Fine Structure of Rickettsia canada in Tissues of Dermacentor andersoni Stiles

Preliminary observations on growth and developmental fine structure of Rickettsia canada in various organs and tissues of the hard tick, Dermacentor andersoni Stiles, are reported. R. canada is typically rod-shaped, being delimited with a three-layered wall having a velvety coating adsorbed to its exterior surface. A finely reticulated cytoplasmic matrix containing prominent ribosomes is delimited with a three-layered unit membrane. Average length and width of these organisms are 1.6 by 0.4 mum. Although R. canada produces a generalized infection in D. andersoni, hypodermal and muscle tissues experience heaviest growth. Three morphologically distinct rickettsial forms were observed in individual hypodermal cells: (i) typical growth forms with a finely reticulated cytoplasmic matrix and distinct ribosomes; (ii) atypical forms with lightly to densely staining cytoplasm and a coagulated appearance in which ribosomes cannot be distinguished from the matrix; and (iii) forms with crystalline bodies that have a striated to beaded lattice structure and, at times, a fibrillar body in the cytoplasm as well. Occasional finger-like to irregular invaginations of the plasma membrane are noted. Intranuclear growth was demonstrated by electron microscopy in gut epithelial cells only. Growth and development of R. canada were manifest in all tissues examined.     

Phase contrast and electron microscopy of the mycetocytes and symbiotes of the pea aphid, Acyrthosiphon pisum

Phase contrast, scanning electron, and transmission electron microscopy of the symbiotes of Acyrthosiphon pisum was undertaken. Some staining properties of the symbiotes were also studied.The symbiotes of the pea aphid were found to be coccoid bodies 2 to 5 μ in diameter, gram negative, stained slightly by Fuelgen's, and stained blue by Machiavello's. The symbiotes appear to be surrounded by three membranes. Ribosomes may occur within the cytoplasm of the symbiotes. The cytoplasm of the mycetocytes contains large numbers of mitochondria, endoplasmic reticulum, ribosomes, and a large nucleus, and nucleolus.A discussion of the classification of the symbiotes is also presented.     

A Novel Rickettsia Species Detected in Vole Ticks (Ixodes angustus) from Western Canada

The genomic DNA of ixodid ticks from western Canada was tested by PCR for the presence of Rickettsia. No rickettsiae were detected in Ixodes sculptus, whereas 18% of the I. angustus and 42% of the Dermacentor andersoni organisms examined were PCR positive for Rickettsia. The rickettsiae from each tick species were characterized genetically using multiple genes. Rickettsiae within the D. andersoni organisms had sequences at four genes that matched those of R. peacockii. In contrast, the Rickettsia present within the larvae, nymphs, and adults of I. angustus had novel DNA sequences at four of the genes characterized compared to the sequences available from GenBank for all recognized species of Rickettsia and all other putative species within the genus. Phylogenetic analyses of the sequence data revealed that the rickettsiae in I. angustus do not belong to the spotted fever, transitional, or typhus groups of rickettsiae but are most closely related to “Candidatus Rickettsia kingi” and belong to a clade that also includes R. canadensis, “Candidatus Rickettsia tarasevichiae,” and “Candidatus Rickettsia monteiroi.”     

Ultrastructure of Rickettsia rhipicephali, a new member of the spotted fever group rickettsiae in tissues of the host vector Rhipicephalus sanguineus.

Rickettsia rhipicephali is similar in ultrastructure to R. rickettsii while differing from other rickettsiae of the typhus group and of Q fever and others by its lack of a prominently reticulated cytoplasmic matrix and in the thickness of the inner osmophilic layer of the cell wall. In tissues of the tick vector Rhipicephalus sanguineus, R. rhipicephali had a mean length and width of 1.2 and 0.46 micrometer, respectively. It possessed a trilaminar cell wall with an adhering capsule-like layer. The trilaminar cell wall was approximately 12 to 18 nm thick; its inner osmophilic layer was thicker than that previously reported for other rickettsiae. The capsule-like layer varied from 7 to 18 nm thick. The plasma membrane was similar in structure, measurement, and appearance to that of other reported rickettsiae. The cytoplasm appeared to be composed of a finely granular, amorphous, ground substance and randomly dispersed ribosomes and lacked a reticular matrix or nuclear fibrils. In massively infected salivary glands and ovarial tissues of its tick vector, R. rhipicephali produced a low degree of histopathology which does not appear to affect the engorgement and egg-laying process of the ticks.     

