Discharge papilla development in the phycomycete Allomyces

The process of discharge papilla (DP) formation in Allomyces macrogynus was studied by light and electron microscopy. The plug of the DP was first deposited between the plasmalemma and the wall of the zoosporangium (ZS). The wall above the plug subsequently was eroded away. Deposition of a new inner wall layer in the sporangium held the plug in place and thickening of the layer formed a collar around the plug. Further deposition of material after this stage resulted in the characteristic pulley-shape. The plug material appeared homogeneous in electron micrographs but there was evidence of an outer layer. Digestion of the plug at the time of spore release was from within.Abbreviations DP discharge papilla - ZS zoosporangium     

STRUCTURE AND COMPOSITION OF THE RESISTANT SPORANGIAL WALL IN THE FUNGUS ALLOMYCES

The fine structure and chemical composition of the wall of resistant sporangia of Allomyces neo-moniliformis were investigated. Studies with the electron microscope showed that the wall is approximately 1.3 μ in thickness and is of complex construction. It consists essentially of three parts: a five-layered outer wall, two layers of “cementing substances,” and a single-layered inner wall. The presence of a highly convoluted cell membrane was also demonstrated. Six structural components were found to make up the walls of the resistant sporangia: glucose, glucosamine, chitin, melanin, protein, and lipids. Comparison of the structure and composition of the walls of resistant sporangia with the walls of hyphae and zoospores of Allomyces as reported by other investigators showed that, while the structure is very different, the composition is quite similar with only melanin and lipids apparently being absent from the zoospore and hyphal walls.     

STRUCTURE AND GERMINATION OF BLASTOCLADIELLA EMERSONII RESISTANT SPORANGIA

Resistant sporangia of Blastocladiella emersonii were induced by the addition of bicarbonate, potassium chloride, sodium chloride, or ammonium chloride to the medium and by the exposure of the zoospores to ultraviolet irradiation. Mature resistant sporangia induced by all of these conditions exhibit similar areolate wall pitting. Under suitable conditions resistant sporangia in all cases examined germinated with the cracking of the outer sporangial wall, with the formation of exit tubes by the inner sporangial wall, and with the cleavage and release of zoospores through discharge papillae formed in the tips of the exit tubes.     

STUDIES ON THE GENETIC CONTROL OF RESISTANT SPORANGIUM FORMATION IN ALLOMYCES

The diploid sporophyte of the phycomycetous fungus Allomyces arbuscula bears two types of sporangia: thin-walled, colorless, ephemeral zoosporangia (ZS) and thick-walled, dark-brown, resistant sporangia (RS). Normal wild-type cultures (strain Portugal IE) under standard conditions produce approximately 90% of their total sporangia as RS. These RS give the cultures a dark-brown color. A mutant was induced with UV irradiation in which the ratio of ZS to RS was shifted so that only 20% of the total sporangia are RS. These cultures are a pale, tan color. Hybrids between the mutants and wild-types produce ca. 65% RS and are also intermediate in the color of the culture. Meiotic segregation in the RS of the hybrid sporophytes gives gametophytes half of which when selfed produce mutant sporophytes and half of which produce wild-type sporophytes. The shift from RS to ZS formation is thus considered to be the result of a one-gene mutation at a locus ‘R.’ The haploid gametophytes of wild-type strains have in addition to male and female gametangia a small number (2-4%) of RS. In mutant gametophytes the percent RS has dropped to 0.1-0.2%. The proposed genotypes at the ‘R’ locus in Allomyces arbuscula are: wild-type sporophytes (RR), hybrid sporophytes (Rr), mutant sporophytes (rr), wild-type gametophytes (R) and mutant gametophytes (r).     

Le corps paranucléaire des gamètes géants d'Allomyces javanicus traité à l'acide borique

Summary The gametes ofAllomyces javanicus exhibit a nuclear cap essentially made of ribonucleoprotein with sulphydryl groups.When dipped in phosphate buffered solutions of boric acid M/20–M/400, the male and female gametangia ofAllomyces liberate giant male and female gametes. Such giant gametes exhibit either a single regular nuclear cap (with 2 or 3 nuclei) or a massive and irregular basophilic formation (with 4–10 nuclei).Boric acid may interfere with ribonucleic acid, essential constituent of the nuclear cap, during the cleavage of the gamete-units in the gametangia.     

