Infection and development of Nosema bombycis (Microsporida: Protozoa) in a cell line of Antheraea eucalypti

Spores of Nosema bombycis derived from diseased insects were highly purified by Urografin density gradient centrifugation. Antheraea eucalypti cells were inoculated with the purified spores primed with 0.1 n KOH solution to start a continuous propagation of N. bombycis in cell culture. The first increase in the number of infected A. eucalypti cells was observed at 48 hr postinoculation, and it was caused by the secondary infective forms of N. bombycis. The secondary infective forms were produced during the course of sporoblast differentiation. The parasites in cell cultures divided synchronously until 36 hr postinoculation. Mature spores were observed initially 6 days postinoculation at 27°C. The infected cultures were subcultured extensively for more than 1 year with the addition of healthy A. eucalypti cells.     

Fine Structure of Bacillus megaterium during Microcycle Sporogenesis

Ultrathin sections were prepared from cultures of Bacillus megaterium QM B1551 undergoing microcycle sporogenesis (initial spore to primary cell to second-stage spore without intervening cell division) on a chemically defined medium. The cytoplasmic core of the dormant spore was surrounded by plasma membrane, cell-wall primordium, cortex, outer cortical layer, and spore coats. Early in the cycle, the coat opened at the germinal groove, the cortex swelled, ribosomes and a chromatinic area associated with large mesosomes (which may later be incorporated into the expanding plasma membrane) appeared in the core, and the cell wall became defined at the site of the cell wall primordium. Poly-β-hydroxybutyrate granules began to appear in the primary cell at about 3 hr. By 7 hr, the forespore of the second-stage spore was delineated by typical double membranes. Between 7 and 12 hr, second-stage cell-wall primordium and cortex developed between the separating forespore membranes. The inner membrane became the plasma membrane of the second-stage spore, and the outer membrane eventually disintegrated within the second-stage spore cortex. A densely staining double layer (spore-coat primordium) developed external to the outer forespore membrane. The inner spore coat and the outer cortical layer of the second-stage spore developed from this primordium. The outer part of the spore coat, probably of sporangial origin, was laid down on the external surface of the inner spore coat. By 12 hr, second-stage spores were almost mature. By 20 hr, the mature endospores, with a thickened outer coat, were often still enclosed by degenerate primary cell wall and by the outer cortical layer and spore coat of the initial spore.     

Effect of weather on a microbial insecticide

A commercial formulation of Bacillus thuringiensis was exposed to the weather in the field at Fargo, North Dakota, during the summers of 1969 and 1970. A serial dilution technique was utilized in counting viable bacterial spores after exposure to the weather. Weather suppresses populations of B. thuringiensis in the field. The greatest effect occurred during the first 24 hr of exposure. Rain and solar radiation caused a significant reduction in the number of viable spores. Solar radiation had the greatest effect. The information has practical value when determining the time of application, the number of applications, and the amount of a microbial insecticide to use to control a pest.     

Characteristics of developing mitochondrial genetic and respiratory functions in germinating fungal spores

Spores of the fungus Botryodiplodia theobromae began a cyanide-sensitive oxygen consumption immediately upon exposure to a liquid medium, and spore germination and respiration were not affected by ethidium bromide, d-threochloramphenicol, and acriflavin until later during germ tube emergence. These inhibitors of the mitochondrial genetic system all inhibited total cell protein synthesis to the same intermediate degree from the outset of incubation. When spores were incubated in water under non-germinating conditions, protein synthesis and oxygen uptake proceeded at initial rates almost identical to those seen in spores germinating in the presence of the three mitochondrial system inhibitors. Although the spores respired at rapid rates from the onset of incubation, no cytochrome absorption peaks could be observed in mitochondrial fractions prepared from ungerminated spores; they were readily observed in germinated spores, however. When the spores were germinated in the presence of inhibitors of the mitochondrial system, an excess of cytochrome c was observed in the near absence of cytochromes a and b. The results indicate that the ungerminated spores of this organism contain a preserved, potentially functional aerobic respiratory system which requires cycloheximide-sensitive ribosome activity to become functional when the spores are inoculated into a liquid medium.     

Evidence that the fertilization envelope blocks sperm entry in eggs of Xenopus laevis: interaction of sperm with isolated envelopes.