Retrograde axonal transport in lateral motor neurons of the chick embryo prior to limb bud innervation

Horseradish peroxidase was injected into the distal limb buds of 3-, 3.5-, and 4-day chick embryos and was allowed to diffuse into the path of outgrowing axons. In the majority of the embryos injected, reaction product was found in cells of the ventral spinal cord ipsilateral to the injection site. The reaction product in the most clearly stained cells appeared smooth and diffuse rather than the typical granular appearance found in later embryos and adults. A granular background was seen in 4-day embryos, however, although distinct granular cell outlines were infrequent. This study indicates that retrograde axonal transport is a very early cellular feature during neurogenesis, demonstrable almost from the inception of neurite outgrowth. It is speculated that this transport capacity might function in relaying positional information from growing fiber tips back to cell bodies to aid in the formation of specific synaptic connections or in guiding directed axonal growth, or it may provide a means for trophic interactions vital to the survival of the young neuroblast.     

Isolation of a Spotted Fever Group Rickettsia, Rickettsia peacockii, in a Rocky Mountain Wood Tick, Dermacentor andersoni, Cell Line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

Lethal Effect of Rickettsia rickettsii on Its Tick Vector (Dermacentor andersoni)

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, was lethal for the majority of experimentally and transovarially infected Rocky Mountain wood ticks (Dermacentor andersoni). Overall, 94.1% of nymphs infected as larvae by feeding on rickettsemic guinea pigs died during the molt into adults and 88.3% of adult female ticks infected as nymphs died prior to feeding. In contrast, only 2.8% of uninfected larvae failed to develop into adults over two generations. Infected female ticks incubated at 4°C had a lower mortality (80.9%) than did those held at 21°C (96.8%). Rickettsiae were vertically transmitted to 39.0% of offspring, and significantly fewer larvae developed from infected ticks. The lethal effect of R. rickettsii may explain the low prevalence of infected ticks in nature and affect its enzootic maintenance.     

Immunological studies of the fragments of bovine cartilage proteoglycan produced by chondroitinase-trypsin digestion.

The peptidyl transferase activity of polysomes from Escherichia coli, rabbit reticulocytes and chick embryos, assayed in the fragment reaction, is 3- to 10-fold lower than the corresponding activity of single ribosomes. The polysomal peptidyl transferase activity is restored in full under conditions of in vitro protein synthesis that result in conversion of polysomes to single ribosomes. Thus, the peptidyl transferase center is masked in translating ribosomes. Unmasking of peptidyl transferase, however, does not require the release of ribosomes from messenger RNA: it is also seen upon treatment of polysomes with puromycin, under conditions in which polysomes remain intact. Apparently, release of nascent polypeptide chains is sufficient to allow access of formylmethionyl hexanucleotide substrate to the peptidyl transferase site.     

Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues

Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host.     

Chick embryos can form teratomas from microinjected mouse embryonic stem cells

We examined whether chick embryos are a suitable experimental model for the evaluation of pluripotency of stem cells. Mouse embryonic stem cells (mESCs) expressing the reporter gene, LacZ or GFP were injected into the subgerminal cavity of blastoderms (freshly oviposited) or the marginal vein of chick embryos (2 days of incubation). Injected mESCs were efficiently incorporated into the body and extra‐embryonic tissues of chick embryos and formed small clusters. Increased donor cell numbers injected were positively associated with the efficiency of chimera production, but with lower viability. A single mESC injected into the blastoderm proliferated into 34.7 ± 3.8 cells in 3 days, implying that the chick embryo provides an optimal environment for the growth of xenogenic cells. In the embryo body, mESCs were interspersed as small clustered chimeras in various tissues. Teratomas were observed in the yolk sac and the brain with three germ layers. In the yolk sac, clusters of mESCs gradually increased in volume and exhibited varied morphology such as a water balloon‐like or dark‐red solid mass. However, mESCs in the brain developed into a large soft tissue mass of whitish color and showed a tendency to differentiate into ectodermal lineage cells, including primitive neural ectodermal and neuronal cells expressing the neurofilament protein. These results indicate that chick embryos are useful for the teratoma formation assays of mESCs and have a broad‐range potential as an experimental host model.     

