Acetabularia Caliculus (Ulvophyceae, Chlorophyta)

Sex-Speciﬁc Cell Fusion Pattern of Isogametes in Marine Green Alga, Acetabularia caliculus (Ulvophyceae, Chlorophyta)

Shinichi Miyamura1* and Tamotsu Nagumo2

1 Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan 2 The Nippon Dental University, Chiyoda-ku, Tokyo 102–8159, Japan

Received November 16, 2015; accepted April 8, 2016

Summary Using field emission scanning electron microscopy (FE-SEM) and fluorescence microscopy, the respective relations between the arrangements of the gamete cell fusion site and the inheritance pattern of chloroplast DNA (cp-DNA) as a sex-specific marker were studied for the isogamous dasycladalean alga Acetabularia caliculus. The gamete had two flagella elongated from the cell anterior. One oval-shaped eyespot situated on the cell posterior was visualized using FE-SEM. When the gametes belonging to the opposite mating types were mixed together, the two gametes aligned side-by-side at their lateral side and fused to form a quadriflagellate planozygote. In the planozygote, the two flagella from the opposite mating type gametes lay side-by-side and two eyespots aligned on the same side of the cell, suggesting opposite positioning of the cell fusion sites between two parental gametes. To confirm whether the gamete fusion pattern observed in the planozygotes was a result of sex-specific fusion or not, the inheritance pattern of cp-DNA was observed using fluorescence microscopy after staining with DAPI concomitantly with the cell fusion pattern. When the eyespots were used as positional marker and were viewed from the surface that included the eyespots, the chloroplast of one parental gamete was found to contain cp-DNAs, although cp-DNAs were not detected in the chloroplast derived from another parental gamete. These results suggest that the gamete fusion pattern observed by FE-SEM was a result of sex-specific fusion.

Key words Acetabularia caliculus, Chloroplast DNA, Fertilization, Gamete, Isogamy, Uniparental inheritance.

Isogamy, a kind of sexual reproduction, involves gam- yamura et al. 2015). It becomes possible to distinguish etes of similar shape and size. Therefore, morphological two gametes belonging to the opposite mating types distinction of two gametes belonging to the opposite morphologically, even in isogamous species, if the latter mating types in isogamous organisms is generally ex- feature is a universal feature in chlorophyte algae. To tremely difficult. However, in some isogamous species, date, sex-specific arrangement of the mating structure/ the morphologies of the two gametes differ slightly. For cell fusion site and the consequent sex-specific cell fu- example, in the chlorophyte alga, Chlamydomonas rein- sion pattern has been reported mainly in isogamous and hardtii, the mating type plus (mt+) gamete elongates the anisogamous species in Ulvophyceae (Nakayama and fertilization tubule from the mating structure (cell fu- Inouye 2000, Miyamura et al. 2003, O’Kelly et al. 2004) sion apparatus), while a mating type minus (mt-) gamete in addition to isogamous species, C. reinhardtii (Chlo- does not elongate the fertilization tubule (Friedmann et rophyceae) (Holmes and Dutcher 1989, Gaffal et al. al. 1968). Another difference is the mating structure’s 1991, Miyamura et al. 2009) and Nephroselmis olivacea sex-specific arrangement. The mating structure of the (Nephroselmidophyceae) (Suda et al. 2004). In the Ulvo- mt+ gamete is located at the cell apex on the opposite phyceae, this phenomenon has been reported for several side of the flagellar beat plane to the eyespot, whereas species in the Ulvales (Miyamura et al. 2003, O’Kelly et that of the mt- is located on the same side as the eyespot al. 2004), the Ulotrichales (Nakayama and Inouye 2000) (Holmes and Dutcher 1989, Gaffal et al. 1991, Miyamu- and the Bryopsidales (Miyamura et al. 2005, Miyamura ra et al. 2009) (Fig. 1a). The former feature is found and Nagumo 2007), but it has not been studied in other only in C. reinhardtii and other closely related species orders. Therefore, it remains uncertain whether this (Harris 1989), whereas the latter feature has been found phenomenon is a universal feature in the Ulvophyceae, also in other chlorophyte algae, irrespective of the mode or not. of sexual reproduction (isogamy or anisogamy) (see Mi- Acetabularia caliculus is a marine giant unicellular alga belonging to the Dasycladales that is a sister to Bryopsidales (Lewis and McCourt 2004). A. caliculus * Corresponding author, e-mail: [email protected]. ac.jp inhabits temperate and tropical coastal areas, often DOI: 10.1508/cytologia.81.215 growing on the remains of shells (Sano et al. 1981). It 216 S. Miyamura and T. Nagumo Cytologia 81(2)

maturation. The matured cysts were placed in the dark for a few hours and were then illuminated to induce gamete liberation. The gametes liberated from the cysts showed positive phototaxis and gathered at the meniscus of the illuminated side of the vessel within ca. 10 min. The mixture of the gametes containing the opposite mating types was collected and was then used for ﬁxation.

