Regular Article
Temperature influences on dimorphism of microthallus in a brown alga Cladosiphon okamuranus
2024 Volume 89 Issue 2 Pages 89-96
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2024 Volume 89 Issue 2 Pages 89-96
The life cycle of edible brown alga Cladosiphon okamuranus is composed of two generations, sporophyte and gametophyte, with parthenogenesis of asexual zoids and gametes. Regardless of the origin of a motile cell, almost all microthalli, except for a prostrate filament, experience prostrate disks with assimilatory filaments as their developmental steps. The disks from different origins are morphologically undistinguishable resulting in confusion of ‘seed’ cultures in factual cultivation scenes. In this study, we therefore attempted to characterize the prostrate disks morphologically by using haploid and diploid strains of C. okamuranus, which were maintained by parthenogenesis. Two types of prostrate disks, a disk with long assimilatory filament (long type) and a disk with short assimilatory filament (short type), were identified in the diploid culture and their appearance ratios were influenced by temperature, although the haploid strain possessed only short type. The long type showed a similar morphology to the disks on culture nets harvested from an actual cultivation site. Our results imply that this morphological differentiation (dimorphism) in the diploid strain may be a divergent step between macrothallus bearing unilocular sporangia or microthallus bearing only plurilocular sporangia, and water temperature may be one of the key regulatory factors for the morphogenesis in dimorphism.
Brown algae involve the species showing the isomorphic or heteromorphic alternation of generations (Nakahara 1984). In a species with the heteromorphic alternation of generation, the differences in thallus sizes between sporophytes and gametophytes are remarkable, although generation and the thallus size are not correlated. The microscopic thallus is likely to occupy a major period of its life in many brown algal species in nature, but the morphology of the microscopic thallus has not been well studied. The microthalli are often responsible for a crucial step in the cultivation of brown algae. For instance, in the case of Cladosiphon okamuranus, the culture of microthalli is utilized as a ‘seed’ culture due to the convenience of the proliferation and maintenance, yet using microthallus culture sometimes confuses laboratories and actual cultivation due to the indistinguishable morphology of the microthallus.
C. okamuranus belonging to Chordariaceae (Ectocarpales sensu lato, Phaeophyceae) indicates the heteromorphic alternation of generations with microscopic gametophyte and edible macroscopic sporophyte (Shinmura 1974, 1975, 1976, 1993) (Fig. 1). The sporophyte bears unilocular sporangia in the bottoms of assimilatory filaments, and a released spore from a unilocular sporangium develops male or female gametophyte as a prostrate disk with assimilatory filaments (microthallus). After maturation of plurilocular gametangia in the upper parts of the assimilatory filaments of the gametophytes, released gametes are fused and then develop a prostrate disk as an initial development resulting in macroscopic sporophyte (Shinmura 1976). All motile cells (released from plurilocular gametangium, unicellular sporangium, and plurilocular sporangium) possess the ability of parthenogenesis generating prostrate disks with assimilatory filaments (microthalli). Even in C. okamuranus showing the heteromorphic alternation of generations, the morphologies of prostrate disks including early developments between sporophyte and gametophyte are indistinguishable (Shinmura 1974, 1975, 1976). Only development to the macrothallus can be a cue for the identification of sporophyte, but appropriate culture conditions for macrothallus (erect thallus) development are still obscure, leading to further confusion in culture.
This is a modified version of Fig. 10 in Shinmura (1993).
Some species in Chordariaceae, historically treated as Chordariales, show heteroblasty and/or dimorphism in their life cycle. The heteroblasty was discovered by Sauvageau in Cladosiphon zosterae (as Coastagnea zosterae) (Sauvageau 1924). In Myrionema strangulans, Loiseaux (1968) described the different manners of germling of zoospores from a plurilocular zoidangia as dimorphism, and developments of distinct morphology by the same manners of germling of the zoospores as dimorphism (Figure 1 in Loiseaux 1968). These morphological variations in microscopic stages cause serious confusion in the understanding of their life cycles. In the developmental process of asexual spores from plurilocular sporangia of C. zosterae, three factors controlling morphological expression were suggested: heteroblasty, nitrogen sources, and morphogenetic factors from bacteria (Lockhart 1979). Furthermore, it has been reported that manners of morphogenesis in some species of Ectocarpales sensu lato are affected by environmental factors like temperature, light periodicity, and light quality (Wynne 1969; Lüning and Dring 1973; Dring and Lüning 1975a, b).