Brilliant Blue as an alternative dye to Fast Green for in ovo electroporation

Chick embryo electroporation is a powerful tool for the introduction of transgenes into tissues of interest for the study of developmental biology. This method often uses Fast Green to visualize the injected area by staining the solution containing DNA green. Here, we show that Fast Green fluoresces in a red color after electroporation, suggesting that researchers need to be cautious when detecting red fluorescence. Fast Green solution did not show any fluorescence before injection into chick embryos, but fluoresced red within 3 min post-injection into chick embryos. We identified Brilliant Blue as suitable alternative dye for use as an indicator of injection sites in ovo electroporation. We found that 0.2% of Brilliant Blue was sufficient to track the area of DNA injection. In addition, this chemical did not show red fluorescence after electroporation. Our findings demonstrate that Brilliant Blue can be used for detecting red fluorescent proteins introduced into chick embryos by electroporation. Our study also shows useful examples for the application of Brilliant Blue for the precise quantification of two fluorescence intensities after EGFP and mCherry co-electroporation.     

Preliminary development of a polymerase chain reaction assay forAnaplasma marginale in ticks

Summary Polymerase chain reaction (PCR) was applied for detection ofAnaplasma marginale in tissues of maleDermacentor andersoni. Primer sequences were derived from the gene for the MSP1β surface protein ofA. marginale (Florida isolate). Optimum PCR conditions were used to detectA. marginale in individual bisected ticks and salivary glands; associated control tissues were negative.     

Dissociation of ribosomes and seed germination

Ribosomes from rice embryos (Oryza sativa) were dissociated into ribosomal subunits in vitro by systematic reduction of the Mg²⁺ concentration. Ribosomes from imbibed (28 C) embryos were more easily dissociated than those from nonimbibed embryos. This was not observed with ribosomes from either imbibed, nonviable embryos, or from embryos imbibed at 0 C. Ribosomes from embryos which had been imbided and subsequently dehydrated resembled ribosomes from nonimbibed embryos in their resistance to dissociation. The change in the resistance to dissociation was essentially complete after the first 20 minutes of imbibition at 28 C, and accompanied activation in vivo of protein synthesis as determined by amino acid incorporation in vitro. Ribosomes from either imbibed or nonimbibed embryos could be dissociated into subunits by 0.5 m KCl. These subunits were separated by density gradient centrifugation, and, if recombined, were active for polyphenylalanine synthesis in vitro. The individual subunits prepared from nonimbibed embryos could be replaced by the corresponding subunit fraction from imbibed embryos without loss of capacity to support polyphenylalanine synthesis. The change in the ease of dissociation of ribosomes appears to be a physiological process, and its possible relationship to the initiation of protein synthesis during seed germination is discussed.     

Protein synthesis induced by heat in an Ixodes tick

We determined whether ambient temperature influences the proteins produced by Ixodes dammini ticks. Nonfed adult females were subjected to temperature pulses, and ³⁵S-labeled methionine was injected into the hemocoel or used as an incubation medium for excised salivary glands. Heat-induced protein synthesis was observed in both whole-body and excised salivary gland preparations from nonattached ticks solely when ambient temperature was raised to 42°C. The approximate M_r of each protein was 88 kilodaltons (kDa), 75 and 74 kDa, and between 21 and 27 kDa for a group of lower molecular weight proteins. In another experimental series, ticks were allowed to attach to rabbits and then were subjected to temperature pulses. The 88 and 74 kDa proteins were present in preparations from nonheated ticks that had attached for 1 h. Only small amounts of these proteins were evident in tissues prepared from ticks attached for 2 days or more. Heat-induced proteins became apparent in ticks that were incubated at 42°C, regardless of time of attachment.     