Life history of Japanese Stypocaulon durum (Sphacelariales, Phaeophyceae)

Phenology, morphology, life history and responses to different temperature and photoperiod conditions were studied in Japanese Stypocaulon durum (Ruprecht) Okamura. Erect thalli of the species were collected year-round, but the mature thalli forming either uniloc-ular sporangia or two different types of plurilocular structures (evidently gametangia) on separate thalli were found only in winter. ln culture, an isomorphic life history is suggested for the species, alternating between a sporophyte forming unilocular sporangia and gam-etophytes forming plurilocular macro- (female) and micro- (male) gametangia. Contents of unilocular sporangia were not released, but germinated in situ, developing into erect thalli forming plurilocular gametangia. Macrogametangia released aplanogametes (oospores), but male gametangia appeared to be non-functional, although flagellated cells were once formed in the loc-uli. This is the first report of plurilocular gametangia in the species. Although the species grew well and matured under considerably lower temperature conditions than European Stypocaulon scoparium (L.) Sauvageau, its temperature requirements showed similarity to northwestern Atlantic Stypocaulon species. This supports the notion that northwestern Atlantic Stypocaulon is conspecific with S. durum.     

Studies in the life cycle of Allomyces javanicus Kniep

Summary The life cycle of Allomyces javanicus was studied with the hanging drop method under laboratory conditions. The isolate has a life cyrle similar to that already described in A. javanicus and A. arbuscula. Planonts from resistant sporangia do not germinate directly to produce gametophytic plants, but on the contrary, the latter germinate to produce plants like their immediate parents. Female and male gametes growing in the gametangia of relatively young sexual mycelia develop into asexual mycelia after conjugation, but some of the female gametes after germination develop into asexual mycelia without conjugation.     

The giant and complex genital plug of the asper group of Bothriurus (Scorpiones, Bothriuridae): morphology and comparison with other genital plugs in scorpions

The genital plugs of two species of the asper group of Bothriurus (Scorpiones: Bothriuridae) are described and compared with other genital plugs reported in the family Bothriuridae. In both species, B. asper and Bothriurus sp., the genital plug is cone-shaped and formed by fusion of the basal lobes of the hemispermatophore. Fusion is complete in B. asper and the surface of the plug has many microspines that anchor it to the female genital atrium. In Bothriurus sp., the basal lobes are partially fused, but free on the dorsal side, and the plug has a smooth surface with a dorsal curvature. Both genital plugs completely fill the genital atrium of inseminated females, pressing against the cuticular wall of the atrium. Given the large size and complex shape, the genital plug of the B. asper group is unique, not only among Bothriuridae, but in the order Scorpiones. This new type of genital plug resembles the genital plugs of the scorpion families Urodacidae and Vaejovidae. A comparison of the four major types of genital plugs reported in Bothriurus species and some other bothriurids is provided, as well as a comparison with other genital plugs reported in more distantly related families of scorpions.     

Morphology and life history of Scytosiphon canaliculatus comb. nov. (Scytosiphonales,Phaeophyceae) from Japan*

Morphology of field material and life history in culture were studied in Scytosiphon canaliculatus (Setchell et Gardner) comb. nov. from northern Japan. Erect gametophytes of S. canaliculatus are cylindrical, tubular, up to 7 mm wide and 40 cm long, and without regular constrictions. S. canaliculatus has pronounced anisogamy and ascocysts accompanied with plurilocular gametangia. The life history of S. canaliculatus showed an alternation between erect gametophytes and crustose prostrate sporophytes bearing unilocular sporangia. Since field sporophytes of S. canaliculatus were found to be identical with Hapterophycus canaliculatus Setchell et Gardner (Ralfsiaceae, Phaeophyceae), it is proposed to transfer H. canaliculatus to the genus Scytosiphon. In the field, gametophytes with plurilocular gametangia appeared in spring and disappeared in summer. Sporophytes with unilocular sporangia were collected in late autumn and winter. Unilocular sporangia were produced at 15°C in short-day culture conditions and unispores developed into erect gametophytes at 5–15°C. It is suggested that the seasonal Occurrence of gametophytes in the field is due to the seasonal formation of unilocular sporangia, which is regulated by temperature and photoperiod.     

