Comparison of restriction fragment length polymorphisms of ribosomal DNA between Diphyllobothrium nihonkaiense and D. latum.

Restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA) were compared between Diphyllobothrium latum and D. nihonkaiense using seven kinds of restriction endonucleases. No intra-specific variation in restriction fragment profiles was shown within both species of Diphyllobothrium. Digestion of the genomic DNA with three endonucleases. SmaI, HinfI and HhaI, provided one or two different bands between two species, although the hybridization patterns generated with the others. HindIII, XbaI, StyI and HaeIII, were the same in both. RFLPs in the digested profiles with SmaI, HinfI and HhaI could be used as species-specific markers even if only fragments of strobilae with morphological similarity were available. Other cestodes, Spirometra erinacei and Taenia saginata, used as controls showed quite different restriction fragment patterns with all the enzymes used.     

Differentiation of staphylococcal species and strains by ribosomal RNA gene restriction patterns

Staphylococcal DNA was digested with endonucleases and probed with labelled ribosomal RNA (rRNA) from Escherichia coli. Reproducible restriction patterns containing between seven and 22 bands were obtained for seven different species of staphylococci. These profiles were species-specific with different strains of a particular species sharing an identical or similar restriction pattern. The results reported here indicate that rRNA gene restriction pattern analyses have an application in the taxonomy of staphylococci.     

The functional morphology of the scolex of Diphyllobothrium Cobbold (Cestoda, Pseudophyllidea). A scanning electron and light microscopical study on scoleces of adult D. dendriticum (Nitzsch), D. latum (L). and D. ditremum (Creplin).

Scoleces of adult D. dendriticum, D. latum and D. ditremum were studied using scanning electron and light microscopy. In D. dendriticum and D. latum the transverse bar between the bothria at the scolex apex always possessed a frontal pit while in D. ditremum no such invagination was seen. The scoleces attach to the host intestine by taking a firm grip with each bothrium around one or two intestinal villi. In D. ditremum and D. latum a layer of secreted substance is present between the scolex apex and the host intestine and between the inner bothrium surface and the host intestinal villi. In D. dendriticum, however, such a secreted layer was only observed at the scolex apex. A general account of the musculature and nervous and excretory systems of a Diphyllobothrium scolex is given.     

Fatty acid makeup of the cestodes, Eubothrium crassum and Diphyllobothrium dendriticum]

The fat-acidic composition of E. crassum and D. dendriticum was investigated. Lipids of E. crassum differ in greater unsaturation as compared to these of D. dendriticum as well as in greater amount of acids of type omega 3, whereas acids of type omega 6 and stearic acid were found in extracts of E. crassum in less quantity than in D. dendriticum. This phenomenon is characteristic of organisms living under conditions of low temperatures.     

Differentiation of mosquito-pathogenic strains of Bacillus sphaericus from non-toxic varieties by ribosomal RNA gene restriction patterns.

DNA from 17 strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, was cleaved with EcoRI or HindIII and fragments were separated by agarose gel electrophoresis. Southern blots of this DNA were hybridized to a radioactively labelled DNA probe prepared from the cloned 16S rrnB ribosomal RNA operon of Escherichia coli. Banding patterns of the chromosomal DNA digests and the autoradiograms were specific to DNA homology groups I (B. sphaericus sensu stricto), IIA (mosquito-pathogenic strains), IIB (B. fusiformis) and V, but groups III and IV were not clearly distinguished. This suggests that the mosquito-pathogenic strains represent a separate subspecies.     

Cestode egg production dynamics and fertility in Diphyllobothrium dendriticum (Cestoda, Pseudophyllidea)

Dynamics of egg production of the tapeworm Diphyllobothrium dendriticum has been estimated experimentally in nestlings of the herring gull Larus argentatus per day and per reproductive period. The number of eggs in strobiles has been estimated for the maturation period. Mean egg production of the tapeworm per day (10.43 +/- 2.72 mln) is two orders of magnitude as high as the maximum number of eggs in a mature strobile (0.206 +/- 0.007). It is proposed to estimate coefficient of reproduction intensity as the ratio of egg output per unit of time and maximum egg number in a mature strobile.     

