Effects of pH on Excystation of Gregarina cuneata and Gregarina polymorpha (Eugregarinida: Gregarinidae)1

ABSTRACT. The mechanisms that signal gregarine excystation are unknown. Previous authors have demonstrated that gregarine sporozoites excyst from their surrounding oocyst in response to stimuli contained in host digestive fluids, but the role of host intestinal pH in this signaling system has not been investigated. In this study, an in vitro assay is used to quantify the effects of 3 pH levels (6.1, 7.0, 8.0) on the excystation of two gregarine species, Gregarina cuneata and Gregarina polymorpha. Both gregarine species excyst at all three pH levels, but there are significant within and among species differences in excystation rate and cumulative excystation over time. Gregarina cuneata excysts more rapidly at pH 6.0 and G. polymorpha excysts more rapidly at pH 8.0. Cumulative excystation is maximized at pH 6.0 for G. cuneata and at pH 7.0 for G. polymorpha. Hydrogen ion mediated excystation may lead to the formation of foci for subsequent establishment or migration and may play a role in parasite site specificity.     

Actin and spectrin-like (Mr= 260–240 000) proteins in gregarines

In Gregarina blaberae a M_r = 47 000 and a M_r = 260–240 000 doublet polypeptides reacted in immunoblotting: i) with a polyclonal monospecific rabbit antibody to frog muscular actin, a monoclonal anti-actin antibody against chicken gizzard; and ii) with polyclonal and monoclonal antibodies to human erythrocyte β-spectrin, respectively. The M_r = 47 000 actin-like protein is associated with the ghost and a contractille cytoplasmic extract. The presence of an actin-like protein in Gregarina and Lecudina and its cellular distribution in the cortex indicated that the gliding movement might involve an actin-myosin system in contrast to previous studies. Immunofluorescence showed clear differences between the anterior part of Gregarina and Lecudina which illustrated the high cell polarity of these protozoa. The M_r = 260–240 000 doublet was detected in SDS-PAGE from G. blaberae trophozoite ghosts but not in the cytoplasmic extracts or in extracts from sexual stages, indicating that the presence of these spectrin-like proteins is stage-dependent. Visualization of the M_r = 260–240 000 by immunofluorescence showed clear species differences, with rings arranged perpendicular to the longitudinal narrow folds of G. blaberae, with longitudinal lines underlying the folds of L. pellucida and with lines separating the large folds of Selenidium pendula. The cellular distribution is consistent with a stabilizer function of the spectrin-like proteins in the scaffolding of the cortex of gregarines according to the high diversity of the cell-shape and the cell motility systems in gregarines. The presence of spectrin-like proteins in protozoa and particularly in parasites from primitive arthropods indicated that ancestral spectrin genes could the M_r = 260–240 000 form.     

Gregarina hylobii and Ophryocystis hylobii n. sp. (ophryocystidae,neogregarinida) parasitizing Hylobius abietis (Curculionidae,coleoptera)

Two sporozoan parasites, Gregarina hylobii and Ophryocystis hylobii n. sp., parasitizing the adults of natural populations of Hylobius abietis are described. The gregarine G. hylobii, a parasite of the gut of H. abietis, has been reexamined and its complete life cycle, including the solitary gamonts, gametocysts, and sporoducts is described. The life cycle of the neogregarine O. hylobii infecting the Malpighian tubules of host animals examined at light and electron microscope levels is discussed. Some data on the prevalence of infections and host-parasite relationships are also discussed.     

Bacteria-like structures in the endoplasm of Gregarina garnhami (Eugregarinida,Protozoa)

Summary An investigation of bacteria-like structures, which are found in large numbers in the endoplasm of the eugregarine, Gregarina garnhami, was carried out using light and electron microscopy. Gram staining indicates that these structures are gram negative; they show orange fluorescence when stained with acridine orange. Ultrastructural observations show that they resemble the endosymbiotic bacteria found in other protozoan species. Some of these structures appear to be dividing, and the possibility that these structures are in fact bacteria is discussed.     