RAPID ASSEMBLY OF A WOUND PLUG: STAGE ONE OF A TWO‐STAGE WOUND REPAIR MECHANISM IN THE GIANT UNICELLULAR CHLOROPHYTE DASYCLADUS VERMICULARIS (CHLOROPHYCEAE)1

Upon injury, selected coenocytic algae are capable of forming temporary wound plugs to prevent detrimental cytoplasmic loss. Wound plugs of Dasycladus vermicularis ([Scropoli] Krasser) were harvested 5 min post‐injury and dried. The plug material contained 94% water and can be considered a hydrogel. The gel plug extended several millimeters from the cut end and filled the space inside the cell wall, which resulted from cytoplasmic retraction. Total organic carbon included 55% sugars, 5%–15% protein, and 0.18% lipids. The major sugars were glucose, galactose, mannose, and galacturonic acid. Fluorescein isothiocyanate‐lectins specific for these sugars were localized around the plug matrix. Sulfur content calculated as sulfate corresponded to 17% of the carbohydrate by weight, and sulfated material was detected in plugs by Alcian Blue staining. Formation of the initial plug occurred within 1 min of injury and was not significantly perturbed by the addition of ionic, antioxidant, or chelating agents to the seawater medium. However, addition of exogenous d (+)‐galactose and d (+)‐glucose prevented formation of the nascent gel plug. Wound plugs that were allowed to form from 10 min up until 24 h post‐injury were isolated and incubated with selected biochemical probes to identify the biochemical processes involved in plug formation. The operative strategy in Dasycladus to prevent “cytoplasmic hemorrhage” required availability of sequestered carbohydrate and lectin precursor components throughout the thallus for plug assembly. Once the initial assembly had commenced, additional biochemical interactions were initiated (as a function of time) to promote structural integrity.     

Actin stringently accumulated in the specifically positioned,differentiating female gametangia of Allomyces

Summary Female gametangia of the normal bisexual Allomyces species are richer in fluorescently probed (FITC) actin, independent of their apical or subapical positioning during differentiation on the fertile hyphae. The anti-actin, cytochalasin D, can selectively suppress male differentiation in both species.     

Early Devonian fungi: a blastocladalean fungus with sexual reproduction

In this paper we describe a fossil fungus–Paleoblastocladia milleri gen. et sp. nov.–from the 400 million-year-old Early Devonian Rhynie chert that shares numerous features with modern zoosporic fungi placed in the order Blastocladiales. The fungus occurs in tufts that arise from stomata or between the cuticle and epidermis of Aglaophyton major axes. Thallus development begins from an irregular bipolar basal cell that produces a system of intramatrical rhizoids and clavate-shaped extramatrical, nonseptate hyphae. These hyphae develop into two types of mature thalli. Sporothalli are characterized by several orders of dichotomous branching and the production of terminal, globose zoosporangia, as well as thick-walled, pitted resting sporangia. On separate dichotomously branched thalli (gametothalli) are terminal chains of two or three gametangia, in which the terminal one is slightly larger. Despite the fact that all of the reproductive organs contain either zoospores or gametes, none show evidence of discharge papillae. The fossil fungus is compared with extant members of the Blastocladiales, and the presence of sexual reproduction is discussed.     

Development of the discharge apparatus in the fungus Entophlyctis

Correlative light and electron microscopic observations were used to reconstruct the morphological events involved in the development of the discharge apparatus of Entophlyctis zoosporangia. A discharge plug formed as vesicles containing fibrillar material fused with the plasma membrane and deposited their matrices between the plasma membrane and zoosporangial wall. At the apex of the enlarging plug, the zoosporangial wall lost its microfibrillar appearance, became diffuse, and left an inoperculate discharge pore. The discharge plug exuded through this pore and then expanded into a sphere which rested at the tip of the discharge papilla or tube. After the release of the discharge plug, the number of fibrilla containing vesicles decreased and abundant endoplasmic reticulum appeared in the cytoplasm below the plug. Granular material then accumulated at the interface of the discharge plug and the plasma membrane. This was the endo-operculum. A single layer of endoplasmic reticulum subtended the area of plasma membrane which the endo-operculum covered. Later, dictyosomes appeared in the cytoplasm below the endo-operculum. Fusion of Golgi vesicles with the plasma membrane below the endo-operculum coincided with the initiation of cytoplasmic cleavage. This sequence of events indicates that, unlike the discharge plug, the endo-operculum does not originate by vesicular addition of preformed material.     