In this study, we aimed to elucidate morphological variations of the microthallus in C. okamuranus and tried to understand the influence of temperature on morphogenesis. First, a haploid strain was established from a natural individual bearing unilocular sporangia, and then a diploid strain was obtained as autogamy by culturing several haploid disks orthotopically. Using these two strains, we observed the morphological characteristics and variations of the prostrate disks that are produced by parthenogenesis. In addition, we examined the effect of incubation temperature on the appearance rates of variations of the prostrate disks in the diploid strain.
Two culture strains were established from a matured thallus harvested at the Shikiya sea farm in Chinen, Okinawa on the 6th of June 2018. Several unilocular sporangia were isolated by a Pasteur pipette and washed several times with distilled seawater under a stereomicroscope. Each unilocular sporangium was separately maintained in a drop of half-strength PES medium (1/2 PES, Provasoli et al. 1957) on a sterilized coverslip, and the cover slip was kept in a petri dish settled at 25°C under long-day condition (L : D=14 : 10) with suitable light intensity (20–30 µmol m−2 s−1) with an incubator, Multi Thermo Incubator MTI-201 (EYELA, Japan). Following the settlement of some spores from the unilocular sporangium on the coverslip, the coverslip was transferred to a new petri dish filled with 1/2 PES. A developed gametophyte as a prostrate disk was scraped off to isolate and maintained as a haploid strain in a few weeks (named CoS1-5H). Meanwhile, several gametophytes were maintained together for a while to originate a diploid strain probably via fusion (named CoS1-5D). Both haploid and diploid strains were maintained by parthenogenesis in the culture condition described above.
Diploid and haploid cells from natural thallusTo evaluate the nuclear phase of the two culture strains described above, the diploid and haploid cells were obtained from a natural sporophyte. The sporophyte was collected at Bise, Okinawa, and a part of the thallus was mashed on a coverslip and prepared as a diploid sample for nuclear staining. Juvenile germlings of zoids from a unilocular sporangium of a natural thallus collected in Bise were used as a haploid sample.
Nuclear staining and measurement of fluorescence intensityThe two culture strains and the natural samples were prepared on coverslips and all procedures for staining and observation were applied to the coverslips with the cells. We followed the staining procedure described in Motomura (1995) which utilized the intensity of mithramycin A for DNA measurement. The cells were fixed with Carnoy’s fixative (ethanol : glacial acetic acid=3 : 1) for 1 h on ice following the initial fixation with 50% (v/v) fixative in the culture medium for 10 min on ice to reduce the damage on the cells. Fixative was replaced with 70% (v/v) ethanol in ultrapure water and the sample was maintained at −30°C for 24 h or more. Then, the samples were washed with sterilized water for 1 h with mild shaking. This step was repeated twice. The ultrapure water was replaced with McIlvaine buffer (pH 7.0) containing 5 mM MgCl2 for 1 h with mild shaking and this step was repeated twice, as well. Each sample was covered with a drop of 25 µg mL−1 mithramycin A (M6891, Sigma-Aldrich, USA) in the McIlvaine buffer (pH 7.0) containing 5 mM MgCl2 and incubated for 1 h in the dark at 27°C. The stained sample on a coverslip was mounted with an antifade reagent (VECTASHIELD Mounting Medium H-1000, Vector Laboratories, USA), and observed with an epifluorescence microscope (BX53F2, OLYMPUS, Japan) equipped with a digital camera (WRAYCUM NOA2000, WRAYMER, Japan). For the detection of Mithramycin fluorescence, a filter system consisting of a BP460-495 excitation filter, DM505 dichroic beam splitting mirror, and BA510IF suppression filter was used. In addition, a 525AF45 filter (XF1074, Omega Optical, USA) was used to reduce the autofluorescence of chlorophyll. All images were taken with identical settings for the microscope and camera, which suppress the background fluorescence (Fig. 2). The fluorescence intensity of mithramycin was measured by Image J (Schneider et al. 2012), and 300 nuclei in the culture strains or 100 nuclei in natural samples were used for the measurement of the fluorescence intensity. For comparison of DNA values indicated by the fluorescence intensity of mithramycin, only interphase nuclei were selected. A nucleus with fluorescence was segmented by a certain threshold, then fluorescence intensity was measured as Mean Grey Value, which indicates the average grey value within the segmentated area. The stainings and measurements of diploid and haploid samples were simultaneously performed to compare them under the same conditions.
