Morphological aspects

The filamentous fungus A. nidulans develops into a multicellular organism. The transition from undifferentiated growth, such as vegetative growth, to multicellular structures requires environmental and endogenous factors. Asexual and sexual development is linked to the formation of different spore-producing structures.

1.2.1. Hyphae

The initial and basic unit of filamentous fungi represents undifferentiated structures named hyphae. The formation of vegetative hyphae facilitates the habitation of various ecological niches. Hyphal growth requires cell surface expansion and cell wall disposition at the hyphal tip. Different types of hypae are known and are formed during

developmental processes.

Ascogenous dikaryotic hyphae are formed within cleistothecia. Through the fusion of two haploid nuclei a diploid meiotic nucleus is formed in an ascus mother cell. The ascus mother cell expands through a swelling process into an ascus (Kirk and Morris

1991).

Subtending hyphae are known to be attached to a spore from which the spore develops as a consequence of a swelling process. This is the case in Hülle cells.

1.2.2. Hülle cells

Eduard Eidam first described Hülle cells in 1883 where he termed Hülle cells as a

“Blasenhülle” or bubble envelope (Eidam 1883). In different species, Hülle cell like structures are known such as in Candida albicans which produce at the very end of the hyphae globose blisters named chlamydospores (Navarathna et al., 2016). Eidam suggested that Hülle cells originate from the tip of “secondary hyphae” which in turn emerge from “primary hyphae” and develop as a consequence of a swelling process (Figure 1).

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Hülle cells and the subtending hyphae are connected via two distinct types of septa (Figure 2A). The inner one is a single perforate septum where woronin bodies can be observed and represents a typical ascomycetous septum. The second septum which separates Hülle cells from the subtending hyphae is unique and named basal septum (Figure 2B). At the basal septum vesicle fusion is observable. Consequently, to this fusion so called lomasome-like accumulations are visible. These lomasome-like structures are membrane-invaginations (Figure 2C). In Hülle cells several nuclei, mitochondria, lipid bodies and storage products can be observed (Ellis et al., 1973).

Figure 1. Adult Hülle cells and the subtending hyphae of Hülle cells.

Hülle cells are globose in shape with a thick cell wall and are often connected to subtending hyphae.

These hyphae are of different size and length between very long and rather short and sometimes show a branch structure. Hyphae also contain subdividing septa. Within the globose structure of Hülle cells, a cytoplasm with metabolic activity is situated. The thick cell wall is ringshaped and open on one side. Two septa are visible in the open part of the ring (Raper and Fennel 1965).

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During initial Hülle cell formation, it was shown that several nuclei fuse to form a marcronucleus (Carvalho et al., 2002). Different species of the Aspergillus genus produce Hülle cells, including A. nidulans and Aspergillus heterothallicus (Bayram and Braus 2012). Hülle cells have an average size of 12-20 µm, are of globose shape with an unusual thick cell wall and are mainly associated with the sexual developmental program. Hülle cells are known for all species in the section Nidulantes (Chen et al., 2016). In different species, Hülle cells vary in shape between the more elongated such as in Aspergillus ustus and the globose version like in A. nidulans. In A. nidulans and Aspergillus heterothallicus Hülle cells associate with the cleistothecia, whereas in Aspergillus protuberus and Aspergillus ustus Hülle cells are not in direct contact with the cleistothecia and are formed in masses (Muntanjola-Cvetkovic and Vukic 1972).

1.2.3. Cleistothecia

In A. nidulans the products of meiosis, the sexual spores, are situated and produced in the cleistothecia. The function of cleistothecia, therefore, is to protect the sexual spores against harsh environmental conditions. During sexual sporulation, certain hyphae develop to ascogenous hypae. Ascogenous hyphae contain two haploid nuclei of opposite mating type. These hyphae form a hook shaped structure, named crozier, their nuclei divide synchronously. Different septae are formed thereby forming a dikaryotic top cell. After fusion of the end cell and the basal cell of the crozier karyogamy and further ascus development take place (Braus et al., 2002, Busch and Braus 2007). Hülle cells are most probably formed through a swelling process of certain vegetative hyphae. The first morphological manifestation of a visible structure is the appearance of cleistothecial initials, 40 hours after germination (Sohn and Yoon 2002). These structures are coiled lumps of cells, which undergo further coiling and become enlarged to approximately 10 µm. This is the stage where Hülle cells first appear (Sohn and Yoon 2002). A. nidulans produces dark reddisch ascospores which are found in globose structures named asci (Dyer and O’Gorman 2012). These asci are protected by dark brown/violet ascomata. The ascomata are named cleistothecia in Aspergillus nidulans, due to its closed conformation (Greek: Kleistos = closed, theke

= case). Cleistothecia are surrounded by numerous hyaline to pale brown globose Hülle cells.

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1.2.4. Conidiophores

The Aspergillus species produces first foot cells from vegetative hyphae, which apically extend into a stalk. The very end of this cell swells to a so-called multinucleated vesicle. The vesicle produces a finger like metula as a first layer and a second layer, the phialides are formed by mitosis (Bayram and Braus 2012). These phialides undergo repeatedly asymmetric mitotically processes which lead to the production of haploid airborne conidiospores. After initial formation, conidiospores

Figure 2. Electron microscope images of the content and septa found in Hülle cells of Aspergillus nidulans.

A) Overview picture of the content and the septa found in Hülle cells. The basal septum (bs) which separates Hülle cells from the subtending hyphae is unique. A second type of septum inside of Hülle cells is a typical ascomycetous septum where woronin bodies (W) and mitochondria (M) are observed.

At the Hülle cell side (cytoplasm) lipid bodies (Lb) and nuclei (N) are visible. Further septa are possible in Hülle cells. B) The unique basal septum (bs) of Hülle cells. At the periphery of this septum (at Hülle cell side) lomasome-like accumulations (La) of the membrane are present (lomasome-like structures are invaginations of the membrane). In between the two septa woronin bodies (W), lipid bodies (Lb) and nuclei (N) are visible. Woronin bodies are close to the basal septum (bs). C) The basal septum (bs) contains globular structures (G). Image modified (Ellis et al., 1973).

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undergo the maturation process. The progression of condiospores is a separate process from the initial phase. The development of condiospores contains several changes in cell wall structures and chemical modifications. During the maturation process, four different cell wall layers emerge. This enables impermeability of condiospores, necessary to protect spores from harsh environmental conditions.

Chemical modifications include the production of trehalose which serves as an energy source and protects against various environmental conditions such as dehydration, cold and oxidation (Elbein et al., 2003). Matured conidia are formed 15 hours after initial sporulation. The conidiophore represents the complete asexual structure which carries asexual spores called conidia.