PHOTOSYNTHETIC CHARACTERIZATION OF DEVELOPING AND MATURE AKINETES OF APHANIZOMENON OVALISPORUM (CYANOPROKARYOTA)1

Akinetes, differentiated resting cells produced by many species of filamentous, heterocystous cyanobacteria, enable the organism to survive adverse conditions, such as cold winters and dry seasons, and to maintain germination capabilities until the onset of suitable conditions for vegetative growth. Mature akinetes maintain a limited level of metabolic activities, including photosynthesis. In the present study, we have characterized changes in the photosynthetic apparatus of vegetative cells and akinetes of the cyanobacterium Aphanizomenon ovalisporum Forti (Nostocales) during their development and maturation. Photosynthetic variable fluorescence was measured by microscope‐PAM (pulse‐amplitude‐modulated) fluorometry, and the fundamental composition of the photosynthetic apparatus was evaluated by fluorescence and immunological techniques. Vegetative cells and akinetes from samples of Aphanizomenon trichomes from akinete‐induced cultures at various ages demonstrated a gradual reduction, with age, in the maximal photosynthetic quantum yield in both cell types. However, the maximal quantum yield of akinetes declined slightly faster than that of their adjacent vegetative cells. Mature akinetes isolated from 6‐ to 8‐week‐old akinete‐induced cultures maintained only residual photosynthetic activity, as indicated by very low values of maximal photosynthetic quantum yields. Based on 77 K fluorescence emission data and immunodetection of PSI and PSII polypeptides, we concluded that the ratio of PSI to PSII reaction centers in mature akinetes is slightly higher than the ratio estimated for exponentially grown vegetative cells. Furthermore, the cellular abundance of these protein complexes substantially increased in akinetes relative to exponentially grown vegetative cells, presumably due to considerable increase in the biovolume of akinetes.     

Effect of nutrients and aeration on O2 evolution and photosynthetic pigments ofAnabœna torulosa during akinete differentiation

The addition of a nitrogen (nitrate) and carbon sources (acetate, citrate and fructose) and phosphate deficiency (nitrate medium deficient in phosphate) under unaerated conditions induced akinete differentiation inAnabœna torulosa. Aerated cultures of this organism in these nutrients did not differentiate akinetes. Oxygen evolution by aerated cultures was higher when compared to unaerated cultures, which concurred with high chlorophyll content of aerated cultures. Nitrate nitrogen supported high phycocyanin content in unaerated cultures, phycocyanin and allophycocyanin contents were low under aerated conditions. The contents of phycocyanin, allophycocyanin, phycoerythrin and carotenoids gradually decreased at the mature akinete phase. Under aerated conditions, chlorophyll content rose and the content of all the pigments increased with the growth rate of the organism.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

AKINETE DIFFERENTIATION IN ANABAENA CIRCINALIS (CYANOPHYTA)1

Three lines of evidence established conclusively that phosphorus limitation triggered akinetes to differentiate in Anabaena circinalis Rabenhorst. First, akinetes differentiated when phosphorus was limited, but not when nitrogen, inorganic carbon, iron, trace elements, or light were limited, or when dissolved oxygen concentration was increased. In the phosphorus limitation experiment, akinetes appeared first in the 0 mg P-L^?1 cultures, and the higher the initial concentration of phosphorus was, the longer it took for akinetes to differentiate. Second, akinete differentiation commenced when Q_p fell to the same critical concentration in all cultures. The critical Q_p for akinete differentiation in A. circinalis was 0.3-0.45 pg P·cell^?1, and there was no significant difference between cultures grown with 0.6, 0.2, 0.06, or 0 mg P · L^?1 (F= 5.48, of = 3, P > 0.05). Similarly, there were no significant differences between P cultures in internal cellular soluble reactive phosphorus (SRP) concentration (F= 0.63, df = 3, P > 0.05) or external SRP per cell in the medium (F= 5.16, df= 3, P > 0.05) when akinete differentiation commenced. Both were between 0.01 and 0.07 pg SRP-cell^?1. A thorough literature search indicates that this information has not been reported previously. The third line of evidence came from electron micrographs, which illustrated that polyphosphate was present in trichomes prior to akinete differentiation but was absent in trichomes with akinetes indicating that phosphorus reserves were depleted when akinetes differentiated. Lipid globules (carbon reserve) and cyanophycin granules (nitrogen reserve) increased in number in trichomes with akinetes, compared to trichomes without akinetes. Thus, the ratio of internal P:C:N was different in trichomes with akinetes compared to trichomes without akinetes and may be important in activating akinete-differentiating genes.     