(A, B) Two-disc thalli from CoS1-5H strain. (C, D) Combined disc thalli from CoS1-5D strain. (A, C) Each spot of the fluorescence signals of mithramycin A indicated a single nuclear and the intensity from each spot was evaluated as a Mean Grey Value. (B, D) DIC images on the right side showed the ideal thalli from the left side. Scale bar=50 µm.
Cultivation net with juvenile thalli of C. okamuranus was kindly offered by a producer at Chinen on the 28th of November 2018. The strain of the microthallus from the cultivation net was acquired by a producer of C. okamuranus at the cultivation site inside of natural lagoon.
Measurements of assimilatory filamentsTwo types of prostrate disks in the diploid culture and some disks on the cultivation net were scratched and collected by a processed Pasteur pipette. One or two disks from each sample were gently crushed on a slide and observed with a microscope (BX53F2, OLYMPUS). Images acquired by a digital camera (WRAYCUM NOA2000, WRAYMER) were used for measurement of the length of assimilatory filaments (n=20) and the width of cells in the assimilatory filaments (n=25) with Image J.
Influence of temperature on prostrate disk morphologyThe effect of temperature on the morphogenesis of two types of prostrate disks was examined with haploid and diploid strains, CoS1-5H and CoS1-5D. The culture condition was the same as described above, except for temperature. Three conditions, 20, 25, and 30°C, were set in a growth chamber (Multi Thermo Incubator, EYELA MTI-201, Tokyo Rikakikai, Japan). A matured disk with plurilocular sporangia (CoS1-5D) or plurilocular gametangia (CoS1-5H) was selected as a mother thallus and transferred to a dish with two coverslips inside. Then, the dish was kept at 20, 25, or 30°C. The culture medium was refreshed once a week. After 23 days, settled prostrate disks, which were larger than 5 mm in diameter, were randomly selected and clarified into three types, long type, short type, and other (premature disk without assimilatory filaments).
StatisticThe numerical data such as fluorescence intensity and morphological measurements were analyzed for statistical significance using Welch’s t-test. The difference was assessed with a two-sided test with a α level of 0.01.
At first, we attempted to confirm the nuclear phase of two culture strains by following the previous study of the nuclear phase with mithramycin A (Motomura 1995). The natural thallus collected at Bise was used as reference cells of diploid and as a mother thallus for getting the haploid cells developed from released spores of the unilocular sporangium. The mean values of fluorescence intensity were 13.74±2.88 (n=100) for the diploid cells and 7.08±2.10 (n=100) for the haploid cells in the natural thallus, and these two groups had significant differences (p=7.28×10−44) (Fig. 3). In the two culture strains CoS1-5D (diploid) and CoS1-5H (haploid), the average values of fluorescence intensity were 10.23±1.14 (n=300) and 5.47±1.16 (n=300), respectively. The haploid and diploid cells indicated significantly different intensities (p=4.98×10−218). These results confirmed that CoS1-5D and CoS1-5H were maintained as diploid and haploid strains.
Haploid and diploid materials from natural thalli were shown in (A) and (B), respectively. For the culture strains, CoS1-5H (C) and CoS1-5D (D) were shown. A hundred cells (A, B) or three hundred cells (C, D) were measured.