Genetic and morphological variability in a population of Diphyllobothrium dendriticum (Nitzsch, 1824)

Plerocercoids of Diphyllobothrium dendriticum were isolated from whitefish (Coregonus clupeaformis) from McGregor Lake, Alberta. Adult D. dendriticum were obtained by infection of golden hamsters (Mesocricetus auratus). Analysis of morphological variables indicated high variability and some overlap with D. latum. Correlations in morphology were due to the covariance of shape variables. Isozyme analysis also showed high variability, with two thirds of the enzymes studied being polymorphic. Correlations with morphology indicate a relationship between rare patterns for malate dehydrogenase and esterase and increased size of adults. Southern hybridisation with ribosomal gene probes produced two common patterns and a rare intermediate form, indicating that the species is variable even within a local population. Correlations between rDNA pattern type and morphological variables supported the hypothesis of a genetic base for the morphological variability.     

Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis

Acidophilic iron-oxidizing microorganisms are important both environmentally and in biotechnological applications. Although, as a group, they are readily detected by their ability to generate ferric iron (resulting in a distinctive color change in liquid media), these microbes highly diverse phylogenetically. Various other characteristics, such as optimum growth temperature, response to organic carbon sources, and cellular morphologies, facilitate, in some cases, identification of isolates to a genus or species level, although this approach has limitations and may give erroneous results. In this study, a combined approach of using physiological traits together with amplified ribosomal DNA restriction enzyme analysis (ARDREA) has been successful in identifying all known acidophilic iron-oxidizing bacteria to the species level. Computer-generated maps were used to identify restriction enzymes that allow the differentiation of the acidophiles, and these were confirmed experimentally using authentic bacterial strains. To test further the validity of this approach, six acidophilic moderately thermophilic iron-oxidizing bacteria isolated from Montserrat (West Indies) were analysed using the ARDREA protocol. Three of the isolates were identified as Sulfobacillus acidophilus-like, and one as Sulfobacillus thermosulfidooxidans-like bacteria. The fifth isolate gave DNA digest patterns that were distinct from all known strains of iron-oxidizing acidophiles. Subsequent sequencing of the 16S rRNA genes of these isolates confirmed the identity of the four Sulfobacillus isolates, and also that the fifth isolate was a novel species. Schematic diagrams showing how ARDREA may be used to rapidly identify all known acidophilic iron-oxidizing bacteria are presented.     

Differentiation of the tegument and associated structures in Diphyllobothrium dendriticum Nitsch (1824) (Cestoda: Pseudophyllidea). An electron microscopical study

Grammeltvedt Anne-François 1973. Differentiation of the tegument and associated structures in Diphyllobothrium dendriticum Nitsch 1824 (Cestoda : Pseudophyllidea). An electron microscopical study. International Journal for Parasitology3: 321–327. The differentiation of the tegument and associated structures of the coracidium, procercoid, plerocercoid and adult is described. The embryophore is composed of four zones and is covered by a fibrous layer resembling a glycocalyx. The oncospheral plasma membrane is extensively folded. A typical cestode tegument, with a distal and perinuclear cytoplasm, is probably already existing in the coracidium. The formation of the microvilli starts after about three days in the copepod host. In young procercoids ribosomes and Golgi complexes were observed in the distal cytoplasm. These organelles disappear at later stages. The infective procercoid has a typical tegument. The microvilli are shaped like a thorn compressed from the sides. They have an electron dense tip and a less dense base in which microfilaments are seen. Bodies, called disc-shaped and lamellated bodies, are described. The microvilli of the plerocercoid are characterized by a great variation in shape. The villi are bounded by two unit membranes. The lamellated bodies are especially well developed. The adult microvilli are uniform in shape. The lamellated bodies are few in young adults and disappear in mature worms.     

Ultrastructural studies on Diphyllobothrium ditremum and D. dendriticum (Cestoda, Pseudophyllidea), with emphasis on the scolex tegument and the tegument in the area around the genital atrium.

