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Live imaging analysis of sexual and asexual reproduction, zygospore and sporangiospore formation, in Gilbertella persicaria
2024 Volume 65 Issue 4 Pages 180-186
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2024 Volume 65 Issue 4 Pages 180-186
Most Mucoromycota fungi form zygospores as sexual reproductive structures. When two colonies of compatible strains meet, zygospores are formed in the area where the colonies meet. The structure and development of zygospores have been studied for a long time by light microscopy and electron microscopy. This study is the first time-lapse report on the dynamic movements of sexual and asexual reproductive processes by live imaging in Gilbertella persicaria (Choanephoraceae, Mucorales). Our live imaging analysis indicated the formation of zygospores begin immediately after two aerial hyphae contact whether at the tip or middle of the hyphae. The early-stage zygospores elongated from the contact site with a rate of 1.2-1.7 µm/s and reach < 200 µm in 2-3 h. Following maturation of zygospores, from progametangia to gametangia and maturation stage, took a few hours, in total 5 to 6 h after the first contact of two hyphae. When a zygospore was formed near the tip of hypha in contact with the partner hypha, the hyphal growth ceased. When zygospore was formed behind the tip of the hypha, the hyphal growth continued without slowing down. This study provides quantitative spatio-temporal information on the dynamics of zygospore formation.
Fungi exhibit dynamic behavior in hyphal growth and development (Riquelme et al., 2018; Takeshita et al., 2017). Reproductive behavior in fungi is usually recorded using light and/or electron microscopy. Such observations are known as the reproductive ontogeny, which is well studied in model fungi such as Saccharomyces cerevisiae (Desm.) Meyen (Guth et al., 1972) and Aspergillus nidulans (Eidam) G. Winter (Sohn & Yoon, 2002). Most fungi in the phylum Mucoromycota except for Glomeromycotina form the sexual zygospores as reproductive structures, which are relatively bigger compared to reproductive structures formed by other fungal groups and can be induced in artificial media easily. Zygospore development was studied by light microscopy and/or by transmission and scanning electron microscopy in Mucor spp. (Schipper, 1969), in Rhizopus sexualis (G. Sm.) Callen (Hawker et al., 1957; Hawker & Beckett, 1971), in Gilbertella persicaria (E.D. Eddy) Hesselt. (O'Donnell et al., 1977), in Mortierella indohii C.Y. Chien (Ansell & Young, 1983) and in R. stolonifer (Ehrenb.) Vuill. (Ho & Chen, 1998). Details of all stages, including dissolution of the fusion wall, delimitation of the gametangia by septa, and formation of the complex wall of the zygospore were described and illustrated (Hawker & Beckett, 1971; O'Donnell et al., 1977). The effects of temperature, light, pH, and type of medium on zygosporogenesis of Mucor piriformis A. Fisch. and G. persicaria were studied as well (Michailides et al., 1997).
A series of snapshot observations indicate the development process of zygospores in Mortierella spp. (Degawa & Tokumasu, 1997, 1998a, 1998b). Observations by snapshots enable us to predict the dynamic movements of reproductive processes, however it is still difficult to record each time point of developmental stages and speeds of the development. To obtain the unrecordable information by snapshots, we observed the dynamic movements of reproductive processes by live imaging in G. persicaria. This species is suitable as experimental material for the live imaging because the development of zygospores is completed in a relatively short time. Our live imaging analysis here provides spatio-temporal information of zygospore formation from initiation to maturation in G. persicaria.
Strains, media. On 9 Apr 2015, a soil sample under Morus alba L. in front of the Ueda city hall, Ueda-shi, Nagano Pref., Japan, was collected and used for induction of G. persicaria in a moist chamber incubated at 25 °C using banana as a bait. After the incubation for a few days, several strains of G. persicaria occurring on the bait were established. Single-sporangiospore-isolation was conducted prior to mating experiments. Two compatible representative strains of G. persicaria used in this study were determined by a mating experiment and deposited in the NARO Genebank (MAFF, Ibaraki Pref., Japan). Two compatible strains of G. persicaria (MAFF 247833; strain name SFC55 and MAFF 247834; strain name SFC57) were point-inoculated in potato dextrose agar medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) with a spacing of approximately 2 cm.
Zoom microscopy. Images were recorded by a stereo microscope (Axio Zoom V16: Carl Zeiss, Jena, Germany) combining a 16x zoom with a high numerical aperture of NA 0.25, with objective lens (PlanApo Z: Carl Zeiss), microscope camera (AxioCam: Carl Zeiss), and reflected light illumination (slit-ring illuminator with cold light source CL 6000 LED: Carl Zeiss). Time-lapse images were taken every 10 min at room temperature and analyzed by using the Zen system (Carl Zeiss) and ImageJ software (Schneider et al., 2012).