In the cultures of these two strains, unilocular sporangia appeared in neither the diploid strain nor the haploid strain. In contrast, a lot of plurilocular sporangia or gametangia were frequently formed around the tip of assimilatory filaments in both strains, so these strains seemed to be maintained with parthenogenesis of the spores from plurilocular zoidangia in the tips of assimilatory filaments (Fig. 4A, B). In the developmental process of the spores, we found that CoS1-5D and CoS1-5H exhibited distinct morphological characteristics of microthalli regardless of nuclear phases. In the process of a prostrate disk development regardless of nuclear phase, after a settlement of a zoospore on a coverslip, the cell began to proliferate two-dimensionally and form a basal disk with heart-shaped cells (Fig. 4C). Then, assimilatory filaments were erected from the basal disk resulting in the formation of a microthallus. In addition to the prostrate disks, prostrate filaments almost always appeared in both strains, and the average ratio of the prostrate filament appearance was 37.5% in the haploid strain and 9.3% in the diploid strain. The absolute dominance of the prostrate disk was always maintained in both strains. Noteworthy, even in the prostrate disks of the diploid strain, there were two morphological types of microthalli, long type and short type, due to the length of the assimilatory filaments (Fig. 4D, E). The length of the assimilatory filaments was significantly different between the two types (p=1.51×10−16) in contrast to their width (p=0.024) (Table 1). Compared to the microthalli on the culture net, the length of assimilatory filaments in the long type was similar to the one of the culture nets rather than the short type (the p-value between the long type and the culture net was 0.024, and the p-value between the short type and the culture net was 4.68×10−15). Furthermore, the shapes of the assimilatory filaments were straight in the long type but wavy in the short type (Fig. 4H, I). Growth direction was also distinct between the two types, radial in the long type and vertical in the short type. The short-type microthallus sometimes showed unique biseriate assimilatory filament (Fig. 4J).
Prostrate disks develop plurilocular sporangia in the upper part of assimilatory filaments in the diploid (A) and haploid (B) strains. Arrows indicate the empty sporangia. Early development of a prostrate disk initiates with two-dimensional cell development resulting in the basal part of the microthallus. The basal disk is initially formed by heart-shaped cells (C). Two types of microthallus in the diploid strain are shown (D–I). Two types of microthallus, the long type (D, F, H) and the short type (E, G, I), were shown. The long-type microthallus (D) and the short-type microthallus (E) possess distinct lengths and erecting directions of the assimilatory filaments. Side views of the microthalli are shown in F and G. The morphology of the assimilatory filament cells is similar (H and I), but the assimilatory filament of the short type is slightly wavy (I). Only in the prostrate disks of the haploid strain, assimilatory filaments often generate biseriate cell arrangement partially (J). Scale bars=100 µm (B, C, H–J) and 200 µm (A, D–G).
| Long type | Short type | Cultivation net | C. okamuranus | |
|---|---|---|---|---|
| Length (µm), n=20 | 201.87±21.35 | 72.82±30.74 | 227.24±41.29 | 150–250 |
| Width (µm), n=25 | 7.82±1.16 | 7.41±1.39 | 7.59±1.12 | 7–10 |
| Shape | Straight | Wavy | Straight | |
| Growth direction | Spread | Vertical | Spread | |
| References | Inagaki (1958) |
The long and short types of microthalli were both from the diploid strain (CoS1-5D).
To understand the influence of temperature on the morphology of the microthalli, the haploid and diploid cultures were maintained in three different conditions, 20, 25, or 30°C. The diploid strain (CoS1-5D) showed two types of microthallus, the long type and short type, in contrast, the haploid strain (CoS1-5H) possessed only the short type (Fig. 5). Interestingly, the ratio of the morphotype appearance of microthallus was drastically changed depending on the temperature. The maximum ratio of the appearance of the long-type microthallus was at 25°C although it disappeared at 30°C. At the same time, the ratio of premature thallus seemed to be affected by temperature.
The long type appeared only in the diploid strain (CoS1-5D) in contrast to the haploid strain (CoS1-5H).
In the species of Ectocarpales sensu lato, clonal spores often produce both filamentous and discoid microthalli in their developmental processes, coincidentally (Peters 1987). Following the definition summarized in Lockhart (1979), this phenomenon is defined as heteroblasty since the filamentous and discoid thalli are developed via the distinct processes of germination. C. okamuranus demonstrated the heteroblasty in the parthenogenetic development, and the ratio of appearance of two morphotypes, filamentous or discoid microthallus, seemed to be constant in both haploid and diploid strains. The morphological fate of these microthalli may be fixed before the second cell division of spores because a disk and a uniseriate filament must have distinct cell arrangements in the four-cell stage (see figures in Shinmura 1974, 1975, 1976). The constant occurrence of the two morphologies indicates that this species is most likely to utilize the heteroblasty proactively. If the cell division occurs at the same frequency both in filamentous and discoid thalli, it will be reasonable to suggest that the period required for maturation of the filamentous thallus is significantly shorter than that of the discoid thallus. In fact, the release of spores from a filamentous microthallus composed of six cells was observed in the present study. Utilizing these two options is surely a positive strategy for the survival of this species.
