Induction of yeast cells from sporangiospores of Rhizopus stolonifer was inhibited by sodium fluoride

This study was aimed at determining the effect of the glycolytic inhibitor, sodium fluoride (NaF), on induced yeast cells. Rhizopus stolonifer was cultivated in synthetic broth for 120 h, at pH 4.5 and ambient temperature of 28 ± 1°C, using sporangiospores as inoculum. Growth was in discrete units, which were sedimented. Microscopic examination revealed induction of neoplasts, protoplasts and yeast cells, but biomass profiles (OD, 625nm) did not exhibit sigmoid pattern in control or glycolytic inhibitor-NaF incorporated broths. A two-way analysis of variance showed that NaF and its interaction with time significantly affected growth (p < 0.05). Means separation was into three subsets, with 20 mM (subset 1) having the highest biomass and least occurred at 5 mM, which was comparable to 30 mM, control and 10 mM (subset 3). A further study revealed effect of application time of NaF at two levels (20 and 10 mM) on morphological expression. Three main effects were observed: (a) complete inhibition of yeast induction, (b) delayed induction of yeast cells and (c) apoptosis of induced yeast cells. In the complete absence of yeast cells, protoplasts were copiously produced. However, when yeast cells were induced prior to NaF challenge, the inhibitor caused death of cells. Hence, it was suggested that induction of terminal budding yeast cells was closely associated with glycolysis. Further studies are recommended in order to examine the contribution of individual inductive enzymes including those of glycolysis, during the transformation process.


INTRODUCTION
Fungal dimorphism is the ability of a fungus to exist in two different morphological forms whereby it exhibits filamentous or yeast-like growth habit. This occurs as a result of changes in environmental conditions (Romano, 1966). The Mucorales contains a number of species that are known to undergo this dual morphogenetic change. They include the much-researched Mucor rouxii (Bartnicki-Garcia, 1968;Bartnicki-Garcia and Nickerson, 1962a, b, c, d;Schulz et al., 1974), Mucor circinelloides (Lubberhusen et al., 2003;McIntyre et al., 2002), Mucor genevensis (Rogers et al., 1974), Mucor racemosus (Mooney and Sypherd, 1976), Mucor pusillus (Fisher, 1977), Mucor hiemalis (Mysyakina and Funtikova, 2000), Cokeromyces poitrasii (Price et al., 1973), Mycotypha spp. (Hall and Kolankaya, 1974). In each of these cases, the alternative to filamentous growth is yeast-like form, which has a globose mother cell that gives rise to numerous buds from different loci by blastic action. Although such units are discrete, the form of growth is different from that of Saccharomyces cerevisiae, which reproduces asexually by terminal budding.
However, M. circinelloides has been shown to exhibit terminal budding yeast growth in well-defined synthetic medium (Omoifo, 2006a, b;Omoifo and Awalemhen, 2012). In glucose substrated minimal medium, the organism exhibited polymorphic existence occurring as *Corresponding author. E-mail: coomoifo@yahoo.com. holothallic-, holoblastic-, thallo-arthric conidia and terminal budding yeast cells, which was preponderant. Myoinositol (myo) enhanced the proportion of yeast cells induced in the multiionic medium, which was optimum at 2 mM myo but growth did not follow a sigmoid pattern (Omoifo et al., 2006). Sigmoid growth was however obtained when zinc was incorporated into the medium of cultivation (Omoifo and Omamor, 2005). Studies had previously shown that the incorporation of zinc in the growth medium led to the conversion of M. rouxii to yeast-like cells (Bartnicki -Garcia and Nickerson, 1962b) and Candida albicans to yeast cells (Yamaguchi, 1975).
Rhizopus stolonifer, which is also a Zygomycete like M. circinelloides, was said to be incapable of morphogenetic inter-conversion (Bartnicki-Garcia and Nickerson, 1962b). We have shown that R. stolonifer also exhibits dimorphism in xylose substrated minimal medium where several morphological forms were induced. These included septate mycelia, monoliform hyphae that apically produced yeast mother cells that ceded, thereafter becoming terminal budding as well as sporederived terminal budding yeast cells; these occurred beside coenocytic mycelia simultaneously expressed on exposure of the broth-bound septate mycelia to the headspace/atmosphere (Omoifo, 2011a). Thus, like M. circinelloides, R. stolonifer exhibits saccharomycetous growth habit at a stage in its life cycle. Perhaps R. stolonifer will exhibit sole yeast morphology in the specific medium used for the induction and growth of terminal budding yeast cells of M. circinelloides by Omoifo (2006a, b). This proved to be the case where induced yeast cells of R. stolonifer was found to assume sigmoidal growth habit at pH 5.0 (Omoifo, 2011b), as distinct from M. circinelloides that exhibited similar morphology and optimum growth at pH 4.5.