Single-celled myxamoebae undergo differentiation into either stalk cells or spore cells during a 24-hr period in Dictyostelium discoideum. This study employed ultramicrochemical techniques and enzymatic cycling to assess the presence of cell-specific events in spore and stalk cells. Freeze-dried sections of one organism were assayed in 0.1 μl of reaction mixture. This method was used to determine the extent of localization of trehalose in spore cells and stalk cells during development.Trehalose was low in the early stages of differentiation to about 20 hr when the level started to increase. In developing spore cells, the trehalose level increased sixfold during the last 5 hr of development. Likewise, the entire stalk contained trehalose when the stalk was first formed. At mature sorocarp, trehalose levels were the same in spores and the apex of the stalk. There was a decreasing gradient of trehalose down the stalk. The bottom one-fourth of the stalk was devoid of this disaccharide. Therefore, trehalose was degraded from an area of the stalk where it was localized earlier in development.The results of this investigation negate the assumption that trehalose is never present in the stalk. Although trehalose was found in spore cells, prestalk cells also contained high trehalose levels. The stalk cell-specific trehalose was not retained during differentiation, however, but was apparently degraded in the mature stalk cell.     

Termite pathogens: Effects of ingested Metarhizium,beauveria, and Gliocladium conidia on worker termites (Reticulitermes sp.)

Separate groups of subterranean termites (Reticulitermes sp.) were exposed to whole cultures of Beauveria bassiana, Gliocladium virens, or Metarhizium anisopliae. Individuals were removed after varying time intervals and hindgut contents were plated onto potato dextrose agar. Viable spores first appeared in the hindguts within 8 hr of exposure. Fungi reisolated from the hindguts of diseased termites were pathogenic of healthy termites. Histological examination showed that invasion of the hemocoel by M. anisopliae occurred exclusively through direct invasion of the integument ca. 24 hr after death. B. bassiana invaded, primarily through the alimentary tract, ca. 12 hr prior to termite death.     

Patterns of primary spore discharge of Entomophthora spp from the blue green aphid,Acyrthosiphon kondoi

Experiments were conducted to study the effects of time, temperature, and light regime on primary spore formation at 100% RH for the three major pathogens of Acyrothosiphon kondoi. Only small differences were detected between the continuous light and continuous dark regimes. Entomophthora obscura produced between 6 and 10 × 10³ primary spores mostly during the first 48 hr. Total primary spore production was similar at the five temperatures tested from 5° to 25°C. Entomophthora planchoniana produced large numbers of primary spores (about 5 × 10⁴ per aphid) only at temperatures between 10° and 20°C. The majority of primary spores were formed during the first 24 hr. Primary spore production with Entomophthora nr. exitialis ranged from about 10⁵ per aphid at 5° and 10°C to 3 or 4 × 10⁵ at 15° to 25°C, with most spores being formed during the first 48 hr. It is suggested that rainfall is more likely to be important for transmission of E. obscura and E. nr. exitialis than for transmission of E. planchoniana, and that E. obscura is likely to be the most important pathogen during cool or cold weather.     

Combined Effects of Ultrahigh Vacuum and Temperature on the Viability of Some Spores and Soil Organisms

Considerably fewer spores of Bacillus stearothermophilus, B. megaterium, and Clostridium sporogenes were recovered than were spores of B. subtilis var. niger and Aspergillus niger after 4 to 5 days at 53 and 60 C in ultrahigh vacuum. There were no significant differences in the recoveries of these five organisms at 25 C and atmospheric pressure, and after exposure to 25 and -190 C in vacuum. At 60 C, a far greater decrease in viability was demonstrated for B. stearothermophilus, B. megaterium, and C. sporogenes in ultrahigh vacuum than at atmospheric pressure. Viable B. subtilis var. niger spores were not detected in an initial 10⁷ spores after retention at 90 C and ultrahigh vacuum, and 10⁴ spores were viable after 5 days at 90 C and atmospheric pressure from an initial 10⁶ spores. Molds and actinomycetes in soil were particularly resistant up to 69 C in vacuum. Actinomycetes were the only soil organisms recovered so far at 120 C.     