In addition, dimorphism was also found in the parthenogenesis of the diploid spores in C. okamuranus. Two morphological types of the discoid microthalli, classified by features of the assimilatory filaments, arose through the same developmental manner. In the genus Cladosiphon, the morphology of the assimilatory filaments is one of the important characteristics for taxonomic classification (Inagaki 1958; Kawai et al. 2016). Although there are significant differences in the length of assimilatory filaments between the two morphological types of the microthalli, the values are still involved in the range of C. okamuranus definition described and summarized in Inagaki (1958) and Kawai et al. (2016). Based on our observations, we generated a hypothesis that only the microthallus with longer assimilatory filaments (long type) possessed the ability to develop the macrothallus resulting in the generation of unilocular sporangia. This hypothesis was supported by the result of assimilatory filament measurement; the value of assimilatory filament length in the long-type microthallus was closer to the one in the natural microthalli developed on the cultivation net, although the values between them slightly showed a significant difference. At this moment, it is hard to make the macrothallus develop in the culture chamber, but the hypothesis needs to be evident in the future. Assuming that the long-type is the prior form of macrothallus, the role of the short-type microthallus might be to stay in the microthallus. In the present study, the higher temperature at 30°C caused only the short-type microthallus in both strains. The short-type microthallus might tolerate stress conditions (e.g., high temperature) and can mature rapidly with plurilocular sporangia generated at the tip of the assimilatory filaments. Brown algae generally stay microthallus under thermal stress conditions (Breeman 1988; Peters and Breeman 1992). This may be due to the higher tolerance to some stresses and the rapid maturation to let zoospores away from the stress conditions. Therefore, the short-type microthallus is probably the morphological type for stress conditions in C. okamuranus.
In this study, we found that culture temperature influenced the dimorphism in the diploid strain. The long-type thallus appeared only at 25°C and 20°C in the diploid strain. This agrees with the temperature when the macrothallus (sporophyte) initiates to germinate in natural sea farms in Bise, Okinawa-Jima (approx. 20–24°C on a daily average from December to January) (Tozaki et al. 2024). In some brown algal species, the effects of environmental factors on morphogenesis were clarified, for instance, a short day was the primary factor inducing the formation of erect thallus, and this response was inhibited by a minute light-break with blue light in Scytosiphon lomentaria (Dring and Lüning 1975b). In the case of Coilodesme japonica, the transition to the higher temperature caused three distinct morphological types of microthallus, two of the three developed into the macrothallus bearing the unilocular sporangium (Deshmukhe and Tatewaki 1993). These examples show the effect of environmental factors on the process of heteroblasty. In contrast, Lockhart (1979) reported that nitrogen source and bacteria contamination were the factors influencing morphological differentiation in C. zosterae. This phenomenon indicates the effect of environmental factors on dimorphism as well as our result in C. okamuranus. Although Loiseaux (1968) pointed out that dimorphism often interferes with heteroblasty and sometimes it is difficult to distinguish each other, these two species of Cladosiphon are certainly regulated in their morphogenesis of the dimorphism processes by environmental factors.
The biological significance of the two morphological types of the microthallus regulated by the temperature is to secure a high possibility of survival in either way of sexual or asexual reproduction. Indeed, all spores in the lifecycle possess the ability of parthenogenesis and morphological variation with heteroblasty in this species. This fact indicates the active strategy of this species for survival, but at the same time, this means how harsh the natural environments are.
We sincerely thank Mr. A. Hayashi of the Chinen Cooperative Association for providing the cultivation nets. We are very grateful to Dr T. Motomura for their valuable insights and suggestions. A part of this study was financially supported by a Grant-in-Aid for Scientific Research (B) (No. 20H03118) to AT from the Ministry of Education, Science, Sports, and Culture, Japan, basic-science research funding of Riken Food Co., Ltd. in 2018–2023 to AT and YS.
Atsuko Tanaka: conception and design of the experiments; Honoka Takara, Seidai Kamata, Yoichi Sato and Ran Ueshiro: acquisition and interpretation of data; Atsuko Tanaka, Yoichi Sato and Honoka Takara: drafting and revision of the paper.