Scanning electron microscopy One volume of suspension of the gamete mixture was mixed with an equal volume of a fixative containing 5% glutaraldehyde, 3% NaCl in 0.1 M cacodylate buffer, pH 7.1, on a polycarbonate membrane (Nuclepore; What- man Japan KK, Tokyo, Japan), which was coated with 0.1% poly-L-lysine (Sigma Chemical Co., St. Louis, MO, U.S.A.), at 15, 60, and 90 min after liberation of the gametes. The cells were fixed at 22°C for 30 min and then at 4°C overnight. After removing the supernatant, the cells were washed in a series of 0.05 M cacodylate buffer so- lutions containing 3, 2.25, 1.5, 0.75, and 0% NaCl, with Fig. 1. Schematic illustration of the isogametes showing the spatial each step taking 15–20 min. Post-fixation was done in arrangement of mating structures/cell fusion sites in Chlam- ydomonas reinhardtii (a) and Acetabularia caliculus (b). The 1% OsO4 dissolved in 0.05 M cacodylate buffer, pH 7.1, mating structure/cell fusion site of the mt + gamete is located overnight at 4°C. After dehydration through a graded at the cell apex on the opposite side of the flagellar beat series of ethanol, the cells were infiltrated with t-butyl - plane to the eyespot (type α gamete), whereas that of the mt alcohol, freeze-dried at 4°C and coated with platinum– is located on the same side as the eyespot (type β gamete) in C. reinhardtii (a) (Holmes and Dutcher 1989). This relation palladium in a sputter-coating unit (Hitachi E-1030 or a with mating type is reversed in A. caliculus (b). Mating Hitachi E-102; Hitachi High-Technologies Corp., Tokyo, types of A. caliculus was defined tentatively as mt + and Japan). Observations were made using a field-emission - mt based on the inheritance pattern of cp-DNA. Because scanning electron microscope JSM 6330F (JEOL, To- the mating structure of A. caliculus was not observed in this study, cell fusion sites are shown as broad dotty regions. #1, kyo, Japan) at 5 kV (Fig. 2a, c, d, g) or S5000 (Hitachi no. 1 flagella; #2, no. 2 flagella. High-Technologies Corp., Tokyo, Japan) at 2 kV (Fig. 2e, f, h). propagates by the formation of planozygotes (motile zygotes) after fertilization of isogametes (Arasaki 1942). Fluorescence microscopy Arasaki (1942) and Shihira-Ishikawa (1994) reported the One volume of the specimens was mixed with 1 fertilization process by which two biflagellate gametes volume of 3% paraformaldehyde dissolved in buffer align side-by-side, fuse at their lateral side and finally containing 50 mM Pipes, 5 mM EGTA, 2 mM MgSO4, become a quadriflagellate planozygote. However, details 0.28 M sucrose, pH 7.0, and 1 volume of 1 µg mL-1 DAPI of the gamete fusion pattern and its sex-specificity have dissolved in a modified S buffer containing 20 mM not been elucidated. Tris–HCl, 0.5 mM EDTA, 0.28 M sucrose, 1.2 mM sper- This study was conducted to observe the gamete fu- midine, 7 mM 2-mercaptoethanol and 0.4 mM phenyl- sion pattern of A. caliculus using field emission scan- methysulfonyl-fluoride (Kuroiwa and Suzuki 1980). ning electron microscopy (FE-SEM) and fluorescence After 2–10 min, the cover slip was pressed gently against microscopy to ascertain whether a sex-specific arrange- the specimens. All observations were made using an ment of the cell fusion site is found in Dasycladales. epifluorescence microscope BHS-RFC (Olympus Opti- cal Co., Ltd., Tokyo, Japan) that was equipped with dif- Materials and methods ferential interference contrast optics. Photographs were taken using color film (Provia 400; Fuji Photo Film Co., Algal materials Ltd.) and were subsequently converted to digital images Acetabularia caliculus was collected at Noto Pen- (iMac; Apple Computer Inc., Cupertino, CA, U.S.A.). insula, Ishikawa Prefecture, on the west coast of Ja- Micrographs were minimally adjusted for brightness pan. Specimens were kindly supplied by Dr. Shihira- and contrast (Photoshop CS6; Adobe Systems Inc., San Ishikawa. The cells were placed in a vessel containing Jose, CA, U.S.A.). They were then cropped (Photoshop artificial seawater (Uminokenkyūsha, Muroto, Kochi, CS6) and reduced from their original size (Illustrator Japan), where they were maintained under a 12 : 12 light/ CS6; Adobe Systems Inc.). darkness photoregime, 20 µmol m-2 s-1 at 22°C until cyst 2016 Gamete Fusion Pattern in Acetabularia 217