Two compatible strains of G. persicaria were point-inoculated in potato dextrose agar medium with a spacing of approximately 2 cm. Each mycelium grew and the periphery of the colony met at 14 h. Three hours later (17 h), a black band appeared at the border of the colonies. The area expanded with darker color as time passed (Video 1; Fig. 1A). By magnification of the black band, the formation of zygospores and sporangia were observed (Video 2; Fig. 1B). Zygospores were formed first where aerial hyphae met (Video 2, t = 0:00-5:00; Fig. 1B, t = 4:00), and then sporangia formed to cover the zygospores (Video 2, t = 6:00-18:00; Fig. 1B, t = 18:00). Zygospores and sporangia are indicated by arrowheads and arrows, respectively (Fig. 1B). First, the formation of sporangia was recorded in time-lapse every hour for 3 d (Video 3; Fig. 1C). The tips of the aerial hyphae expanded and became a sphere, and the color changed from yellow to black. This structural change was monitored at higher magnification in time-lapse at 10-min intervals (Video 4; Fig. 1D). It took 70 ± 10 min for the tips of the aerial hyphae to expand and become a sphere 80 ± 12 µm diam (mean ± SD, n=20, respectively). The color and shape remained the same for 110 ± 10 min, then the pigments spread out in the sphere over the next 70 ± 10 min (mean ± SD, n=20, respectively). The pigmented area appeared outside the sporangia (Fig. 1D, arrowheads) and from there the pigment spread to cover the entire area. The darkening during sporangia maturation is expected to take place as a consequence of the buildup of strongly hydrophobic pigments in a specific region of the hydrophilic sporangia surface. Once a certain quantity of pigment is generated, it could rapidly encircle the entire sporangia surface, owing to surface tension.
Next, we recorded the formation of zygospores by live imaging every 10 min (Video 5; Fig. 2A). The zygospore formation began as soon as the elongating aerial hypha came into contact with another hypha (Fig. 2A, t = 1:19-1:39). The early-stage structure, called progametangia (Hawker & Beckett, 1971), grew at a constant rate of about 170 µm in 120 min (Video 5; Fig. 2B). Video 5 shows that the early-stage structures (blue and orange lines) elongate almost simultaneously at a rate of 1.2 and 1.3 µm/s, respectively, during 120 min. In this case, the tips of contacted hyphae did not elongate (asterisks), suggesting that the cytoplasm and other materials used for hyphal growth are used for zygospore formation.
After the early sexual structure elongated vertically between two parallel hyphae, a horizontal constriction was clearly visible in the center (Video 6, 7; Fig. 3A, t = 4:40, 2B, t = 1:30, arrows). This stage is called early gametangia, which is separated by cross-septa or called fusion wall (Hawker & Beckett, 1971) or fusion septum (O'Donnell et al., 1977). Approximately 50 µm region above and below the constriction showed dark color. Within one hour, the fusion wall became indistinct, then two constrictions appeared above and below each other. (Fig. 3A, t = 4:50-5:30, 3B, t = 1:30-3:00). This stage is gametangia separated with two gametangial septa (Fig. 3A, B, arrow heads) and suspensors (Fig. 3A, B asterisks) (O'Donnell et al., 1977). The initialization of zygospore begins by destruction of fusion wall and mingling of contents (Hawker & Beckett, 1971). The zygospore trapped between the two gametangial septa and suspensors expanded perpendicular to the suspensors next 3 h. The expansion is shown in the kymograph (Fig. 3C, the width of 45 µm at t = 3:00 reached 65 µm at t = 6:00). During the zygospore development, the zygospore became spherical and darker as it expanded.
These formation times can be summarized as follows. Immediately after two aerial hyphae contact, the progametangia begin to elongate from the contact site with rate of 1.2-1.7 µm/s and reach < 200 µm in 2-3 h. When the elongation of progametangia slows down, a fusion wall appeared in the center of progametangia. Within an hour, the fusion wall disappears and gametangia separated with two gametangial septa. The gametangia between suspensors expanded perpendicular to the suspensors following 2-3 h, which is development and maturation of zygospore with spherical shape, dark pigment and specific wall. The zygospore formation completes in 5 to 6 h after the first contact of two hyphae. While this could be faster than that in Mortierella spp. (Degawa & Tokumasu, 1998b), the duration for zygospore formation could vary depending on factors such as the nutrient composition of the medium, temperature, and humidity. Additional research is necessary to gauge the extent of variation among distinct parental strains and various species in different environments.
We observed that zygospores are formed by contact between near the hyphal tips (Fig. 2A), where the growth of original hyphae are arrested. We observed that a zygospore was also formed by contact between one hyphal tip and 300 µm distant from the tip of another hypha (Video 8; Fig. 4A). In this case, both hyphae continued to grow without slow down (Fig. 4A, asterisks). The zygospore elongated at a rate of 1.7 µm/s, while the hypha elongated at a rate of 4.0 µm/s (Fig. 4B).