However, several transient morphologies have been observed in the conversion of spores to yeasts. These include growth/germ spheres which thereafter lyse thus releasing granular /neoplastic units and protoplasts. The occurrence of the transient forms was said to be prelogarithmic (Omoifo, 2009). It was also proposed that spore-to-yeast conversion occurred at acidic level where an electrogenic biphasic pattern is established, controlled by H + release ability of the intracellular medium of the transforming microorganism (Omoifo, 2012;Omoifo and Awalemhen, 2012). Thus, a sequential sporangiosporeyeast transformation (SSYT) hypothesis was proposed as a format for studying the conversion process (Omoifo, 2003). This led to the model development where the transient form, protoplast, assumed central significance (Omoifo, 2009). Studies in our laboratory have shown that potassium (K + ) was neccessary for the formation of protoplasts since a thorough examination of control cultures did not reveal these units. But this was possible only in proton-directed intermedial communicating multiionic system. Protoplasts when formed could be cylindrical, ovoid, short rod and in single or double and, or bacterial-like. From these yeast cells evolve, which could be globose, subglobose, pyriform, obpyriform or cylindrical (Omoifo, 2009). The yeast cells constituted the dominant morphology in the 1.0 K + to 0.10 Na + g/L modulated broth during the conversion of M. circinelloides (Omoifo and Awalemhen, 2012). In the aforementioned study, it was shown that protoplasts originated from two structures: growth/germ spheres and ruptured hyphal cells. When protoplasts, which were copiously obtained at 15°C, were transfered to solid cultures and incubated on the side bench, 28 ± 1°C, filamentous growth was induced (Omoifo, 2003). A role for aerobiosis was therefore implicated. Such physiological import had been suggested by other researchers, including Hall and Kolankaya (1974) and Schulz et al. (1974), but in each of the studies, multipolar budding yeastlike cells was obtained from sporeinoculums. It was therefore neccessary to explore the physiological direction that led to terminal budding yeast development.
The objective of this study, beside further confirmation of the induction of terminal budding yeast cells from spores of R. stolonifer, was to examine the effect of the glycolytic inhibitor, sodium fluoride (NaF), on induced yeast cells. It showed that several transient morphologies occurred during the transformation process and that adoption of the yeast morphology was inseparable from the physiological process of glycolysis.

Fungal strain and maintenance
The organism, R. stolonifer, used in this study was a gift from Mr. Frank Wizuzu, Principal Technologist, Plant Pathology Division, Nigerian Institute for Oil Palm Research, Benin City, Nigeria. It was maintained as glucose-yeast extract peptone (GYP: 10:03:5 g/L) solid cultures where it exhibited coenocytic filamentous growth habit. A fresh culture was prepared after seven days.

Inoculum preparation for growth studies
Inoculum was obtained by pouring sterile deionized distilled water over aerobic growth cultures and a sterile glass rod gently passed over the surface so as to dislodge the spores. The suspension was poured into sterile centrifuge tubes and spores were washed by centrifuging at 5000 rmp for 7 min at 25°C in an MSE 18 centrifuge. The supernatant was decanted, sediment re-suspended and further washed with two changes of deionized sterile distilled water. Spore count was taken with Neubauer Haemocytometer (BSS No. 784 Hawksley, London Vol. 1/4000) and was adjusted to 1 million spores per ml in deionized sterile distilled water with the aid of tally counter.