Scanning Electron Microscopy of Gregarines (Protozoa, Sporozoa) and Its Contribution to the Theory of Gregarine Movement

SYNOPSIS. Scanning electron microscopy was used to reveal detailed surface structure of 4 septate ( Gregarina cuneata, G. steini, G. rhyparobiae, Pileocephalus blaberae ) and one aseptate species ( Nematocystis elmassiani ) of eugregarines. The epicyte of all these gregarines is differentiated into a system of regular longitudinal folds. In the septate species these folds undulate so that these organisms glide along. The undulatory pattern is absent from Nematocystis , which does not glide. The theories and the mechanism of gregarine gliding are discussed.     

Substrate Contact,Mucus, and Eugregregarine Gliding1

ABSTRACT. Experiments have been carried out on Gregarina garnhami to examine some of the factors that may be significant in gliding. Suspension of gregannes in Ficoll showed that substrate contact is essential. Reflection interference microscopy shows that there are fluctuating surface/substrate contacts, but it is not necessary for the whole of the undersurface of the cell to be in close contact with the substrate. Gliding is always accompanied by the formation of a mucus trail. The effects of the drugs amphetamine and ephedrine on mucus trail formation and gliding have been examined. Lateral undulations of the epicyte folds have previously been implicated in grcgarinc gliding, but G. garnhami does not exhibit lateral undulations of the epicyte folds as it moves. The folds are predominantly straight with indications of varicosities or swellings along the length of the folds. These observations are discussed in relation to gliding movement.     

The Site Specificity of Two Species of Gregarina in Tenebrio molitor Larvae

The site specificity of the apicomplexans Gregarina cuneata and Gregarina sleini , in larval Tenebrio molitor was investigated. Gregarina cuneata was found to inhabit the anteriormost region of the larval midgut, while G. steini was restricted to the posterior portion of the intestine. The site specificity of the pair was conserved in single and concurrent infections. Interspecific interactions do not seem to be presently responsible for the resource partitioning by the 2 gregarine species. Key words. Gregarina, site specificity, Tenebrio molitor.     

Chronologie des Modifications Ultrastructurales au Cours de la Croissance de Gregarina blaberae*

SYNOPSIS. In an ultrastructural study the development of the sporozoite as well as the growth and development of the trophozoite of Gregarina blaberae were followed in the course of experimental infections of larvae of the cockroach Blaberus craniifer. The spectacular growth involved the transformation within 18 days of the sporozoite, measuring 15 × 1 μm, to a cephaline—trophozoite affixed to the intestinal epithelium—of 250 μm length and 65 μm diameter. The sporozoite's ultrastructure is not different from that of sporozoites of other Sporozoa studied to date—the conoid and dense bodies are present. The pellicle consists of 3 membranes, but there are some interruptions in the internal membrane complex. The first dictyosomes are formed from the nuclear envelope. The migration of the nucleus and of the dense bodies, followed by the regression of all the structures characteristic of the sporozoites, and the establishment of a cortical zone that comes to cover the epimerite, take place within 48 hr after infection and mark the transformation of the sporozoite into the trophozoite. Development of the cephaline involves the formation of the epicytic folds, which occurs at the base of the deutomerite, starting on the 3rd day of development. A regular system of longitudinal or epicytic folds is formed over the entire surface of the gregarine. On the 4th and 5th days of development, a vacuolar system and a chondriome become differentiated in the epimerite, while a fibrillar septum separates the protomerite from the deutomerite. The next stage, starting on the 6th day, is characterized by distribution of polysaccharide reserves between these 2 segments. The model studied allows us to determine the role of the epimerite in the parasite's nutrition, as well as the development of the chondriome and of the cortical membranes in the course of the vegetative growth phase of the cephaline gregarine.     

