Effects of Colchicine and Light on Cell Form in Fern Gametophytes. Implications for a Mechanism of Light-induced Cell Elongation

Spores of the fern, Onoclea sensihilis L., suffer a disruption of normal development when they are cultured on media containing colchicine. Cell division is inhibited, and the spores develop into giant spherical cells under continuous white fluorescent light. In darkness only slight cell expansion occurs. Spherical cell expansion in the light requires continuous irradiation. Photosynthesis does not seem to be involved, since variations in light intensity do not affect the final cell diameter; the addition of sucrose to the medium does not permit cell expansion in darkness; and the inhibitor DCMU does not block the light-induced cell expansion. Continuous irradiation of colchicine-treated spores with blue, red or far-red light produces different patterns of cell expansion. Blue light permits spherical growth, similar to that found under white light, whereas red and far-red light promote the reestablishment of polarized filamentous growth. Although ethylene is unable to induce polarized cell expansion in colchicine-treated spores in darkness or white and blue light, it enhances filamentous growth which already is established by red or far-red irradiation. Both red and far-red light increase the elongation of normal filaments (untreated with colchicine) above that of dark-grown plants, but under all 3 conditions the rates of volume growth are identical. Light, however, does cause a decrease in the cell diameters of irradiated filaments. These data are used to construct an hypothesis to explain the promotion of cell elongation in fern protonemata by red and far-red light. The model proposes light-mediated changes in microtubular orientation and cell wall structure which lead to restriction of lateral cell expansion and enhanced elongation growth.     

Unikaryon phyllotretae sp. n. (Protista,Microspora), a new microsporidian pathogen of Phyllotreta undulata (Coleoptera; Chrysomelidae)

The microsporidium Unikaryon phyllotretae sp. n., a new pathogen of Phyllotreta undulata, is described based on light microscopic and ultrastructural characteristics. Microscopic examination of parasitized individuals revealed two types of spores. The majority of the spores were of the first type, which are oval and measured 2.74±0.17×1.93±0.17 μm when fresh. Fresh spores of the second type (very rare) are elongated and measured 4.39±0.18×1.61±0.20 μm. All life stages have single nuclei. Sporogony ends with uninucleate single sporoblasts and spores. The spores were only observed in Malpighian tubules. The isofilar polar filament of the parasite has six to eight coils, and a well-developed polaroplast was of the lamellated type, with closely packed anterior lamellae and loosely packed posterior lamellae.     

Photocontrol of the germination of onoclea spores: I. Action spectrum

Light stimulates the germination of spores of the fern Onoclea sensibilis L. At high dosages, broad band red, far red, and blue light promote maximal germination. Maximal sensitivity to these spectral regions is attained from 6 to 48 hours of dark presoaking, and all induced rapid germination after a lag of 30 to 36 hours. Maximal germination is attained approximately 70 hours after irradiation. Dose response curves suggest log linearity. The action spectrum to cause 50% germination shows that spores are most sensitive to irradiation in the red region (620-680 nm) with an incident energy less than 1000 ergs cm⁻²; sensitivity decreases towards both shorter and longer wavelengths. Although the action spectrum is suggestive of phytochrome involvement, photoreversibility of germination between red and far red light has not been demonstrated with Onoclea spores. An absorption spectrum of the intact spores reveals the presence of chlorophylls and carotenoids. Since the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea does not inhibit germination, it is concluded that photosynthesis does not play a role in the germination process.     