Photochromic pigments in akinetes and pigment characteristics of akinetes in comparison with vegetative cells of Anabaena variabilis

Since akinete germination is triggered by light and the action spectrum for this process has features in common with the spectra of the two photochromic pigments, phycochromes b and d, a search was made for the presence of these phycochromes in akinetes of the blue-green alga. Anabaena variabilis Kützing. Allophycocyanin-B was also looked for, since the action spectrum for akinete germination points to a possible participation of this pigment too. Isoelectric focusing was used for purification of the pigments. The different fractions were investigated for phycochromes b and d by measuring the absorbance difference spectra: for phycochrome b. 500 nm irradiated minus 570 nm irradiated, and for phycochrome d, 650 nm irradiated minus 610 nm irradiated. For determination of allophycocyanin-B. fourth derivative analysis of absorption spectra was made for some of the fractions from the isoelectric focusing column. Phycochrome b was also assayed for by measuring in vivo absorption difference spectra. The assays were positive for all three pigments. The complete photosynthetic pigment systems were also studied by in vivo fluorescence measurements on both akinetes and vegetative cells of Anabaena variabilis. Fluorescence emission and excitation spectra at selected emission wavelengths were measured at room temperature and liquid nitrogen temperature. The energy transfer from phycoerythrocyanin to phycocyanin is very efficient under all conditions, as is the energy transfer from phycocyanin to allophycocyanin at room temperature. At low temperature, however, phycocyanin is partly decoupled from allophycocyanin, particularly in the akinetes; the energy transfer from allophycocyanin to chlorophyll a is less efficient at low temperature in both types of cells, but especially in akinetes. Delayed light emission was measured for both types of cells and found to be very weak in akinetes compared to vegetative cells. From this study it would seem that akinetes lack an active photosystem II, although the 691 nm peak in the 570 nm excited low temperature fluorescence emission spectrum proves the presence of photosystem II chlorophyll, and also its energetic connection to the phycobilisomes.     

Potassium deficiency triggers the development of dormant cells (akinetes) in Aphanizomenon ovalisporum (Nostocales,Cyanoprokaryota)1

Akinetes are spore‐like nonmotile cells that differentiate from vegetative cells of filamentous cyanobacteria from the order Nostocales. They play a key role in the survival and distribution of these species and contribute to their perennial blooms. Various environmental factors were reported to trigger the differentiation of akinetes including light intensity and quality, temperature, and nutrient deficiency. Here, we report that deprivation of potassium ion (K⁺) triggers akinete development in the cyanobacterium Aphanizomenon ovalisporum. Akinetes formation is initiated 3 d–7 d after an induction by K⁺ depletion, followed by 2–3 weeks of a maturation process. Akinete formation occurs within a restricted matrix of environmental conditions such as temperature, light intensity or photon flux. Phosphate is essential for akinete maturation and P‐limitation restricts the number of mature akinetes. DNA replication is essential for akinete maturation and akinete development is limited in the presence of Nalidixic acid. While our results unequivocally demonstrated the effect of K⁺ deficiency on akinete formation in laboratory cultures of A. ovalisporum, this trigger did not cause Cylindrospermopsis raciborskii to produce akinetes. Anabaena crassa however, produced akinetes upon potassium deficiency, but the highest akinete concentration was achieved at conditions that supported vegetative growth. It is speculated that an unknown internal signal is associated with the cellular response to K⁺ deficiency to induce the differentiation of a certain vegetative cell in a trichome into an akinete. A universal stress protein that functions as mediator in K⁺ deficiency signal transduction cascade, may communicate between the lack of K⁺ and akinete induction.     