Control of protein synthesis in brine shrimp embryos by repression of ribosomal activity

Undeveloped encysted embryos of the brine shrimp, Artemia salina, contain a large quantity of metabolically repressed 80S ribosomes. These ribosomes appear to be inactive or nonfunctional due to the presence of an inhibitor protein on their 60S subunit. During development the inhibitor is released or inactivated and the 80S ribosomes and their constituent subunits become fully functional in a poly(U)-directed protein-synthesizing system. The inefficiency of most 80S ribosomes from undeveloped Artemia embryos appears to be due to their inability to form stable complexes with poly(U) and phe-tRNA in the presence of elongation factor, EF-1. A potent inhibitor of protein synthesis has also been found in the 105,000g supernatant fraction from undeveloped Artemia embryos. The exact nature of this inhibitor has not been ascertained but it appears to be a heat-labile protein devoid of RNase and protease activity. It is not known whether this inhibitor is the same as that associated with 60S ribosomal subunits of undeveloped cyst ribosomes.     

POLYAMINES and NUCLEIC ACID METABOLISM DURING DEVELOPMENT OF CHICK EMBRYO BRAIN

Abstract— Spermine and spermidine reach maximum concentrations in the chick embryo brain between the 12th and 14th day of incubation. Sucrose-density-gradient analysis of polyribosome distribution in the developing chick embryo brain, showed the presence of polyribosomal aggregates in the regions of 147 S and 206 S between the sixth and eighth day of incubation. After the 16th day of incubation the presence of heavier polyribosomal aggregates in the region of 259 S and 280 S was found. The injection of spermine or spermidine into the air space of embryos on the tenth day of incubation leads to a remarkable increase in the incorporation rate of [³H]formate into the ribosomes. Studies under similar experimental conditions, showed an increased radioactivity in the region of 147 S, 206 S, 259 S and 280 S in embryos injected with spermine or spermidine.     

Maintenance of aphid cells and the intracellular symbiotes of aphids in vitro

The behavior of both cells and intracellular symbiotes of the cabbage aphid, Brevicoryne brassicae, in primary culture in three arthropod tissue culture media is described. The insect cells usually survived for 2–3 wk, but in one culture cells survived for 16 wk. The symbiotes survived for approximately the same period as the cells, and probably multiplied slowly, at least in some of the cultures. A preliminary experiment with tissues from the green peach aphid, Myzus persicae, gave similar results.     

Embryonic expression of beta-actin-lacZ hybrid gene injected into the fertilized ovum of the domestic fowl.

An experiment was carried out to investigate the expression of cloned DNA injected into the germinal disc of the chick fertilized ovum. The beta-actin-lacZ hybrid gene, MiwZ, was injected, in the closed circular form, into the cytoplasm of the germinal disc at the single-cell stage. The embryos were cultured in vitro, then in recipient eggshells up to day 4 of incubation. The survival rate of the embryos at day 4 was 42% (55/130), and the rate of embryos expressing MiwZ was 64% (35/55). Twenty-two embryos expressed the MiwZ in both embryonic and extraembryonic tissues, while the remainder expressed the MiwZ in only extraembryonic tissues. Mosaic expression was observed in most of the embryos expressing MiwZ in embryonic tissues. Expression throughout all tissues of the embryo including blood cells occurred in one case. In this case, the injected DNA was assumed to have integrated at an earlier stage. The results indicate that it is now possible to investigate the promoter activities of introduced exogenous genes as well as the effect of introduced genes on embryogenesis in early chick embryos. This technique may also facilitate the production of transgenic chicks.     

Artificially Induced Symbiosis Between Marine Flagellates and Vertebrate Tissues in Culture*

SYNOPSIS. Algal symbiotes isolated from marine invertebrate hosts, and grown axenically in culture, have been successfully combined in artificial associations with chick fibroblasts and grunt (Haemulon sp.) fin explants. Growth (measured by cell numbers and chlorophyll a) is stimulated in both symbiotes and host cells. Studies of nitrogen uptake and exchange suggest that these artificial associations are similar to other, natural symbioses involving invertebrate hosts, e.g. the hermatypic corals.