Abnormal spore morphology and wall ultrastructure in Anemia tomentosa var. anthriscifolia and A. tomentosa var. tomentosa (Anemiaceae)

The spores of Anemia tomentosa var. anthriscifolia and A. tomentosa var. tomentosa were studied focusing the attention on their abnormalities. The study was based on fresh and herbarium material and the spores were examined with light, scanning and transmission electron microscopy. Normal, abnormal and abortive spores were observed in both taxa. The normal spores were trilete, triangular in polar view, and the ornamentation consisted of parallel ridges separated by narrow and smooth grooves. The spores were observed in monads, dyads, triads and tetrads. The abnormal spores were monolete, trilete, tetralete or alete with great variations in size. In fact, some spores were almost double the size of the normal ones. Some differences were also found in the ornamentation of the spores. Aborted and not completely developed spores were also observed in the specimens. The wall ultrastructure of the taxa was studied for the first time. The exospore was two-layered with numerous cavities inside its structure, and the perispore was also two-layered. The results revealed that the sporoderm ultrastructure of both normal and abnormal spores of the taxa analyzed was very similar.     

Photodynamic Inactivation of Bacillus Spores, Mediated by Phenothiazinium Dyes

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.     

REPRODUCTION IN ZONARIA FARLOWII II. CYTOLOGY AND ULTRASTRUCTURE1

The development of spores and eggs of Zonaria farlowii was studied with light and electron microscopy. Oogonia require a lunar month to develop and are produced in periodic crops. The sporangia, on the other hand, are longer in developing and on a particular plant do not become mature all at the same time. Differences in size, content, and products of sporangia and oogonia have been found. One of the quantifiable differences is in the amount of osmiophilic substance, presumably oil, present in the 2 cells. This substance is more abundant in the sporangium, a cell which produces 8 reproductive spores. These differences are particularly interesting since the embryology of sporic and zygotic germlings has been found to be identical, while ontogeny of spores and eggs differs. The findings of this study are discussed in view of possible relationships between microstrtrclitral features of spores and fertilized eggs and their subsequent development.     

Blue Light Interference in the Phytochrome-controlled Germination of the Spores of Cheilanthes farinosa

Short exposure of the spores of Cheilanthes farinosa to low intensity red light promotes their germination, which is not reversed by a subsequent exposure to far red light. Germination is, however, inhibited by blue light administered before or after red light. Inhibition of germination by blue light is annulled by exposure to a higher intensity of red light, and germination of the repromoted spores is inhibited by far red light. Mutual photoreversibility of germination is also observed in repromoted spores irradiated successively with far red and red light. Although germination appears to be basically under phytochrome control, it is postulated that the presence of a blue light-absorbing pigment interferes with phytochrome transformations in the spores.     

Description of Hyalinocysta expilatoria n. sp., a microsporidian parasite of the blackfly Odagmia ornata

Hyalinocysta expilatoria n. sp. is described from a larva of Odagmia ornata collected in Sweden. Infection was restricted to the adipose tissue which was transformed into a syncytium. The earliest stage observed was diplokaryotic merozoites, which mature directly into diplokaryotic sporonts. Each sporont produces a sporophorous vesicle (pansporoblast), which persists, also enclosing mature spores. Usually nuclear divisions result in a plasmodium with 8 nuclei, which fragments into 8 sporoblasts, each of which develops into a spore without further division. Occasionally an aberrant number of spores (2, 4, 6) is formed. The spores are pyriform with a flattened area at the posterior pole. Spores in sporophorous vesicles with 8 spores are 4.0–6.0 μm long, in vesicles with 4 spores 4.0–5.0 μm, and in vesicles with 2 spores 7.0–8.0 μm. In some vesicles the spores develop asynchronously, and 2, 4, or 6 mature spores are found together with 6, 4, or 2 immature. There was also a small number of vesicles with supernumerary spores, less than 8 normally developed. The 325–350 nm thick spore wall is composed of three layers. The polar filament is anisofilar with 7 coils in a single layer. The anterior 5–6 coils are wide, the posterior 2-1 thin. The angle of tilt of the anterior filament coil is approximately 50°. The spore has a single nucleus. The sporophorous vesicle is delimited by a thin membrane, also visible in haematoxylin stained preparations. Vesicles with mature spores are void of metabolic inclusions.     