Development of Bacillus thuringiensis in Galleria mellonella larvae exposed to gamma radiation

A suspension of Bacillus thuringiensis was inoculated at 24 and 72 hr into the oral cavity of Galleria mellonella larvae following exposure to 20, 50, and 70 Kr of gamma radiation, respectively. The cytopathology was conducted after B. thuringiensis had developed for 3, 5, and 7 hr and after radiation damage had developed for 27, 29, 31, 75, 77, and 79 hr in the larvae exposed to 20, 50, and 70 Kr, respectively.B. thuringiensis spores appeared in the midgut lumen from 3 to 7 hr after inoculation of 20 Kr irradiated larvae. At 7 hr after B. thuringiensis infection, and 79 hr after 20 Kr irradiation, the following changes were seen: B. thuringiensis rods appeared adsorbed onto the walls of epithelial cells, a few spores appeared in hemolymph, epithelial cells developed vacuoles, and villi appeared detached from the basement membrane.Within a period ranging from 3 to 5 hr after infection, B. thuringiensis rods attacked vacuolated epithelial cells of most of the 50 and 70 Kr irradiated larvae. At 7 hr after infection and at 31 hr after 70 Kr irradiation, the spores reached the interior of some epithelial cells and were also seen concentrated near the basement membrane.In general, the midgut epithelial cells of the 70 Kr-irradiated groups of larvae appeared highly vacuolated, badly disrupted, and in most cases undistinguishable as a result of attack of B. thuringiensis.In short, B. thuringiensis did not show a characteristic pattern of pathology on 20 and 50 Kr-irradiated midgut cells. The problem of permeability of B. thuringiensis toxin into the irradiated cells needs further investigation.     

Characterization of Bacillus anthracis Persistence In Vivo

Pulmonary exposure to Bacillus anthracis spores initiates inhalational anthrax, a life-threatening infection. It is known that dormant spores can be recovered from the lungs of infected animals months after the initial spore exposure. Consequently, a 60-day course antibiotic treatment is recommended for exposed individuals. However, there has been little information regarding details or mechanisms of spore persistence in vivo. In this study, we investigated spore persistence in a mouse model. The results indicated that weeks after intranasal inoculation with B. anthracis spores, substantial amounts of spores could be recovered from the mouse lung. Moreover, spores of B. anthracis were significantly better at persisting in the lung than spores of a non-pathogenic Bacillus subtilis strain. The majority of B. anthracis spores in the lung were tightly associated with the lung tissue, as they could not be readily removed by lavage. Immunofluorescence staining of lung sections showed that spores associated with the alveolar and airway epithelium. Confocal analysis indicated that some of the spores were inside epithelial cells. This was further confirmed by differential immunofluorescence staining of lung cells harvested from the infected lungs, suggesting that association with lung epithelial cells may provide an advantage to spore persistence in the lung. There was no or very mild inflammation in the infected lungs. Furthermore, spores were present in the lung tissue as single spores rather than in clusters. We also showed that the anthrax toxins did not play a role in persistence. Together, the results suggest that B. anthracis spores have special properties that promote their persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence.     

Effect of ultraviolet radiation on the microsporidian Octosporea muscaedomesticae with reference to protectants provided by the host Phormia regina

The effect of ultraviolet light on the microsporidian Octosporea muscaedomesticae in relation to infection in the adult black blowfly, Phormia regina, was investigated. A 30-Watt germicidal lamp, 253.7-nm wavelength, was used as source of uv light in five investigations. In addition, sunlight served as a uv source in two studies. Viable naked dried spores exposed to the uv lamp at a distance of 10 cm were killed after 15 min. Viable naked spores in an aqueous suspension were killed after 30 min of exposure to the uv lamp and after 3 hr of exposure to bright sunlight, respectively. Daily 30-min uv lamp exposures on living hosts harboring all life phases of the parasite did not interfere with the ensuing infection in the blowfly's midgut and the pathogen's developmental cycle. Spores harvested from uv-treated infected hosts were found to be as infective as spores retrieved from hosts not treated with uv. Spores contained in dried fecal droplets and exposed up to 3 hr to the uv lamp, or 12 hr to bright sunlight, respectively, remained infective. Addition of uric acid to a preparation of naked spores prior to 15- and 30-min uv irradiations yielded 100% infection in both host groups. A uv-protective function is ascribed to components provided by the host's tissues and feces.     

Differentiation of Various Cell Types during Fruiting Body Formation of Dictyostelium Discoideum*

The processes of differentiation of the presumptive cells (prespore and prestalk cens) into mature spores, stalk and basal-disc cells in Dictyotelium discoideum was investigated. The number of stalk and disc cells in pre-labeled culminating cell masses was estimated by determining the radioactivity of the undissociable fraction separated by filtration from the dissociable fraction containing presumptive cells and spores. Changes in the proportion of amoeboid cells stainable with fluorescein-conjugated antispore serum and encapsulated spores were also followed in the dissociable fraction. Formation of stalk and disc cells began at 17 hr of development and was completed at 26 hr, while formation of morphologically identifiable spores began at 18 hr and was completed at 20 hr, long before completion of stalk formation. At the onset of culmination, unstained cells abruptly increased with an accompanying decrease of stained cells, when unstained rear-guard cells appeared in the hind region. Although some of the rear-guard cells soon differentiated into basal-disc cells, the rest remained amoeboid in the upper part of the spore mass (sorus) after complete formation of a fruiting body. Despite the presence of the amoeboid cells in mature sori, the proportion of the sorus to the stalk and disc of a fruiting body was approximately equal to that of stained (prespore) to unstained (prestalk) cells in a migrating slug.     