Meiosis in the aquatic fungusCatenaria allomycis

Summary Catenaria allomycis Couch (Blastocladiales) is an endobiotic fungal parasite primarily of species of the genusAllomyces. The life cycle ofC. allomycis contains both sexual and asexual phases. Synaptonemal complexes have been found in young developing resistant sporangia (RS) suggesting that meiosis occurs within the thick walled RS prior to syngamy. Ultrastructural evidence suggests that meiosis proceeds through pachytene in the developing RS and is arrested in diplotene of prophase I until the sporangia are induced to germinate at which time the meiotic process is completed. Quantitative nuclear counts in developing RS support the ultrastructural observations. Meiotic nuclei are characterized by polar fenestrae in the nuclear envelope and intranuclear plaque-like microtubule organizing centers (MTOC).Portion of a Ph.D. dissertation submitted by the senior author to the Graduate School, University of Georgia.     

A mechanism of propagule release from unilocular reproductive structures in brown algae

Summary The structure of unilocular sporangia inP. littoralis was investigated along with several other species of brown algae in order to study the mechanism by which propagules are released from unilocular reproductive structures. Unilocular sporangia inP. littoralis are composed of a spherical cell wall of two distinct layers and contain a number of zoospores. The mass of spores is surrounded and permeated by mucilaginous carbohydrates. It is suggested that the production of these carbohydrates generates the necessary pressure to weaken the sporangial wall. In addition, ultrastructural observations indicate that further weakening seems to occur due to digestion of the inner wall layer. Walls of sporangia were mechanically broken just prior to normal spore release in order to investigate whether internal pressure exists, and if it can effect spore discharge. Results show that an internal pressure does exist prior to normal spore discharge and that this pressure is not generated by turgor pressure of the spores themselves or by a semi-permeable wall osmoticum system. The discharge of spores seems to occur when the carbohydrate around the spores swells. The adsorption of water when plants are immersed by the incoming tide thus seems a likely mechanism of spore discharge. The similarities of unilocular reproductive structures and spore release in several brown algal species suggests common mechanisms of propagule discharge for members of thePhaeophyta.     

A Virus Infection in the Marine Brown Alga Ectocarpus siliculosus (Phaeophyceae)

Laboratory cultures of Ectocarpus siliculosus originating from New Zealand showed a defect in gametangium formation. Nuclear divisions in gametangium initials are not followed by cell wall formation. In the resulting multinucleate cells nuclear DNA increases dramatically, and nuclear membranes disintegrate. Eventually, the entire structure is filled with hexagonal particles of approximately 130 nm diameter. These were isolated and shown by EM to consist of a dense core surrounded by a 3-layered shell. They are released into the culture medium when the host cells burst. Ectocarpus gametes from healthy cultures could be infected by these particles. The resulting partheno-sporophytes developed pathological symptoms, suggesting that the particles are viruses. The expression of the defect is temperature dependent. At 10°C all gametangia are abnormal, while between 15 and 20 °C defective and normal gametangia and gametes are formed on the same plant. Partheno-sporophytes developing from such gametes carry the viral particles expressed in deformed unilocular and plurilocular sporangia.     

PH-DEPENDENCE OF CHLORTETRACYCLINE(CTC)-INDUCED PLUG FORMATION IN NITELLA FLEXILIS (CHARACEAE)1

The formation of chlortetracycline(CTC)-induced wall appositions (callose plugs) in Nitella flexilis (L.)Ag. was pH-dependent in the range between 4.3-8.3. Plug number and plug diameter increased with the pH of the CTC solution. At pH 4.3 plug formation was light-dependent and occurred below the alkaline regions of the cell surface which form during photo synthetic assimilation of HCO₃^?. Inhibition of photosynthesis by 3–(3′,4′-dichlorophenyl)-1, 1-dimethylurea prevented plug formation in the light. Dark-treated cells could be induced to form plugs by raising the pH of the CTC solution. The formation of large but incomplete plugs in the presence of cytochalasin B is explained by the formation of numerous weak alkaline sites. I suggest that CTC enhances locally the Ca²⁺content at the cytoplasm near the plasmamembrane. The ionophoric character of CTC is probably more pronounced at high pH mainly because of a weaker binding with cations and a closer contact with the membrane.     