Occurrence of pathogens in Ips typographus (Coleoptera: Curculionidae) and in other spruce bark beetles from the wilderness reserve Dürrenstein (Lower Austria)

The study presents new data on spatial distribution of bark beetle pathogens, on changes in frequency over several years and on their prevalence during different time periods within a year from several locations within the wilderness reserve Dürrenstein (Lower Austria). The occurrence of pathogens was investigated in Ips typographus (during five years), in Pityogenes chalcographus (during two years) and in Ips amitinus (in one year). In total, seven pathogen species could be detected in I. typographus. The most dominant pathogen species were the Ips typographus-Entomopoxvirus (ItEPV), the sporozoan species Gregarina typographi and the microsporidium Chytridiopsis typographi; the latter two pathogen species were recorded every year and at about similar high (G. typographi) or low (C. typographi) rates, the ItEPV in strongly varying rates. The neogregarine Mattesia cf. schwenkei and the two microsporidia Nosema typographi and Unikaryon montanum were found in I. typographus only sporadically and the rhizopodan species Malamoeba scolyti was found once. The number of infected males and females was relatively similar with almost all pathogen species in most of the years except U. montanum, which occurred exclusively in females. Three pathogen species were recorded in P. chalcographus which were Gregarina typographi, Mattesia cf. schwenkei and Chytridiopsis typographi. Two pathogen species were observed in I. amitinus, Gregarina typographi and Chytridiopsis typographi.     

Host stadium specificity in the gregarine assemblage parasitizing Tenebrio molitor.

Reciprocal cross-stadia experimental infections were used to demonstrate stadium specificity within the gregarine assemblage parasitizing Tenebrio molitor, the yellow mealworm. Gregarina cuneata, Gregarina polymorpha, and Gregarina steini are characteristic parasites of larval T. molitor. Gregarina niphandrodes is a characteristic parasite of adult T. molitor. Experimental infections were produced in all homologous host-parasite combinations. No infection was produced in heterologous or cross-stadia combinations. This study introduces the concept of separate, distinct parasite niches corresponding to separate life cycle stages and established by known, predictable life cycle events within a single host species.     

Morphological and molecular characterizations of the Gregarina sp. (Apicomplexa: Protozoa) parasitizing on Phaedon brassicae (Coleoptera: Chrysomelidae)

A gregarine parasite (Eugregarinida: Gregarinidae) was observed in the population of daikon leaf beetle, Phaedon brassicae Baly, (Coleoptera: Chrysomelidae) in Korea. Gregarines are well known species-specific parasites of various Arthropoda. This Gregarina sp. also confirmed a species-specific parasite in P. brassicae. Based on 1.727 kb of 18S rDNA sequence (FJ481523), this gregarine species was grouped in eugregarine and a 5.258 kb of full length rDNA replicon was cloned (JF412715). We also observed interaction of trophozoite or gamonto of gregarine and epithelium of a host midgut using a light microscope and a scanning electron microscope. Although the developmental period of the infected host is delayed half a day in every larval stage, there was no significant difference in the developmental period of P. brassicae whether Gregarina sp. was infected or not. Gregarina sp. was a kind of facultative parasite from P. brassicae. This is the first report of a gregarine parasite in P. brassicae.     

The structural gene for the ribosomal protein S18 in Escherichia coli. II. Chemical studies on the protein S18 having an altered electrophoretic mobility

A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.     

The Electrophoretic Velocity of Human Red Cells, of Their Ghosts and Mechanically Produced Fragments, and of Certain Lipid Complexes

Ghosts prepared in CO₂-saturated water from unwashed human red cells can be fragmented mechanically, but ghosts from thrice washed cells cannot. If the ghosts are prepared by freezing and thawing, this difference is not observed. The electrophoretic velocity varies also with the way in which the ghosts are prepared. The pH-mobility dependence of washed red cells flatten off to a plateau at pH 9, and the electrophoretic velocity is zero at about pH 2. Ghosts prepared by freezing and thawing have almost the same pH-mobility dependence, but if the ghosts are prepared in CO₂-saturated hyptonic saline, the mobility at pH 9.4 is 0.75 times that of washed cells. Fragments of ghosts of unwashed red cells have a smaller mobility than that of the red cells. Trypsin reduces the mobility of washed red cells and of ghosts. Sols of lipid complexes (lecithin, cephalin, and lipositol), at varying pH's, have a mobility 1.2 times that of the washed red cell. The pH-mobility relation is otherwise similar. These complexes can be coated with dextran and trypsin.     