Ultrastructural Study and Description of Ovavesicula popilliae N. G., N. Sp. (Microsporida: Pleistophoridae) from the Japanese Beetle,Popillia japonica (Coleoptera: Scarabaeidae)1

A new genus and species of microsporidia, Ovavesicula popilliae n. g., n. sp., is described from the Japanese beetle, Popillia japonica, on the basis of studies by light and electron microscopy. Parasite development primarily occurs within the Malpighian tubules of larvae, and spores are formed in a sporophorous vesicle. Meronts have diplokaryotic nuclei, develop in direct contact with the host cell cytoplasm, and divide by binary fission. Sporonts have unpaired nuclei, develop within a thick sporophorous vesicle, and undergo synchronous nuclear divisions producing plasmodia with 2, 4, 8, 16, and 32 nuclei. Cytokinesis of sporogonial plasmodia does not occur until karyokinesis is complete with 32 nuclei. Intact sporophorous vesicles are ovoid, containing numerous secretory products, and are surrounded by a persistent two-layered wall. The uninucleate spores are regularly formed in groups of 32, and the polar tube in each has six coils.     

Temperature and photocontrol of onoclea spore germination

Germination of Onoclea sensibilis L. spores is controlled by light and temperature. Temperatures of 30 C can induce maximal germination in the dark to a level of 60 to 95% of that induced by a saturating dose of red light (0.38 joules/square meter) providing the spores are placed at the elevated temperature immediately after being sown. Maximum dark germination occurs with a minimum exposure of 16 to 24 hours at 30 C, suggesting that the temperature treatment is required for the induction of germination rather than for the germination process per se. Interaction of temperature and light for induction of germination shows nonadditive behavior. Germination induced by light and temperature applied consecutively never exceeded that which could be induced by a saturating dose of red light alone. Imbibition of the spores at 25 C in the dark for 12 or more hours prior to incubation at 30 C results in a loss of thermosensitivity. Dose response curves for red light induction of germination after varying times of imbibition at 25 C show no concomitant loss of sensitivity of the spores to red irradiation. This suggests that the mechanism and/or pathway of thermoinduction of germination differs from that of photoinduction. The loss of thermosensitivity as a result of presoaking at 25 C can be prevented if the spores are imbibed at 25 C in osmotic agents such as 0.3 molar mannitol or 0.1 gram per liter of polyethylene glycol 400 or in 0.08% dimethylsulfoxide or 10 micrograms per milliliter of herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl-3-(2H)pyridazinone). The latter two substances are hypothesized to act upon membranes. These results suggest that the degree of hydration and possibly changes in membrane properties play a role in the change in sensitivity of Onoclea spores to temperature.     

Microsporidium goeldichironomi n. sp. and Microsporidium chironomi n. sp. (Microsporida: Apansporoblastina): Two new microsporidia from Florida chironomids

Two new species of Microsporida belonging to the genus Microsporidium are described. Microsporidium goeldichironomi n. sp. parasitizes the fat body of Goeldichironomus holoprasinus and Microsporidium chironomi n. sp. infects Chironomus attenuatus. Both microsporidia form uninucleate spores from rosette-shaped sporonts. M. goeldichironomi sporonts form 4, 6, 8, 10, 12, 16, and possibly more spores. Two shapes of spores are produced, oval, or slightly pyriform spores measuring 3.70 ± 0.09 × 2.49 ± 0.13 μm and pyriform spores measuring 3.74 ± 0.44 × 2.04 ± 0.17 μm. Electron micrographs show that both types of spores are uninucleate, have 8 to 11 polar filament coils and a lamellate polaroplast showing several distinct regions. M. chironomi spores are pyriform and are often joined at the posterior end in groups of two or four. They measure 4.12 ± 0.37 × 2.45 ± 0.26 μm. The spores are uninucleate, have six to seven polar filament coils and a lamellate polaroplast showing two distinct regions. Neither species can be transmitted per os and thus are assumed to be transovarially transmitted. No pansporoblastic membrane is present in either species.     