Potential triggers of akinete differentiation in Nodularia spumigena (Cyanobacteriaceae) isolated from Australia

Nodularia spumigena, like many cyanobacteria, produces specialised reproductive structures, known as akinetes, which are believed to allow survival under unfavourable conditions. This study investigated the effects of salinity, nitrogen and phosphorus concentration at two irradiances on akinete differentiation in a N. spumigena isolate from the Gippsland Lakes, Victoria, Australia. A computer image analysis program was used to photograph filaments and assess production of akinetes over time in separate experiments for each environmental parameter. Heterocyst production and cell morphology were also examined. The results suggest that akinete production increases over time. Production of akinetes is further increased at low and high salinities and with the addition of nitrate. Higher irradiance increases akinete differentiation, although in combination with different phosphorus concentrations causes varied effects. The development and sedimentation of akinetes may provide an inoculum for reoccurring blooms. Heterocysts were only observed during experiments with varying salinity and nitrogen exposures. Light quantity appeared to play a large role in heterocyst production. The ability of N. spumigena to produce akinetes and heterocysts is likely to be part of the reason for its success and continual occurrence in estuarine environments low in nitrogen, such as the Gippsland Lakes, Victoria, Australia. Factors known to reduce heterocyst and akinete production will provide new insight to possible management controls for this species.     

Phosphorus deficiency,nitrogen assimilation and akinete differentiation in the cyanobacteriumAnabaena torulosa

Anabaena torulosa is unable to fix N₂ and to differentiate akinetes in a P-deficient nitrate-free medium. In a P-deficient medium with nitrate, the NO₃ ^? assimilation and period of akinete differentiation are of the same order of magnitude as in a P-containing nitrate medium. It is suggested that regulation of akinete differentiation in P-deficient organism proceeds through the regulation of the N-assimilating system. At the time of akinete differentiation, cellular P is excreted into the medium which leads to a decrease of cellular P.     

Pattern of akinete differentiation in the cyanobacteriumScytonema fritschii

The differentiation of akinetes inScytonema fritschii occurred adjacent to the newly developed heterocysts in late exponential phase. The filaments exhibited cell division leading to the formation of heterocysts, interspersed by the potential akinetes which could be identified by the accumulation of a large number of granules. Upon maturity, the akinetes acquired thick envelopes and were seen in elongated series interrupted by dead necridia which resulted from crumpling of the newly developed heterocysts. The formation of akinetes was accompanied by a change in color of cultures from blue-green to brown. Of the inorganic nitrogen sources tested, ammonium nitrate supported the formation of maximum percentage of akinetes. The incorporation of 7-azatryptophan and rifampicin in nitrate-free and nitrogen sources resulted in the production of heterocysts at a very high frequency in the late-exponential phase coinciding with akinete formation but the frequency of the latter was reduced. The activity of nitrogenase, nitrate reductase and glutamate-ammonia ligase was absent in mature akinetes. The absorption spectra of chlorophylla and phycobiliproteins revealed the presence of negligible amounts of the former white the latter were absent. The dry mass steadily increased during akinete differentiation with a concomitant decrease in C/N ratios.     

Nutrient stress causes akinete differentiation in cyanobacterium<Emphasis Type="Italic">Anabaena torulosa</Emphasis> with concomitant increase in nitrogen reserve substances