SEPTAL PLUGS IN A GREEN ALGA

Septal plugs, resembling those found in red algae, occur in the transverse wall between all cells in a newly discovered marine green alga, Pilinia earleae Gallagher & Humm.³ No plasmodesmata traverse the cross-wall, and the septal plug blocks cytoplasmic continuity between cells. The septal plug consists of an electron-translucent core bordered at each end by two electron-opaque caps. Cytochemical procedures demonstrate that the plug consists of protein and polysaccharide, but lacks peroxidase. The outer cap is highly proteinaceous while the inner cap is composed primarily of polysaccharide. The plug core is not routinely stained by Coomassie Blue but it is pronase sensitive and probably proteinaceous. Historically, septal plugs have been considered unique to the red algae and the fungi, but ultrastructural and biochemical data provide no support for derivation of the septal plug in this green alga from a symbiotic relationship. The discovery of septal plugs in a green alga makes the hypothesis of an independent origin of this structure in a number of plant groups more likely.     

Zoosporic fungi of Oceania. IV. Additional monocentric chytrids

Summary Several monocentric chytrid species of the generaPhlyctochytrium,Rhizidium,Chytridium,Chytriomyces andKarlingia were isolated on various substrata from soils of Oceania. Among these are two new species,Phlyctochytrium megastomum andRhizidium endosporangiatum. The former species is characterized chiefly by sporangia with an usually large apical, subapical, or a lateral exit orifice, the persistence of remnants of the pailla wall at the edge of the exit orifice, and by small zoospores. Rhizidium endosporangiatum is characterized at first primarily by predominantly spherical sporangia which develop 1 to 8 broad papillae. At maturity these papillae give them an angular shape. The exit papillae deliquesce at the tip, and through them protrude prominent arms of an endosporangium which expands and in which the zoospores usually swarm briefly. Willoughby's Chytridium parasiticum occurred abundantly as a parasite ofNowakowskiella sp.,Pythium sp., andPhytophthora sp. and developed epibiotic resting spores like those ofChytriomyces. Accordingly, this parasite is transferred to this genus and renamedChytriomyces willoughbyii because the nameparasiticum is preempted by the author's previously describedChytriomyces parasiticus.This study has been supported by a grant from the National Science Foundation, Washington, D.C.     

The morphology of mating plugs and its formation in scorpions: Implications for intersexual participation

Mating plugs have been proposed as a mechanism that has evolved to avoid sperm competition. Their structure and composition vary across taxa and are related to the effectiveness of its function. This effectiveness could be related to different evolutionary interests of the sexes. Urophonius brachycentrus and Urophonius achalensis (Scorpiones, Bothriuridae) are highly suitable species to study mating plugs because both are monandrous species with specific morphological and physiological responses in the female's genitalia. Here, we analyze (a) the morphology and fine structure of the mating plugs of both species, (b) the site of production in males and the formation process of the mating plug, and (c) the changes that it undergoes over time in the female's reproductive tract. In both species, a complex mating plug obliterates the female's genital aperture and fills the genital atrium. We observed considerable interspecific variation in the mating plug morphology. A mating hemi-plug was found surrounding the capsular lobes of the hemispermatophore, which could have a mixed composition (involving portions of the hemispermatophore and glandular products). The glandular portion was transferred in a semi-solid state filling the female's genital atrium and then hardening. Changes that the plug undergoes in the female's genitalia (darkening and increase of the “distal” area of the plug) indicate a participation of the female to the formation of this type of plug. Our study provides new insights into the plugging phenomenon in scorpions, and we discussed the adaptive significance as a post-copulatory mechanism to avoid sperm competition.