Histopathology of a nuclear polyhedrosis infection in Aedes epactius with observations in four additional mosquito species

Aedes epactius larvae were utilized to study the infection sequence of the nuclear polyhedrosis virus (NPV) from Aedes sollicitans. From 30 min to 6 hr postinoculation, polyhedra and many free virions were observed in the larval midgut lumen. Penetration of the midgut cells by virions was not observed. The first infected nuclei were observed 12 hr postinoculation. Nucleocapsids initially exhibited electron translucent cores which became electron dense before the nucleocapsids acquired an envelope. Envelope acquisition occurred through a process of de novo membrane morphogenesis. Occlusion of the singly embedded virions began by 18 hr postinoculation with the mature rough-surfaced polyhedra averaging approximately 1 by 2 μm. Unusually long nucleocapsids (approximately two or three times the length of other nucleocapsids) were only observed in late infection period nuclei. There was no evidence that long nucleocapsids represented an early developmental stage for nucleocapsids of standard length. Infection was restricted to midgut nuclei and gastric caecae cells. Infected early instar A. epactius larvae became moribund 36 to 40 hr postinoculation and infected midgut nuclei were observed to undergo lysis. The late stages of NPV infection were observed in larvae of A. annandalei, Wyeomyia smithii, Toxorhynchites brevipalpus, and Eretmapodites quinquevittatus. Virion development and occlusion in these species was basically identical to the sequence observed in A. epactius larvae.     

ENHANCEMENT OF INVERTASE PRODUCTION BY Aspergillus niger OZ-3 USING LOW-INTENSITY STATIC MAGNETIC FIELDS

The aim of this study is to investigate the effect of low-intensity static magnetic fields (SMFs) on invertase activity and growth on different newly identified molds. The most positive effect of SMFs on invertase activity and growth was observed for Aspergillus niger OZ-3. The submerged production of invertase was performed with the spores obtained at the different exposure times (120, 144, 168, and 196 hr) and magnetic field intensities (0.45, 3, 5, 7, and 9 mT). The normal magnetic field of the laboratory was assayed as 0.45 mT (control). Optimization of magnetic field intensity and exposure time significantly increased biomass production and invertase activity compared to 0.45 mT. The maximum invertase activity (51.14 U/mL) and biomass concentration (4.36 g/L) were achieved with the spores obtained at the 144 hr exposure time and 5 mT magnetic field intensity. The effect of low-intensity static magnetic fields (SMFs) on invertase activities of molds was investigated for the first time in the present study. As an additional contribution, a new hyper-invertase-producing mold strain was isolated.

Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

Biochemical Alternations in Bacillus megaterium as Produced by Aflatoxin B1

Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 μg of aflatoxin B₁ per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 μg of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.     

Bioassay of Entomophthora against the spotted alfalfa aphid Therioaphis trifolii f. maculata

A method for the bioassay of Enthomophthora spp. against aphids is described. Twenty-four isolates comprising five species of fungi were screened for activity against Therioaphis trifolii f. maculata. The only isolates with a high level of activity were those of E. sphaerosperma obtained from aphids. The initial bioassays with E. sphaerosperma indicated that aphids, starved for 24 hr during inoculation with E. sphaerosperma primary spores, were less susceptible than those removed from the plants, for just the period of exposure to the primary spore shower. Using the latter procedure, five assays of the most pathogenic isolate gave a mean LC₅₀ of 11.3 primary spores/mm² while bioassays of four other isolates gave LC₅₀ values ranging from 15.7 to 27.3 spores/mm². The potential of E. sphaerosperma as a microbial control agent for T. trifolii f. maculata in Australia is discussed.     