Fig. 2. Gametes and planozygotes of Acetabularia caliculus viewed by FE-SEM (a, c–h), differential interference contrast (b, j) and fluorescence microscopy (i). (a) Biflagellate gamete. Two flagella elongated from the cell apex. (b, c) Gamete cell body. An oval-shaped eyespot is located at cell posterior. (d) Enlarged image of the eyespot. (e) Quadriflagellate planozygote 15 min after mixing the gametes. (f) Planozygote 15 min after mixing the gametes. The cell was viewed from the opposite side of the eyespots. (g) Planozygote 90 min after mixing the gametes. The cell was viewed from the same side as the eyespots. Two eyespots aligned on the same side of the cell adjacent to the cell fusion plane (black arrowheads). (h) Enlarged image of the two eyespots of the planozygote 15 min after mixing the gametes. (i, j) Planozygote 90 min after mixing the gametes. The cell was stained with DAPI and viewed from the same side as the eyespots. The cp-DNAs (double arrowhead) in the chloroplast from one parent had disappeared (left half in Fig. 2i), whereas mitochondrial DNAs (arrows) were present in both parent cytoplasm. #1, no. 1 flagella; #2, no. 2 flagella; C, chloroplast; E, eyespots; N, nuclei. Scale bars=10 µm (a, e) and 1 µm (b–d, f–j).

Results and discussion algae (Holmes and Dutcher 1989, Melkonian 1989) and was used to number the flagella of the gametes in this Because all gametes shared similar morphology, we study according to previous studies (Nakayama and were unable to distinguish the two gametes belonging Inouye 2000, Miyamura et al. 2005). The flagellum near to the opposite mating types under light microscopy, the eyespot is designated as no. 2; the opposite flagellum as reported previously (Arasaki 1942). The gamete of is no. 1. Acetabularia caliculus has two flagella elongated from In the quadriflagellate planozygote, the two gametes the anterior end of cell (Fig. 2a) and one chloroplast (Fig. lay side-by-side with their longitudinal axes nearly par- 2b). The eyespot was visualized using light microscopy allel (Fig. 2e, f). The two flagella each from the opposite (Fig. 2b) and FE-SEM as the oval-shaped structure situ- mating type gametes lay side-by-side and became a pair ated on the cell posterior (Fig. 2c, d). The long axis of (Fig. 2f). Two no. 1 flagella from the different parental the eyespot always lay parallel to the longitudinal axis gametes became nearly parallel and always pointed to of the gamete. The eyespot always occupied the position the same direction (Fig. 2f). Two no. 2 flagella from both close to one of the two flagella (Fig. 2c). This feature is gametes aligned themselves similarly. The two eyespots probably consistent with those of advanced chlorophyte lay side-by-side on the same side of the planozygote 218 S. Miyamura and T. Nagumo Cytologia 81(2) adjacent to the cell fusion plane (Fig. 2g, h). This fusion suggesting that cp-DNA transmits to the next genera- pattern was observed in 58 mating pairs (n=62) 90 min tion from one of the two mating types in the same way after release of the gametes. The mating pairs consist- as C. reinhardtii (Kuroiwa et al. 1982). We observed the ing of three gametes aligned side-by-side were rarely fate of cp-DNA in the planozygotes using fluorescence observed in two mating pairs in which three eyespots microscopy after staining with DAPI. Figure 2i and j aligned on the same side of the cell (n=62). The align- show fluorescence and differential interference contrast ment of two eyespots on the same side of the planozy- images of planozygotes 90 min after mixing the gametes. gote was also reported in A. acetabulum (Crawley 1966) The nuclei, mitochondrial DNA and cp-DNA emitted and other chlorophyte algae (Miyamura et al. 2015), and a strong blue-white fluorescence after DAPI staining. is explained as a consequence of sex-specific cell fu- When the eyespots were used as the positional marker sion between the two gametes belonging to the opposite and viewed from the surface containing the eyespots mating types: one gamete fuses on the opposite side of (Fig. 2j), the chloroplast of one parent contained cp- the flagellar beat plane to the eyespot (type α gamete), DNAs (right half in Fig. 2i), although cp-DNAs were whereas the other gamete fuses on the same side as the not detected in the chloroplast derived from another eyespot (type β gamete) (Holmes and Dutcher 1989, parent (left half in Fig. 2i). The left and right halves Miyamura and Nagumo 2007) (Fig. 1). The left and right of the planozygote respectively correspond to type α halves of the planozygote in Fig. 2g are derived, respec- and β gametes, which suggests that cp-DNA of type α tively, from type α and β gametes if the same thing were gamete preferentially disappeared in the planozygote. possible in A. caliculus. This inheritance pattern was observed in 78.5% of the To ascertain whether the gamete fusion pattern ob- planozygotes (Table 1). These results suggest that the served in the planozygotes was a result of gamete type gamete fusion pattern observed using FE-SEM (Fig. 2f, (type α and β) specific fusion or not, we studied the rela- g) was a result of sex-specific fusion. Therefore, gam- tion between the gamete fusion pattern and mating types etes of A. caliculus are suggested to be divided into two of the gametes. For this purpose, we used the inheritance types (type α and β) based on the cell fusion site posi- pattern of chloroplast DNA (cp-DNA) as a mating type tion, as reported previously in other chlorophyte algae specific marker because cp-DNA is generally transmit- (Miyamura and Nagumo 2007) (Fig. 1b). The relation ted to the offspring from one of the two mating types in between the gamete types (type α and β) and the inheri- green algae (Miyamura 2010). For A. caliculus, Kuroiwa tance pattern of cp-DNA has been reported in several et al. (1985) already reported that cp-DNA derived from ulvophycean species and C. reinhardtii (Table 2). It is one of the two parents disappears after gamete fusion, particularly interesting that cp-DNA is always transmit-