Inoculation, growth conditions and sample collection
In brief, 1 ml of spore suspension was drawn and inoculated into each broth flask using a 0.5-ml rubber suctioned pipette. The inoculum flask was shaken at each operation so as to keep the spores well distributed in suspension. Each culture flask was then shaken for 30 s and thereafter incubated at 28 ± 1°C. At 24 h intervals the culture flasks were transferred to the sterile inoculating chamber, each shaken to keep the sediment in suspension and 10 ml of broth was withdrawn and deposited into factory sterilized plastic sample tubes, pre-labeled for each experiment. The culture flasks were returned for further incubation. The samples were kept at -18°C until analysis. Slides preparation was then made from each of the sample tubes for subsequent microscopic examination. Two experiments were conducted: Experiment 1: To demonstrate the effect of NaF on biomass and the form of growth of R. stolonifer in synthetic broth. The inhibitor, NaF, at concentrations of 0, 5, 10, 20, 30 and 50 mM was incorporated into duplicate flasks containing 100 ml synthetic broth and flasks were labeled Run 1 and Run 2. The inhibitor was prepared into 100 ml flasks. Experiment 2: To show the effect of application time of NaF on the form of growth of R. stolonifer in synthetic broth. Two levels of NaF, 10 and 20 mM were used in this study. Slides were prepared for viewing by putting one or two drops of lactophenol in cotton blue on a glass slide and with the aid of a flamed wire loop; one drop from the culture flask properly shaken was then added and covered with a glass cover slip and viewed under a binocular microscope at × 400 magnitude. The morphologies at different hours were recorded.

Biomass determination
Culture broth samples were thawed up to room temperature before biomass determination. This was done by measurement of optical density at 625 nm (Sharma et al., 1984). Absorbance was determined with a Camspec M105 spectrophotometer CE 303 (Cambridge, UK). Triplicate determinations were made.

Statistics
Results were subjected to a two-way analysis of variance (ANOVA) test for the single factor and combined interactions, using the Genstat Discovery Edition statistical package. The combined factors are considered significant if p0.05; comparison between means was performed at p0.05.

Effect of NaF on growth
The effect of NaF on biomass of the microorganism and characteristics manifested under the different treatments are shown in Table 1. The least amount of growth occurred at 5 mM NaF, while the highest mean biomass was obtained at 20 mM NaF. As shown in the table, the control broth (s.e., 0.00014) had the least variability and the form of growth was solely terminal budding yeast cells. This level of variability was followed by the 20-mM NaF incorporated broth (s.e., 0.007); protoplasts were the main growth forms manifested. The boxplot of optical density measurement (Figure 1) shows the concise population of the microorganism; the data components were devoid of outliers and such population was symmetrical around zero in each of the NaF-treated broths. This implies that in this study, the parameters obtained followed the normal population distribution.
Furthermore, the median value in the 20-mM NaF treatment was higher than the others but lowest with the 5mM NaF treatment, which was apparently comparable with the control treatment. To test the significance of this, an analysis of variance of the growth data was obtained. A 2-way analysis of variance showed that NaF at p0.05, and NaF and its interaction with time at p0.001, had significant impact on the growth of the microorganism. Means separation, using l.s.d. at p0.05, 0.0325 gave  three homogenous subsets (Table 2). Treatment with 20 mM NaF, which had the highest mean biomass, was distinct and this was followed by treatment at 50 mM (subset 2), while all the other treatments had comparable means (subset 3). Figure 2 shows the biomass profiles of R. stolonifer cultivated in synthetic broth incorporated with varying levels of NaF. Although the growth profile in each of the treatments did not show a sigmoid growth pattern, yeast cells were induced. In the control broth, yeast cells were observed right from day 1 till termination of experiment.
The terminal budding yeast cells varied in shape; they were ellipsoidal, ovoidal and obpyriform. They also occurred at 20 mM NaF, but the terminal budding yeast cells diminished in number; in addiction, protoplasts were observed. Moreover, they appeared purplish in contrast to the blue stain taken up by the yeast cells. As the concentration of NaF increased from the lowest level, protoplasts became more preponderant. In the media with 20 mM NaF treatment, mainly nucleates/neoplasts and protoplasts were observed up till 72 h but by 120 h, yeast cells of the various aforementioned shapes were

Effect of application time of NaF on growth
It became necessary to find out what constituted the growth characteristics of the two extreme groups, that is, subsets 1 and 3 as reflected in Table 3. The boxplots of optical density at the two NaF levels is shown in Figure 3. It also showed that there was normal distribution of population during the period of growth. In order to find out at what stage in the conversion process of the microorganism that NaF was most effective, the inhibitor was incorporated into the broth flasks at different stages of growth of the microorganism. Analysis of variance of growth data showed that the individual treatments as well as interaction of the inhibitor with application time, and application time × sampling time had significant impact on growth of the organism at p<0.001 (Table 4). Separation of  the means obtained was into three subsets; simultaneous application gave the highest biomass. This was followed by application after 6 and 72 h as shown in subset 2, while the least biomass was obtained at application after 24, 36 and 48 h (Table 5). Table 6 shows the growth rates of the organism when NaF was incorporated at different application times in the broths. The maximum growth rate was achieved when 10 mM NaF was applied 72 h after inoculation, but the least was at 24 h after inoculation. On the other hand, at 20 mM NaF maximum rate of growth occurred on application 72 h after inoculation and this was higher than the rate at 10 mM NaF comparable application time. However, the least growth occurred 36 h after inoculation, and this was negative in value; a similar rating with 6 h after inoculation.