The scolices of D. ditremum and D. dendriticum are similar in regard to general morphology and ultrastructure of the tegument. At the scolex apex and along the upper bothrial edges sensory endings are numerous. The inner bothrium surface of D. ditremum appears to be plain or smooth, the distal cytoplasm is only about 0.6 mum thick and covered with long and slender microtriches. When D. ditremum is attached to the host gut an adhesive layer of secreted substance exists between the inner bothrium surface and the host intestinal villi. In D. dendriticum, however, no secreted layer exists between the inner bothrium surface and the host intestinal villi and the inner bothrium surface of this species appears to be lobed or lappet formed. The lobes are protrusions of the distal cytoplasm and are covered with long and slender microtriches. Large conglomerations of secretion globules appearing to be enclosed by a double membrane, are observed in the scolices of D. dendriticum and D. ditremum, although more frequent in the latter. Differences existing between the distal cytoplasm of the scolex and the gravid proglottid are described. The papillae around the genital atrium in mature and gravid segments were studied. Each papilla appears as a thickening of the distal cytoplasm, which in this region is 10-12 mum thick, when studied with transmission electron microscope. From the area between the papillae sensory endings appearing in sections to be either single, double or triple are described.     

Comparison of electrophoretic distribution patterns of ribosomal RNA gene restriction fragments and of ribosomal subunit proteins of Lactococci, Streptococci, and Pediococci.

Comparison of electrophoretic distribution patterns of ribosomal RNA gene restriction fragments and of ribosomal subunit proteins are equally effective procedures for detecting differences and similarities in the Lactococci, Streptococci and Pediococci examined. Electrophoretic distribution patterns of ribosomal subunit proteins may be a useful tool in taxonomic studies.     

The abundance and distribution of Diphyllobothrium dendriticum (Nitzsch) and D. ditremum (Creplin) in the char Salvelinus alpinus (L.) in Sweden

A total of 654 char, Salvelinus alpinus (L.) and 33 brown trout, Salmo trutta L., were examined for the plerocercoids of Diphyllobothrium dendriticum (Nitzsch, 1824) and D. ditremum (Creplin, 1825). Only char were found to be infected. Both parasitic species were abundant. The incidence and mean intensity of infection for D. dendriticum were 83.2% and 8.8, respectively; for D. ditremum 93.7% and 45.0. There was no variation in the intensity of infection between different parts of the lake. The incidence and intensity of infection increased with the age of the fish to age 8+ for both species. From this age the intensity of infection leveled off or decreased. No differences in intensity of infection between the sexes were found. The frequency distribution of parasite counts were overdispersed and fitted the negative binomial distribution even within single age-groups. The two parasite species showed a high positive correlation in intensity of infection, D. ditremum was concentrated in the stomach and pyloric caeca. D. dendriticum was more widely distributed within the host and was supposed to kill heavily infected fish.     

Organization of the yeast ribosomal RNA gene cluster via cloning and restriction analysis.

The restriction endonuclease EcoR1 cleaves Saccharomyces cerevisiae DNA, which codes for ribosomal RNA (rRNA), into seven fragments, A second restriction endonuclease, HindIII, cleaves the same yeast ribosomal DNA into two fragments. These two restriction enzymes each yield DNA segments that total about 5.9 megadaltons. The "repeat unit" of the yeast genes coding for rRNA is thus about 5.9 megadaltons or about 9000 base pairs long. The two HindIII-cleaved DNA fragments as well as one of the EcoR1-cleaved DNA fragments were purified and amplified by cloning in Escherichia coli. Three of the seven EcoR1-generated DNA fragments could then be ordered by treating the two cloned HindIII DNA fragments with EcoR1. This led the assignment of the two HindIII restriction sites. The various restriction DNA fragments were hybridized directly from the gel utilizing 32P-labeled 5 S, 5.8 S, 18 S, and 25 S rRNA. Identification of the various DNA restriction segments then led to the final ordering of the DNA fragments. The gene coding for the 5 S RNA is adjacent to the gene coding for the 35 S precursor rRNA. These two groups of genes thus occur as a cluster in the following sequence: [5 S-spacer]-[spacer-18 S-5.8 S-25 S-spacer]-[spacer-5 S]. The actual map of the DNA restriction fragments is presented.     