Entomophthora lampyridarum,a fungal pathogen of the soldier beetle,Chauliognathus pennsylvanicus

In 1974 and 1979 in Clemson, South Carolina, adults of the common soldier beetle, Chauliognathus pennsylvanicus, were found to be infected by the fungal pathogen, Entomophthora lampyridarum. After infected beetles died they remained attached by their mandibles to foliage and flowers. The wings of infected beetles remained open, allowing conidiophores to develop on the upper surface of the abdomen. Primary conidia were elongate and measured 36.5 × 17.1 μm. Two types of secondary spores were formed: Type I spores were similar in form to primary conidia, but were somewhat smaller; Type II spores were formed at the apex of slender stalks and measured 37.7 × 15.3 μm. Resting spores were spherical, hyaline, and 22.3 μm in diameter.     

Vairimorpha invictae N. Sp. (Microspora: Microsporida), a Parasite of the Red Imported Fire Ant,Solenopsis invicta Buren (Hymenoptera: Formicidae)12

ABSTRACT. Vairimorpha invictae n. sp. infects the red imported fire ant, Solenopsis invicta Buren, in Brazil. The parasite is dimorphic, producing two morphologically distinct types of spores, which develop sequentially in the same fat cells or oenocytes in the fat body. The binucleate free spores develop from disporous sporonts; the uninucleate octospores develop from multinucleate sporonts within a sporophorous vesicle. Infected cells are transformed into large sacs which contain both types of spores in mature adult hosts. Mature free spores are often present by the time the larvae pupate, but mature octospores are found only in adult hosts. Masses of spores may be seen through the intact cuticle by low power phase-contrast microscopy; there are no other physical signs and no behavioral signs of infection. Attempts to transmit the infection in the laboratory failed.     

Life cycle of tetrodotoxin-producing <Emphasis Type="Italic">Bacillus</Emphasis> sp. on solid and liquid medium: Light and electron microscopy studies

The lifecycle of the Bacillus sp. 1839 cultivated during a long period on solid and liquid Youschimizu-Kimura medium was investigated, and then bacteria and spores were studied by light and transmission electron microscopy. Sporulation in this strain is distinguished by engulfment of forespore by mother cell. In the liquid medium, bacteria have the decondensed nucleoid and the loose granular component of cytoplasm; bacteria and spores are generally smaller; the outer coat of spores includes 2 concentric rings. On the solid substratum, the nucleoid is condensed, and the cytoplasmic region is extensive and dense; a longer cultivation stimulates transition of vegetative cells into the spore form; spores have a thicker outer coat with 3–5 rings. On the solid substratum, sporulation in Bacillus sp. 1839 is spontaneous, without additional stimulation; spores have a larger diameter and thicker layers than those in the liquid medium. This research contributes to the current understanding of biotechnological tetrodotoxin production from a bacterial raw material.     

Ultrastructural study of the microsporidian chytridiopsis typographi (Chytridiopsida: Microspora) infecting the bark beetle,Ips typographus (Scolytidae: Coleoptera), with new data on spore dimorphism

Two types of sporogony of the microsporidian Chytridiopsis typographi in the midgut of adult bark beetle, Ips typographus, have been examined by means of light and electron microscopy. New data are reported on spore dimorphism and on the formation of pansporoblasts in two types of sporogony. Thin-walled spores, larger in size, are formed in a parasitophorous vacuole in the host columnar cells. Thick-walled spores are formed in a minimal vacuole in the host. The ultrastructure of the spore walls and the cyst wall are different from the organization in other microsporidia. Both spore types have identical internal structures and viable spores.     