Effect of application time of NaF on the occurrence of yeast cells
Although simultaneous application with inoculum had the  highest biomass in the 10 mM NaF broths, microscopic examination did not reveal yeast cells throughout the period of observation. Morphological entities were cytosolic-derived neoplasts and protoplasts. At 6 h application time, morphological entities were mainly neoplastic units and well-rounded protoplasts, but the former was dominant. At 24 h application time, protoplasts were well-formed 72 h after inoculation and were the sole morphology recorded by 120 h of cultivation. Four morphological forms were observed 72 h after inoculation when application time was 36 h; these included neoplasts, protoplasts, thin monolithic filaments with apparent septation and yeast cells. The yeast cells were globose and scanty, but protoplasts dominated the broths even after 120 h of growth. Furthermore, at 48 h application time, hyaline protoplasts and globose/ovoid terminal budding yeast cells although scanty, were observed 48h after inoculation. By 120 h, the yeast cell shape became more varied as obpyriform shape was also observed. However, neoplasts and protoplasts dominated the medium of growth. When the application time was 72h, terminal budding yeast cells in single or short chains, and spheroidal, globose, or rod shaped were preponderant 72 h after inoculation. By 120 h of cultivation, some yeast cells appeared collapsed, and some of such remained as frass in the medium. There was apparent reduction in cell numbers. It was noteworthy that growth at 6 h application time had similar biomass with 72 h application time (Table 4, subset 2). Yet, there was no yeast cell observed at 6 h application time. When the growth rates for the two application times were compared, that at 72 h application time was higher (Table 6), indicating that the higher rate of growth was as a result of the proliferation of induced yeast cells therein. Therefore, unlike the induced yeast cells, protoplasts were non-proliferating. This probably accounted for the lower growth rate obtained for the simultaneous application where protoplasts were predominant.
Microscopic examination also failed to reveal induced yeast cells in broths with simultaneous application time when 20 mM NaF was incorporated. Figure 4 compares the mean biomass in broths incorporated with 10 and 20 mM NaF at different application times. Although biomass at 10 mM NaF was higher at simultaneous application, curiously its growth rate, μ = log 10 /h (Gibson et al., 1998), was less than that at the 20mM level, as shown in Table  6. The most interesting observation here was that yeast cell was not induced in either media throughout the cultivation period. This indicated that both levels of the inhibitor were equally effective in preventing yeast induction. Except for the simultaneous application time and 72 h application time, the mean biomass at 20 mM NaF was greater than that at the other application times (Figure 4). Figure 4 (upper boxes) also showed the yeast induction possibilities for the 2 different broth media, using 72 h after inoculation as a basis for comparison. Yeast cells were not induced within the first 72 h in the media up till 24 h application time at both 10 and 20 mM levels; but at application time as from 36 h, yeast cells were induced within the period. This indicated that early incorporation of NaF prevented early induction of yeast cells.
Further examination was done at the 20mM level because of the higher biomass generated as from 6 h application time, as seen in Figure 4. Thus, Figure 5 shows that yeast cell density built up as from 6 h application time, reaching a maximum at 36 h application time and this diminished thereafter, although cell density was still higher than that at 6 and 24 h application times. At 48 h application time and 72 h application time, shrunken and collapsed yeast cells, or their carcasses were observed. This also indicated that the inhibitor, NaF, became death-wise effective on the matured yeast cells.