The immune response of wild rainbow trout, Salmo gairdneri Richardson, to naturally acquired plerocercoid infections of Diphyllobothrium dendriticum (Nitzsch, 1824) and D. ditremum (Creplin, 1825)

Natural infections of rainbow trout with two species of Diphyllobothrium result in a host inflammatory response encapsulating the plerocercoid. The encapsulating cyst, observed by light and electron microscopy, comprises leucocytes, fibroblasts and collagenous connective tissue and is infiltrated with a blood vascular network. An indirect immunofluorescence technique and enzyme-linked immunosorbant assay (ELISA) have shown that specific antibodies are elicited by the fish host to these Diphyllobothrium spp. These antibodies have been semi-quantitatively measured by ELISA and correlated with worm burdens in individual fish.     

The dynamics of infection of Diphyllobothrium dendriticum (Nitzsch) and D. ditremum (Creplin) in the char Salvelinus alpinus (L.) in Sweden

A total of 654 char, Salvelinus alpinus (L.), from Lake Bjellojaure, Sweden, were examined for plerocercoids of Diphyllobothrium dendriticum (Nitzsch, 1824) and D. ditremum (Creplin, 1825) from September 1969 to December 1974. Plankton samples showed that the dominating copepod species in the lake were Eudiaptomus graciloides (Lilljeborg), Cyclops scutifer Sars and Heterocope appendiculata Sars. E. graciloides passed the winter as adults and C. scutifer as copepodids while H. appendiculata was restricted to a short period in the summer. Copepods occurred in the stomachs in September-October and to a certain extent in April-May, but only cyclopids and Heterocope sp. were found. C. scutifer is proposed to be the main first intermediate host of both D. dendriticum and D. ditremum . The potential final hosts were thecommon gull, Larus canus L., for D. dendriticum and the black-throated diver, Gavia arctica (L.), for D. ditremum . One common gull was experimentally successfully infected with plerocercoids of D. dendriticum . The parasites showed no seasonal cycle in incidence of infection.     

Differentiation of the endemic New Zealand greenheaded and brownheaded leafroller moths by restriction fragment length variation in the ribosomal gene complex

This study identified markers in the ribosomal RNA gene complex (rDNA) and evaluated their utility in the differentiation of four endemic New Zealand species of leafroller moth, Planotortrix excessana (Walker), P. octo (Dugdale), Ctenopseustis herana (Felder and Rogenhofer) and C. obliquana (Walker). Pooled genomic DNA from laboratory-reared specimens of C. herana and C. obliquana could be distinguished with a single restriction enzyme (e.g. ClaI), while P. excessana and P. octo could be satisfactorily distinguished using double digestions with either ClaI/PvuII or ClaI/BglII. It is concluded mat these markers may be used to identify moths from wild populations.     

Two-dimensional restriction analysis of the Bacillus subtilis genome: gene purification and ribosomal ribonucleic acid gene organization.

With two-dimensional restriction enzyme analysis we have been able to cleave the Bacillus subtilis genome and resolve the resulting deoxyribonucleic acid (DNA) segments into discrete bands on agarose gels. A general procedure for gene purification has been developed by coupling multidimensional restriction analysis with a biological assay for gene detection. The organization of ribosomal ribonucleic acid (rRNA) genes was studied by hybridizing 16S and 23S rRNA probes to the two-dimensional DNA banding patterns.     

Mouse histidyl-tRNAs during pregnancy. Differentiation of activity profiles within and between organs.

Optimal conditions for in vitro formation of His-tRNA were established. Transfer RNA of maternal mouse organs and total embryo at 13 and 17 days of pregnancy was acylated in vitro with [3H] or [14C] histidine and examined by reversed-phase plaskon chromatography. Most tissues show different radioactive profiles reflecting a varying activity of six to eight isoaccepting His-tRNA species. Quantitative differences in profile were observed for liver tRNA during pregnancy. Profiles of embryo and uterus, kidney, heart and muscle changes less, and that of brain did not change during pregnancy. The significance of these observations with respect to molecular differentiation of His-tRNAs during pregnancy is discussed.