poikilothermic hosts (first on crustaceans, later on fishes or squids of various size) and as adults on homeothermic hosts (marine mammals or fish-eating birds). Horizontal starch gel electrophoresis was performed on single specimens, both larvae and adults, for the following enzymes: octanol dehydrogenase (ODH), a-glycerophosphate dehydrogenase (a-GPDH), sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), malic enzyme (ME), isocitrate dehydrogenase (IDH), 6-phosphogluconate dehydrogenase (6-PGDH), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldheyde-3-phosphate dehydrogenase (G3PDH), xanthine dehydrogenase (XDH), superoxide dismutase (SOD), nucleoside phosphorylase (NP), glutamate oxaloacetate transaminase (GOT), hexokinase (HK), adenylate kinase (ADK), phosphoglucomutase (PGM), esterase (EST), acid phosphatase (ACPH), leucine aminopeptidase (LAP), aldolase (ALD), carbonic anhydrase (CA), triose phosphate isomerase (TPI), mannose phosphate isomerase (MPI), and glucose phosphate isomerase (GPI). The buffer systems used were: 1) tris-versene-borate (Brewer and Sing, 1970) for ODH, G6PDH, SOD, NP, CA, MPI, and GPI; 2) tris-versene-maleate (Brewer and Sing, 1970) for LDH and PGM; 3) phosphate-citrate (Harris, 1966) for a-GPDH, MDH, 6-PGDH, and ACPH; 4) discontinuous tris-citrate (Poulik, 1957) for EST and LAP; 5) continuous tris-citrate-II (Selander et al., 1971) for SDH, ME, IDH, G3PDH, XDH, GOT, HK, ADK, ALD, and TPI. The staining techniques were, with minor modifications, those described by Brewer and Sing (1970; LDH and PGM), Shaw and Prasad (1970; SDH, MDH, IDH, 6-PGDH, G6PDH, and XDH), Selander et al. (1971; SOD, GOT, and GPI), Ayala et al. (1972; ODH, a-GPDH, ME, G3PDH, HK, ADK, EST, LAP, ALD, and TPI), and Harris and Hopkinson (1976; NP, ACPH, CA, and MPI). Each species was tested for a number of loci ranging from 18 to 28, 12 of which (Ldh, Mdh-1, Idh-1, 6-Pgdh, G3pdh, Got-i, Adk-2, Pgm-1, Est-], Est-2, Mpi, and Gpi) were the same for all the species studied. The following parameters were used to estimate genetic variability: expected mean heterozygosity per locus (He); proportion of polymorphic loci, according to

Centromere ultrastructure in germ-line chromosomes of Parascaris.

Ultrastructural analysis of the centromere in germ-line mitotic chromosomes of Parascaris univalens and Parascaris equorum revealed that these chromosomes are holocentric. In thin longitudinal sections of both species the kinetochore appeared as a continuous plate (up to 3.8 micron long) and displayed a layered structure. This structure consisted of electron-dense inner and outer layers (average width 10 nm) separated by a less dense middle layer (25 nm wide), which had transverse electron-dense bars (10 nm wide) regularly spaced every 25-30 nm. Thus the ladderlike kinetochore profile observed in Parascaris gonial mitotic chromosomes represents a different type of organization from that of the classical trilaminar kinetochore found in both holocentric and monocentric chromosomes.

Chromosome Organization and Heterochromatin Elimination in Parascaris

A cytological analysis by modern banding techniques of gonial metaphases in two Parascaris forms that have been considered varieties but now seem to be two species [P. univalens (karyotype 2n = 2) and P. equorum (karyotype 2n = 4)] reveals a different chromosome organization in each. Parascaris univalens chromosomes contain only terminal heterochromatin, while P. equorum chromosomes also contain intercalary heterochromatin. In the somatic cells of both species during early embryogenesis, chromatin diminution occurs in and consists of the elimination of all heterochromatin independent of its localization in the chromosomes.