DISCUSSION
The study of Omoifo (2011a,b) demonstrated that sporangiospores of R. stolonifer were transformed to terminal budding yeast cells in synthetic broth. The present results confirmed this. Earlier studies showed that sporangiospores of another Zygomycete, M. circinelloides, underwent lateral morphogenetic transformation in media of similar elemental composition (Omoifo, 2006a, b;Omoifo and Awalemhen, 2012). However, our results are different from that of Bartnicki-Garcia and Nickerson (1962b) who cultivated strains of R. stolonifer under CO 2 pressure and concluded that unlike Mucor species, R. stolonifer was not dimorphic. While M. circinelloides exhibited sigmoid growth pattern in zinc and myoinositol supplemented broths (Omoifo, 2006b) and K + and Na + -modulated broths (Omoifo and Awalemhen, 2012), such regular pattern was not shown in the present study, although the aforementioned elements were incorporated in the growth media. Perhaps this was affected by other conditions of growth. The present study was conducted at 28 ± 1°C, while M. circinelloides was cultivated at 20°C (Omoifo, 2006a, b;Omoifo and Awalemhen, 2012). Several studies have shown that temperature can affect morphological expression of dimorphic fungi, including Blastomyces dermatitidis (Levine and Ordal, 1946), Paracoccidioides brasiliensis (Edward and Nickerson, 1949) and Cladosporium werneckii (Hardcastle and Szaniszlo, 1974). Although the growth of R. stolonifer in the present study was purely in discrete units in submerged cultures, the temperature of growth could have affected the growth pattern. Furthermore, unlike the study of Omoifo (2011b) where R. stolonifer exhibited pH-related sigmoid growth in a Ca 2+ resplendent medium, Ca 2+ was not incorporated into the growth medium in the present study. The role of Ca 2+ in signals transduction circuit, the phosphoinositide cascade, and intracellular ion homeostasis in eukaryotic cells is well known, as it complexes with calmodulin which in turn stimulates many enzymes, ion transport pumps as well as kinases and phosphotases, thus participating in many metabolic and physiological processes, including cell cycle progression in mammals (Kahl and Means, 2003), plants (Hepler, 2005) as well as glycosylation, sorting and protein degradation in yeasts (Durr et al., 1998). Perhaps, it is also required in processes inuring the sigmoid growth pattern during yeast induction from spores of R. stolonifer.
The transient morphologies in the sequential conversion sion to yeast cells include growth/germ spheres, neoplasts and protoplasts (Omoifo, 2003(Omoifo, , 2009(Omoifo, , 2011bOmoifo et al., 2006;Omoifo and Omamor, 2005), and this was confirmed in the present study. Growth spheres were not observed in the control broth; conditions probably existed that facilitated such transition that usually occurred prior to the release of cytosolic nucleates/neoplasts (Omoifo, 2003). Therefore the sequential sporrangiospore -yeast transformation (SSYT) hypothesis earlier proposed for such conversion processes (Omoifo, 2003), was presumed to be fundamental to this study. The normal distribution of the morphological entities expressed in this study confirmed the fitness to this hypothesis. The absence of neoplasts and protoplasts in the control broth proved decisive to this study. That the control broth which induced only terminal budding yeast cells had similar mean biomass with the 5 mM NaF incorporated broth in which the exhibited morphologies were neoplasts, protoplasts and yeast cells, indicated that the inhibitor repressed the conversion of neoplast to protoplast on the one hand and of protoplast to yeast cell on the other. This was probably true of the other levels of NaF where similar transient morphologies were induced. Yet, the mean biomass at 20 mM NaF incorporation was outstanding; the induced morphologies were protoplasts, which predominated and yeast cells. It appeared that at this inhibition level, all neoplastic units converted to protoplasts (note that although internal cell measurements were not obtained in this study, protoplasts had internal dimensions and measurable sizes in contrast to neoplasts which had extreme sizes and undefined shapes) and these contributed to the high biomass recorded at this level, although they were nonproliferating.
In the second study, when 10 mM NaF was applied at the same time as sporangiospore-inoculum, no yeasts were induced except protoplasts even after 120 h of growth. Similar observation was made at 6 h application time. While the same was true of simultaneous application at 20 mM NaF, the effect of 10 mM NaF at preventing yeast induction was more profound as only the former induced yeast cells, albeit scantily at 6 h application time, and after 120 h of growth. This was in spite of the lower growth rate at 6 h application time. With 10 mM NaF incorporation at 36 h application time, some protoplasts were converted to yeast cells by 72 h of growth. Here, scanty moniliform filaments with apparent septation (Omoifo, 2011a), were part of the morphological expressions. However, if it was realized that at 72 h application time yeast cells had expressed as from 48 h after inoculation, then inhibitor effect was on actively proliferating yeast cells. However, rather than cell density build up with time, there was actual reduction in yeast cell count by 120 h.