Table 1. Relation between the inheritance pattern of the chloroplast DNA and parental gamete types in planozygotes of A. caliculus.

Presence (+) or absence (-) Inheritance pattern of cp-DNA Parental gamete type Frequency (%) of cp-DNA

1 Type α - 78.5±0.7 Type β + 2 Type α + 5.0±0.0 Type β - 3 Type α + 16.5±0.7 Type β +

Two hundreds planozygotes were counted at 90 min after mixing the gametes of opposite mating types. Data are presented as the mean±standard deviation of two independent observations.

Table 2. Relation between the inheritance pattern of the chloroplast DNA and parental gamete types in planozygotes of chlorophyte algae.

Type of sexual Origin of Species Mode of inheritance Source reproduction cp-DNA

Acetabularia caliculus Isogamy Uniparental (mt +)** Type β Kuroiwa et al. 1985, This study Ulva partita* Slightly anisogamy Uniparental (mt +)** Type β Kagami et al. 2008, Mogi et al. 2008 Caulerpa brachypus Markedly anisogamy Maternal Type β Miyamura and Nagumo 2007 C. okamurae Markedly anisogamy Maternal Type β Miyamura and Nagumo 2007 C. racemosa var. laetevirens Markedly anisogamy Maternal Type β Miyamura and Nagumo 2007 C. serrulata var. serrulata f. lata Markedly anisogamy Maternal Type β Miyamura and Nagumo 2007 Bryopsis maxima Markedly anisogamy Maternal Type β Kuroiwa and Hori 1986, Kuroiwa et al. 1991, Miyamura et al. 2005 Chlamydomonas reinhardtii Isogamy Uniparental (mt +)** Type α Sager 1954, Holmes and Dutcher 1989

* U. partita was previously identified as U. compressa based on morphological features (Ichihara et al. 2015). ** Uniparental inheritance from mt + gametes. 2016 Gamete Fusion Pattern in Acetabularia 219 ted preferentially from type β gametes to the offspring in rescent microscopic evidence for maternal inheritance of chloro- ulvophycean algae, suggesting that the spatial arrange- plast DNA. Nature 298: 481–483. Kuroiwa, T., Kawano, S., Watanabe, M. and Hori, T. 1991. Preferen- ment of the mating structure/cell fusion site has a close tial digestion of chloroplast DNA in male gametangia during the relation to the specific mating type in the Ulvophyceae, late stage of gametogenesis in the anisogamous alga Bryopsis although this relation is reversed in C. reinhardtii. maxima. Protoplasma 163: 102–113. In conclusion, the results of this study suggest that the Kuroiwa, T. and Suzuki, T. 1980. 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