Addition of nitrogen source (nitrate), carbon sources (acetate, citrate and fructose), depletion of nutrients (phosphate-free nitrate medium), dilution of medium (2, 4 and 8 times diluted nitrate medium) under unaerated conditions induced akinete differentiation in Anabaena torulosa. Aerated cultures under the same conditions did not differentiate akinetes. The amounts of reserve metabolites--glycogen and cyanophycin (multi-L-arginyl-poly-L-aspartic acid) granule polypeptide (CGP)--were determined in unaerated and aerated cultures, and at different stages of growth and akinete differentiation. The addition of nitrate, acetate, citrate and fructose under unaerated conditions resulted in the accumulation of glycogen and CGP in higher amounts after 4 d (akinete initiation); the CGP content further changed at mature free akinetes phase. Higher accumulation of reserve products was also observed under nutrient deficiency (phosphate-depleted or diluted media) after 4 d of cultivation. Under aerated conditions reserve product accumulation was considerably lower. Thus a low accumulation of reserve products in aerated cultures showed that aeration probably somehow relieves the organism from a nutritional stress.     

Cyanobacterial akinete induction and its application as biofertilizer for rice cultivation

Nostoc sp. VICCR1-1 was induced in order to form akinetes on the basis of nutrient modification. Phosphorus and iron were found to be the critical for akinete differentiation, especially when both elements were omitted. The number of akinete cells increased up to 20% when compared with culturing in BG11₀ medium (without N source). In addition, CaCl₂ played a role in heterocyst differentiation, and was able to induce heterocyst ranging between 30% and 46%. In order to prepare akinetes as inoculum, the dried form of akinetes was prepared by mixing it with montmorillonite clay. The inoculum with the amount of 2.8 × 10⁶ cells m⁻² was applied to rice (Oryza sativa) fields. After harvesting, the grain yields from chemical N fertilizer, vegetative cells, and akinete inoculum treatments were not significantly different. To monitor the persistence of Nostoc sp. VICCR1-1 after harvesting, the most probable number-denaturing gradient gel electrophoresis technique using 16S rRNA gene was employed. The results indicated that the remaining population is at 2.5 × 10⁵ and 1.62 × 10⁶ cells m⁻² in treatments supplied with vegetative cells and akinete inocula, respectively. Akinete induction might be one of the appropriate approaches for producing cyanobacterial inoculum.     

Akinete Formation in Tribonema bombycinum Derbes et Solier (Xanthophyceae) in Relation to Freezing Tolerance

Tribonema bombycinum (Xanthophyceae), was examined. T. bombycinum shifted from vegetative cells to akinetes with starving by a prolonged batch culture, by culture with a diluted medium, or by culture with a single nutrient-deficient medium. In addition, akinetes developed by desiccation, but cold treatment at 4 C did not facilitate akinete formation. During starving, the vegetative cells, which had a large central vacuole in the protoplasm and thin cell walls, finally changed to akinetes, which had many small vacuoles and oil droplets in the protoplasm and thick cell walls. During akinete formation by starving, the freezing tolerance (LT₅₀) increased gradually from −3 C in vegetative cells to far below −30 C in akinetes. When vegetative cells were subjected to equilibrium freezing, their size shrank greatly and aparticulate domains accompanied by fracture-jump lesions developed in the plasma membranes. Akinetes subjected to equilibrium freezing showed little shrinkage, and freezing-induced ultrastructural changes did not occur in the plasma membranes. The morphological changes in the process of akinete formation and the responses to equilibrium freezing resembled those of cold-acclimated terrestrial plants. Received 24 November 1998/ Accepted in revised form 1 February 1999     

Akinete differentiation in phototrophic,photoheterotrophic and chemoheterotrophic conditions inAnabaena torulosa

Akinete differentiation inAnabaena torulosa occurred under phototrophic, photoheterotrophic and chemoheterotrophic conditions at the expense of fructose in nitrate-free and nitrate media. Maximum frequency of akinetes was recorded in nitrate-free cultures in photoheterotrophic condition followed by chemoheterotrophic condition. The activity of glucose-6-phosphate dehydrogenase (GPDase) and the levels of glycogen exhibited a correlation with the presence and absence of nitrate and maximum activity was detected in photoheterotrophic condition at the maturation phase. The cells in nitrate-free medium exhibited enhanced levels of GPDase, nitrogen fixation and low levels of glycogen in the presence of fructose. A lowering of GPDase activity in nitrate grown cells was found to be associated with the accumulation of glycogen. The results presented here further suggest that a critical balance of the C:N ratio triggers akinete differentiation.     