Three effects of NaF were deduced from this study. They were (a) complete inhibition of yeast induction (b) delayed induction of yeast form and (c) apoptosis of wellformed and asexually proliferating yeast cells. These events were completely dependent on the time of exposure of the transforming microorganism to the inhibitor. NaF is a well-known inhibitor of the glycolytic process (Voet and Voet, 1995); the key control step in the pathway is the conversion of 2-phosphoglycerate to phosphoenolpyruvate whereby the fluoride ion interacts with the Mg 2+ -requiring enolase enzyme (Witters and Foley, 1976;Bassetti et al., 2004). Warburg and Christian (1942) were the first workers to show in vitro that the inhibitor forms a complex with magnesium ion, which competitively locates at the active site of the enzyme enolase (Cinnasoni, 1972), an effect that is enhanced with high levels of phosphate (Bassetti et al., 2004) thus preventing the execution of dehydration of 2phosphoglycerate (dehydroxylation at the carbon-3 position) and hence formation of PEP (Voet and Voet, 1995;Christophe et al., 2001). Shearer and Suttie (1970) provided the first direct evidence for fluoride inhibition of enolase in whole (rat liver) cells. They additionally found in their study that although depression of activities of pyruvate kinase also occurred, such effect was attributed to secondary actions of enolase inhibition that caused a change in the concentration of some other metabolites in the glycolytic pathway which they tested, posting negative results therefrom in vitro. This result is similar to that of Bucher and Pflerderer (1970) who had earlier shown that NaF failed to inhibit pyruvate kinase in vitro. On the other hand, in Streptococcus salivarius, a 2.4 mM NaF dose on metabolizing whole cells resulted in decreased glucose uptake and immediate fall in cellular glucose-6phosphate content (Kanapka and Hamilton, 1971). York and Van Denmark (1978) obtained copious growth of Streptococcus mutans and Leuconostoc mesenteroides on glucose and lactose substrates. As these workers found out in their study, the catabolic process of the two substrates, which involve enolase reaction steps, was prevented by NaF but it had no effect on arginine or pyruvate utilization, which excludes enolase activity. Similar results were obtained by Curran et al. (1994) who found that using concentrations beyond 0.001 mM NaF inhibited the enzyme enolase in a quasi-irreversible manner. If we assume the primary and secondary inhibitory effects of NaF on enolase during the conversion of sporangiospores to terminal budding yeast cells in this study, the physiological effect of the enzyme inhibition by NaF would not be limited to the single-step conversion of 2-phosphoglycerate to PEP product, as affectivity of the enzyme could be transcendent. In illustration of this, we referred to the work of Lemaire and Wesolowski-Louvel, 2004) that showed that at the molecular level disruption of the gene that encodes enolase in Kluyveromyces lactis caused severe reduction in the transcript levels of the organism's two glucose transporter genes.
Furthermore, transcriptions of hexokinase and pyruvate decarboxylase genes, which encode the enzymes of the glycolytic and fermentative segments respectively, were also impaired. The study of Haarasilta and Oura (1975) showed that in Baker's yeast S. cerevisiae, pyruvate carboxylase is expressed maximally under anaerobic fermentation conditions, thus heightening the first step in the production of ethanol from pyruvate, that is, decarboxylation to acetaldehyde and CO 2 . At the protein level in rat hepatocytes, fluoride binding to enolase and phosphoglucomutase inhibit the enzymes' activity while the lowered level of pyruvate kinase observed was in this case attributed to the effect of cAMP which plays a critical role in the phosphorylation of pyruvate kinase (Shahed et al., 1980). Assuming that these activities occurred at the molecular and physiological levels in our study with NaF challenge, then the transient discrete units, neoplasts and protoplasts, both distinct morphologies in the transformation process to the yeast form (Omoifo, 2003(Omoifo, , 2006a(Omoifo, , b, 2009(Omoifo, , 2011aOmoifo and Awalemhen, 2012) would probably be unable to sequester energy from the pyruvate kinase step. This was probably reflected on the differing depressive effect on the relative growth rate by the varying levels of NaF tested in this study. If high energy bonds were thus limited, perhaps biosynthetic pathways inducible at and including the latter phase of substrate levels phosphorylation, as well as fermentation could be hampered.