Major proteins of the outer cell envelope membrane of Escherichia coli K-12: multiple species of protein I.

Summary Protein I, one of the major outer membrane proteins ofE. coli, in a number of strains exists as two electrophoretically separable species Ia and Ib. Two phages, TuIa and TuIb, have been found which use, as receptors, proteins Ia and Ib, respectively. Selection for resistance to phage TuIb yielded mutants still possessing protein Ia and missing protein Ib (Ia⁺ Ib^-). Selection in this background, for resistance to phage TuIa yielded one class of mutants missing both species of protein I and another synthesizing a new species of protein I, polypeptide Ic.Tryptic fingerprints of Ia and Ic are very similar and the sequence of 8 N-terminal amino acids is identical for Ia and Ic. Yet, Ic showed an entirely different pattern of cyanogen bromide fragments than that of protein Ia. With another example (cyanogen bromide fragments of protein II^*, with and without performic acid oxidation) it is shown that protein modification can lead to gross changes of the electrophoretic mobility of cyanogen bromide fragments. It is not unlikely that all protein I species observed so far represent in vivo modifications of one and the same polypeptide chain.A genetic analysis together with data from other laboratories revealed that at least 4 widely separated chromosomal loci are involved in the expression of the protein I species known to date.     

Polymorphism of blood plasma proteins in theAnser andBranta genera

An electrophoretical analysis of blood plasma proteins of eightAnser and twoBranta species was performed. Ten polymorphic proteins in blood plasma pattern were distinguished and described: four prealbumin proteins, albumin, three postalbumin proteins, transferrin, and a single posttransferrin protein. Genus-specific and species-specific variants of Pr-1, Al, Pa-3, Pa-X, and Tf proteins were found. The species ofBranta differed inPr-1,Pa-3,Pa-X, andTf loci. TheAnser species differed, apparently, in allele frequencies of described gene loci. A single species-specific protein marker was found in swan geese only. The electrophoretic mobilities of Pr-1, TfB, and PtfA, B, and C were similar for several species ofAnser andBranta genera.     

Molecular Phylogeny and Ultrastructure of Caliculium glossobalani n. gen. et sp. (Apicomplexa) from a Pacific Glossobalanus minutus (Hemichordata) Confounds the Relationships Between Marine and Terrestrial Gregarines

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a “trophozoite”, that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU rDNA sequence derived from two single‐cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors.     

Composition en glucides conjugués de l'hémolymphe de Blaberus craniifer: Variations au moment de l'ecdysis imaginale

Analysis of macromolecular carbohydrates performed by gas liquid chromatography on Gregarina blaberae haemolymph showed the presence of hexoses (galactose, mannose, glucose), methyl-pentose (fucose), and hexosamines (N-acetylgalactosamine, N-acetylglucosamine). No trace of pentose or sialic acid or uronic acid was found. Mannose was the main neutral sugar. A change in the molar ratio of mannose consisting of an enrichment of female haemolymph occurred during larval-adult ecdysis. There was a parallel increase in glycoprotein staining with periodate-fuchsin after cellulose acetate electrophoresis of female haemolymph.     

Phylogenic studies on ribosomal proteins

Two-dimensional electrophoresis of ribosomal proteins makes it possible to evaluate the phylogenic distance between any set of two species. The evaluation is based on the number of spots having the same electrophoretic mobility in the two species. The two-dimensional finger-prints of ribosomal proteins from mammals, reptiles and birds, which have diverged 300 million years ago, are identical. For more remote species with respect to mammals, the number of comigrating spots gradually decreases. For as remote species from mammals as plants, one third of the spots have still the same mobility. Only five proteins from E. coli comigrate with ribosomal protein from mammals. The low evolution of ribosomal proteins indicate the high degree of internal organization of the ribosome.