Synthesis and deposition of yolk protein in adult Drosophila melanogaster

Newly eclosed Drosophila melanogaster females contain only previtellogenic stage oöcytes and no immunologically detectable female specific haemolymph protein. During the subsequent 48 hr the concentration of female specific protein in the haemolymph rises to a plateau value of 21 μg/μl; at this time yolk protein represents about one third of the total haemolymph protein in adult females. The first mature (stage 14) oöcytes are observed at 48 hr post eclosion. The female specific haemolymph protein and the major protein from mature oöcytes are electophoretically and immunologically the same or very similar. Injection of alpha amanitin into newly eclosed females inhibits the development of mature oöcytes and the degree of inhibition depends on the age of the female at the time of injection. Phenocopies of non-vitellogenic mutants result when alpha amanitin is injected into newly eclosed females; after 36 hr post eclosion no visible inhibition of vitellogenesis (as observed morphologically at 72 hr post eclosion) can be produced by alpha amanitin.     

Fine Structure of Encephalitozoon cuniculi from Rabbits,Mice and Hamsters*

SYNOPSIS. Fine structure and development of Encephalitozoon cuniculi from rabbits were studied in rabbit choroid plexus (CP) cell cultures and were compared to hamster and mouse microsporida. Sporoplasms had a single limiting membrane and contained a large nucleus. Proliferative forms (schizonts) had double outer membranes, the outermost being associated with the formation of the limiting membrane of vacuoles formed within the host cell cytoplasm. These organisms were often binucleate and divided to form sporonts. Sporonts divided once to form 2 sporoblasts which developed into electron-dense spores. Spores had a thick, 3-layered wall and contained a polar filament. The developmental cycle of E. cuniculi in rabbit CP cultures progressed rapidly. Sporoplasms were observed in host cells at 3 hr postinoculation (PI). By 24 hr PI proliferative forms were associated with host cell cytoplasmic vacuoles which contained developing organisms. Mature spores were present in vacuoles by 2 days PI, indicating that the life cycle in the CP system is ∼ 48 hr. The fine structure and the sequential developmental cycle of the mouse and hamster isolates were observed to be identical to those of the rabbit isolate and different from those of the genus Nosema. It is proposed, therefore, that the 3 organisms represent the same species, Encephalitozoon cuniculi.     

The rate of development of a microsporidan in moth cell culture.

A microsporidan parasite of the forest tent caterpillar Malacosoma disstria infected cells and replicated in vitro in a line from the moth Heliothis zea. After spore germination, the incidence of infected cells increased with time until leveling off with sporulation. During the first 24 hr, there was a static number of parasites, followed by a 2-day logarithmic growth phase during which the population doubled five to six times. The growth rate was 9 to 11 hr per population doubling. Sporulation commenced on day 3, and 40 to 50 spores were recovered from each infected cell. The life cycle was completed within 6 days, culminating in spores that were infectious for cultured cells. The antibiotic fumagillin at a dose of 1 ppm in the culture medium was microsporida-static.     

Reduction of Pulmonary Toxicity of Stachybotrys chartarum Spores by Methanol Extraction of Mycotoxins

The fungus Stachybotrys chartarum has been implicated in cases of nonspecific indoor air quality complaints in adults and in cases of pulmonary hemorrhaging in infants. The effects that have been described have been attributed to mycotoxins. Previous dose-effect studies focused on exposure to a single mycotoxin in a solvent, a strategy which is unlikely to accurately characterize the effects of inhaled spores. In this study we examined the role of mycotoxins in the pulmonary effects caused by S. chartarum spores and the dose dependency of these effects. S. chartarum spores were extracted in methanol to reduce the mycotoxin content of the spores. Then either untreated (toxin-containing) or methanol-extracted S. chartarum spores were intratracheally instilled into male 10-week-old Charles River-Dawley rats. After 24 h, the lungs were lavaged, and the bronchoalveolar lavage fluid was analyzed to determine differences in lactic dehydrogenase, albumin, hemoglobin, myeloperoxidase, and leukocyte differential counts. Weight change was also monitored. Our data show that methanol extraction dramatically reduced the toxicity of S. chartarum spores. No statistically significant effects were observed in the bronchoalveolar lavage fluids of the animals that were treated with methanol-extracted spores at any dose. Conversely, dose-dependent effects of the toxin-containing spores were observed when we examined the lactic dehydrogenase, albumin, and hemoglobin concentrations, the polymorphonuclear leukocyte counts, and weight loss. Our findings show that a single, intense exposure to toxin-containing S. chartarum spores results in pulmonary inflammation and injury in a dose-dependent manner. Importantly, the effects are related to methanol-soluble toxins in the spores.