It is necessary to point out that at simultaneous application time at 10 or 20 mM NaF a stable or persistent proliferating morphology, that is, yeast form was never observed except the transient morphologies, neoplasts and protoplasts. In some cultures where proto-plasts had just been converted to yeast cells, debilitating effect of some levels of NaF caused negative cell growth rate, meaning death ensued, and carcasses left over. This is in sharp contrast to the finding that when protoplasts are transferred to aerobic environment, filamentous growth ensued (Omoifo, 2003). The difference, as we suspect in the present study, was that NaF treatment severely hampered the ability of transforming protoplasts to adopt substrate-level phosphorylation as a process for obtaining high energy bonds in the synthetic broth. While as earlier reported, on transfer to aerobic environment, protoplasts could obtain copious energy supply through oxidative phosphorylation, hence filament formation. This notion was reinforced by the fact that in rapidly growing HeLa cells, NaF insult forces a rapid decrease in cellular ATP content while 14 CO 2 production level from the TCA cycle activity (oxidative respiration) was unaffected (Carlson and Suttie, 1967). Conversely, glucose uptake in Baker's yeast, Saccharomyces cerevisiae D2373-10D, was completely blocked at 50 mM NaF challenge, thus preventing glycolysis; this provided a basis for its (concentration) use in glucose invertase assay as the inhibitor has no effect on the sucrose splitting enzyme, [beta-fructofuranosidase, EC 3.2. 1.26] (Silveira et al., 1996), which in S. cerevisiae 303-67 may be cell wall-bound, or intracellular (Gascon and Lampen, 1968) just like glycolytic enzymes (Alberts, 1987;Dawes, 1986;Soumalainen and Nurminen, 1973). We point out that this level of concentration (50 mM NaF) inhibited or 10 and 20 mM NaF simultaneous application time completely prevented the induction of terminal budding yeast cells in our present study. Since proliferating yeast cells occurred in control cultures and late phase NaF-application time cultures in the present study, it was not illogical to deduce that the physiological process that led to the induction of the yeast form R. stolonifer was partly substrate-level phosphorylation. Carlson and Suttie (1967) found that NaF challenge on HeLa cells imposed rapid decline in glucose utilization, followed by a slower growth rate. Our study using NaF corroborated the latter claim. In their study, Otsuki et al. (2005) showed that glucose utilization by HeLa cells almost stopped just 4 h after the inhibitor treatment; its action was most profound at 5 to 10 mM NaF. They further showed that NaF treatment induced apoptotic activities thus eliciting enhanced BAD -a pro-apoptotic protein expression, decreased mitochondrial membrane potential, as well as triggered caspase activation, DNA fragmentation and production of apoptotic bodies. In the present study, we found that NaF treatment was inhibitory to yeast induction in the concentration range (5 to 50 mM). Specifically, it caused the collapse of mature yeast cells with conspicuous carcass left-over. These cryptic observations tend to suggest that the metabolically degenerating activities in the NaF-disrupted enolase effects in HeLa cells, as observed by Otsuki and his colleagues, were involved in the yeast apoptosis in our study. Such cathartics end resulted in the diminished cell count at 48 and 72 h application time with 20 mM NaF incorporation.
Earlier studies showed that the transient morphologies occurred at the pre-logarithmic growth phase (Omoifo and Awalemhen, 2012). Their formation was said to conform to the sequential-sporangiospore-yeast transformation (SSYT) hypothesis (Omoifo, 2003) whereby chemiosmotic transport mechanisms (Albert et al., 1994), also known to control cell volume (Dawes, 1986;Jain, 1972;Bartnicki-Garcia and Lippmam, 1977) was thought to regulate sizes of the inoculated spores, converting them to germ spheres (Bartnicki-Garcia and Lippman, 1977) within which simultaneously, as has been argued (Omoifo, 2003) that synchronous DNA replication occurred and this gave rise to cytoplasmic granulation (Omoifo, 2003(Omoifo, , 2009. It was assumed that when equilibrium state was reached between the cell interior and the bulk medium, conditions were ripe for activities of lytic enzymes (Maurel and Douzou, 1978). When cell wall lysed, cytosolic neoplasts are exposed to the bulk medium; these subsequently evolve to protoplasts (Omoifo, 2003(Omoifo, , 2009. It is thought that a vector ally directed electrogenic ion transport mechanism, as occurs in mitochondria Mitchell, 1972, 1973;Dixon and Hopkin, 1980), in this case initialized by transmembrane-pH-gradient, underlined biochemical and physiological activities leading to structural modifications in the transforming morphologies observed in this study.