When multiple hyphae touched one hypha, multiple zygospores were formed on the same hypha at the same time (Video 9; Fig. 4C). Because of the complexity of hyphal movement, each hypha is indicated in different colors (Fig. 4D). The blue hypha formed zygospores with the orange hypha at 1 and 2, and with the gray hyphae at 4. The orange hypha formed zygospores with the blue hypha at 1 and 2, and with the green hypha at 3. The zygospores were formed simultaneously on the same hyphae in the vicinity within 100 µm.
We observed a zygospore was formed by contact between 170 µm distant from the tip of one hypha and 240 µm distant from the tip of another hypha (Video 10; Fig. 4E, white arrow). In this case also, both hyphae continued to grow (Fig. 4E, asterisks). Another zygospore began to form after contact with another hypha in the vicinity of 60 µm (Fig. 4E, black arrow). The contact site is 277 µm and 79 µm distant from the tips of the hyphae respectively, and both hyphae continued to grow.
Morphological analysis of zygospores has been done for a long time (Degawa & Tokumasu, 1997, 1998a, 1998b; Hawker & Beckett, 1971; Ho & Chen, 1998; Michailides et al., 1997; O'Donnell et al., 1977). This first time-lapse report provides quantitative spatio-temporal information on the long-known dynamic phenomenon of zygospore formation. Our movies allows us to share the dynamic of zygospore formation widely and objectively. The early sexual structure of G. persicaria elongate from the contact site with rate of 1.2-1.7 µm/s and reach <200 µm in 2-3 h. Following initiation of zygospores, from progametangia to gametangia, the fusion wall disappears and the gametangia is separated with two gametangial septa within 1 h. The maturation stage takes a few hours. Zygospore formation in G. persicaria takes total 5 to 6 h after the first contact of two aerial hyphae. Here the maturation of zygospore refers to a stage where its size reaches its maximum and remains constant. It is not confirmed whether the zygospores actually germinate and produce the next generation.
When two colonies of different mating types grow in close proximity before forming zygospores, the mycelial growth stops (Drinkard et al., 1982). The mutant screening analysis has shown that carotene-derived pheromones contribute to mating type recognition and the growth arrest in Phycomyces blakesleeanus Burgeff (Sutter, 1975). The pheromone system of zygomycetes utilize a volatile organic molecule, trisporic acid and its precursors, which are cleaved from β-carotene via several enzymatic steps in a cooperative synthetic pathway (Lee & Idnurm, 2017; Schimek & Wöstemeyer, 2009). Our movies clearly indicate that one aerial hypha touches another hypha immediately the early-stage zygospores begin to elongate from the contact sites. It is possible that initiation of zygospore formation is triggered by accidental contact between hyphae in G. persicaria, rather than two branching tips from two partner hyphae seek each other's tips and fuse (Banbury, 1955). Accidental contact around the tip or middle of the hyphae would occur more easily and frequently than the fusion of tips of aerial hyphae in three dimensions. The pheromones are required for recognition between mating partners and zygospore morphogenesis and development (Burmester et al., 2007; Schimek & Wöstemeyer, 2009). That function could be necessary for the process to proceed properly after hyphal contact. This may be the reason why zygospore formation begins in closely related species (Kuhlman, 1972; Stalpers & Schipper, 1980), although zygospore formation is not completed if the species are different.
Our results that the tip growth of contacted hyphae stop during the early-stage zygospores elongate (Fig. 2A) suggest that the cytoplasm and other materials required for hyphal growth are used for zygospore formation. On the contrary, we found the example that the contacted hyphae continue to grow during the early-stage zygospore elongation (Fig. 4A, E). Whether hyphal elongation continues during zygospore elongation may depend on the distance of the contact site from the hyphal tip and the amount of material supplied by the hyphae, i.e., its elongation rate. With an equal supply of cytoplasm from the two hyphae, the early-stage zygospores appear to be elongated and separated by fusion wall (Hawker & Beckett, 1971) or fusion septum (O'Donnell et al., 1977) (Fig. 3A, B, arrows) where the two streams meet. Transmission and scanning electron microscopy analyses indicate in detail changes in organelle distribution, septal structure, and surface wall during zygospore development and maturation (Hawker & Beckett, 1971; Ho & Chen, 1998; O'Donnell et al., 1977). Additionally, possible cytoplasmic flow to induce the structural changes was expected at each stage (Hawker & Beckett, 1971). Our live imaging analysis adds information of dynamics to the valuable records of the past, helping us understand how the sequence of processes occurs.
The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.
Supplementary online materials (Video 1~10) are available at https://doi.org/10.47371/mycosci.2024.03.002
This work was funded by MEXT KAKENHI grant number 21H02095, 22H04878, 21H02095, 22H04878. We thank you Dr. K. Seto for his assistance for the isolation of strains used in this study.