The Fission Yeast Synaptobrevin Ortholog Syb1 Plays an Important Role in Forespore Membrane Formation and Spore Maturation

Synaptobrevin, also called vesicle-associated membrane protein (VAMP), is a component of the plasma membrane N-methylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which plays a key role in intracellular membrane fusion. Previous studies have revealed that, similar to synaptobrevin in other organisms, the fission yeast synaptobrevin ortholog Syb1 associates with post-Golgi secretory vesicles and is essential for cytokinesis and cell elongation. Here, we report that Syb1 has a role in sporulation. After nitrogen starvation, green fluorescent protein (GFP)-Syb1 is found in intracellular dots. As meiosis proceeds, GFP-Syb1 accumulates around the nucleus and then localizes at the forespore membrane (FSM). We isolated a syb-S1 mutant, which exhibits a defect in sporulation. In syb1-S1 mutants, the FSM begins to form but fails to develop a normal morphology. Electron microscopy shows that an abnormal spore wall is often formed in syb1-S1 mutant spores. Although most syb1-S1 mutant spores are germinated, they are less tolerant to ethanol than wild-type spores. The syb1-S1 allele carries a missense mutation, resulting in replacement of a conserved cysteine residue adjacent to the transmembrane domain, which reduces the stability and abundance of the Syb1 protein. Taken together, these results indicate that Syb1 plays an important role in both FSM assembly and spore wall formation.     

Ecology of Bacillus thuringiensis in storage moths

A disease-free stock of Plodia interpunctella was produced by a continuous rearing technique. In dense populations of this stock, 10⁴ or more spores of H serotype V Bacillus thuringiensis applied at one point on the surface of 200 g of food were required to cause epizootics, compared with 10⁷ or more when spread evenly over the surface. In infected populations, spores contaminated the surfaces of all stages of the insect. In diseased larval cadavers there were 5.6–42.2 × 10⁸ spores/g of dry insect (P. interpunctella, Ephestia cautella, Anagasta kuehniella, Ephestia elutella, and Galleria mellonella). Larvae did not cannibalize live larvae while food was present though they sometimes ate cadavers. This is the most potent means of natural spread of the disease. Occurring mainly in protected situations such as food stores, natural infections are usually light, but occasionally spectacular surface accumulations of dead larvae occur, possibly associated with stress, physiological condition of the larvae, serotype of the bacterium, or behavior pattern such as migration. Natural disease may curb infestations in debris, but it attacks too late to prevent excessive damage to stored food. A prophylactic, even admixture of 2 × 10⁹ spores/200 g of food is required for effective insect control.     

Detection of B. anthracis Spores and Vegetative Cells with the Same Monoclonal Antibodies

Bacillus anthracis, the causative agent of anthrax disease, could be used as a biothreat reagent. It is vital to develop a rapid, convenient method to detect B. anthracis. In the current study, three high affinity and specificity monoclonal antibodies (mAbs, designated 8G3, 10C6 and 12F6) have been obtained using fully washed B. anthracis spores as an immunogen. These mAbs, confirmed to direct against EA1 protein, can recognize the surface of B. anthracis spores and intact vegetative cells with high affinity and species-specificity. EA1 has been well known as a major S-layer component of B. anthracis vegetative cells, and it also persistently exists in the spore preparations and bind tightly to the spore surfaces even after rigorous washing. Therefore, these mAbs can be used to build a new and rapid immunoassay for detection of both life forms of B. anthracis, either vegetative cells or spores.     

Morphology and ultrasctructure of spores Klukia tyganensis Krassilov (Schizaeaceae, Filicales) from the Berriassian of the Tyrma Depression (Russian Far East)

In situ spores of the fern Klukia tyganensis Krassil. from the coal-bearing deposits on the right bank of the Tyrma River (Berriassian, Bureya River Basin, Tyrma Depression) have been examined using light, scanning and transmission electron microscopy. The spores of K. tyganensis are demonstrated to be similar in the ultrastructure of sporoderm to that of some species of the extant genus Anemia and to differ from the spores of Lygodium in the ultrastructure of perispore and exospore.