The abundance of the terminal budding yeast form in the control cultures in the present study is similar to that reported for M. circinelloides Tieghem cultivated in media of similar elemental composition (Omoifo, 2006b). Ionic movements involving K + and Na + ions, have been recorded during sporangiospore -yeast transformation of M. circinelloides (Omoifo and Awalemhen, 2012). It is well known that Na + is electrogenically interchanged with K + ions through the membrane bound Na + -K + ATPase (Lingrel and Kuntzweiler, 1994;Tonomura, 1986). Observation showed that commencement of Na + ion influx into the intracellular medium coincided with the occurrence of the yeast form (Omoifo and Awalemhen, 2012). In the present study, it was suggested that arising from H +glucose symport, a fact demonstrated for the yeast, Metschnikowia reukaufii (Alderman and Hofer, 1981), the fungus, Neurospora crassa (Slayman and Slayman, 1974) and arthrosporal fungus, Geotrichum candidum (Amrane et al., 1999), the carbon substrate became available in the protoplast interior and subse-quently catabolism through glycolysis occurred, using cytoplasmbound enzymes of glycolysis thus induced as a result of prolonged exposure to the substrate (Merrill and Pitot, 1986;Jansen et al., 2004;Cabib et al., 1988). The absence of yeast cells in NaF-inoculum simultaneous application time -cultures could be reasoned as ab initio prevention of induction of enolase dehydrolytic activities as a result of NaF inhibition of the enzyme even in the possible presence of symported glucose. Assuming this occurred, it could also lead to non-induction of and prevention of activities of beta-glucanase, mannanase and carbon disulphide reductase and other hydrolases, all of which are needed for biosynthesis of the cytoskeletal structure (Soumalainen and Nurminen, 1973;Griffin, 1981;Cabib et al. 1988) that could structurally provide the protoplast the yeast cell wall. The ability of protoplasts of the dimorphic fungus, Histoplasma capsulatum, to regenerate the yeast cell form in liquid medium has been demonstrated (Carbonell et al., 1973).
For some experiments, inhibitor incorporation was perhaps done when some early generated neoplasts or protoplasts had progressed beyond critical steps in the yeast induction process, while the effect of the inhibitor was exerted in the majority of protoplasts. If we recognize the transient protoplast as the archithetural format for biophysical structuring of nascent yeast form, that is, cytoskeletal construction which is a high energy demanding process, then this form was the main target for positive impact by NaF. This probably explained the scanty presence of yeast cells in some media. Perhaps still, energy sequestration occurred albeit slowly, even in the presence of inhibitor, bordering on enolase enzyme activation potential. The yeast cells were scanty but observed only at the late phase of cultivation.

Conclusion
This study showed that NaF could prevent the induction of yeast cell, slow down the rate of induction of yeast cell or cause the loss of budding capability and viability and subsequent death of yeast cell induced from sporangiospores of R. stolonifer cultivated in synthetic broth. This effect exhibited was dependent on the time of exposure of the microorganism to the glycolytic inhibitor, NaF. This study also showed that sodium fluoride had no effect on protoplast formation, indicating that its (protoplast) induction was not a function of glycolysis, but as previously shown, a function of K + influx into intracellular medium of the transforming microorganism (Omoifo, 2006a;Omoifo and Awalemhen, 2012). This study as well as literature referred suggested that the induction of terminal budding yeast cells from sporrangiospores of R. stolonifer was intimately associated with the glycolytic process. Nevertheless, further studies are needed to examine the contribution of the various inductive enzymes during the transformation process; critical to such evidence would be the genome sequencing for such enzymes.

ACKNOWLEDGEMENT
This work was done in the Agronomy Laboratory of the Faculty of Agriculture, Ambrose Alli University, Ekpoma,