Factors affecting akinete differentiation in Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria)

1. Cylindrospermopsis raciborskii is a potentially toxic freshwater cyanobacterium which can produce akinetes (reproductive spores) that on germinating can contribute to future populations. To further understand factors controlling the formation of these specialised cells, the effects of diurnal temperature fluctuations (magnitude and frequency), in combination with different light intensities and phosphorus concentrations were investigated under laboratory conditions. 2. Akinete differentiation was affected by the frequency of temperature fluctuations. Maximum akinete concentrations were observed in cultures that experienced multiple diurnal temperature fluctuations. 3. Akinete concentrations increased with increasing magnitude of temperature fluctuation. A maximum akinete concentration was achieved under multiple diurnal temperature fluctuations with a magnitude of 10 °C (25 °C to 15 °C). 4. A fourfold increase in light intensity (25–100 μmol m^?2 s^?1) resulted in an approximate 14‐fold increase in akinete concentration. 5. High filterable reactive phosphorus (FRP) concentrations (>70 μg L^?1) in the medium, combined with a multiple diurnal temperature fluctuation of 10 °C, supported the development of the highest akinete concentration.     

Seasonal dynamics of akinetes of Anabaena flos-aquae in bottom sediments and water column of small Siberian reservoir

Seasonal dynamics of Anabaena flos-aquae (Lyngb.) Breb., including vegetative cells, akinetes and akinete envelopes, in bottom sediments and water column at both littoral and deeper central stations of a small Siberian reservoir was studied. Two types of akinetes were observed: in the first half of summer Anabaena formed akinetes, which served for vegetative reproduction and germinated in water column soon after differentiation, while in the second half of summer the akinetes produced served as a resting stages, which were deposited to bottom sediments. Canonical correlation analyses revealed that decrease of water temperature was the main environmental factor that stimulated the akinete formation. In contrast to the general opinion, concentration of inorganic phosphorus slightly, but positively influenced the akinete formation. Thus, akinetes formed in response to the temperature decrease, needs a certain level of this nutrient. At littoral and open-water stations abundance and seasonal dynamics of akinetes in water column and their sinking pattern were very similar. However, seasonal dynamics of abundance of akinetes in sediments in these two reservoir locations differed: whereas the abundance of akinetes in open water increased permanently during the summer, that in the littoral decreased soon after their sedimentation. The cause for decrease in abundance of akinetes in bottom sediments in winter is unknown.     

Vegetative survival, akinete formation and germination in three blue-green algae and one green alga in relation to light intensity, temperature, heat shock and UV exposure

The mere vegetative survival was not sufficient but suitable growth conditions were required for akinete formation to occur in the blue-green algaeAnabœna iyengarii, Westiellopsis prolifica, Nostochopsis lobatus and in the green algaPithophora oedogonia. In all algae, akinetes were neither formed nor germinated in darkness, and while dim light of 300 lx was sufficient for most of akinetes to germinate and also to maintain vegetative survival, it was not adequate for optinum akinete formation. Although akinetes of all algae could germinate at 35°C, both the vegetative survival and akinete formation were markedly suppressed at this temperature. Heat or UV shock of any level, whether ineffective or effecting vegetative survival, did not promote akinete formation or germination in any alga tested. Akinetes of all algae under study were relatively tolerant to heat and also to some extent to UV. Both wet and dried akinetes of all algae were equally UV tolerant. In all algae, the viability of both wet and dried akinetes decreased more or less equally with storage time, but the decrease was more drastic when storage temperature was progressively lowered from 20 to 0°C. Hence the akinetes can tolerate dryness but not frost.     

Survival of Akinetes (Resting-State Cells of Cyanobacteria) in Low Earth Orbit and Simulated Extraterrestrial Conditions

Cyanobacteria are photosynthetic organisms that have been considered for space applications, such as oxygen production in bioregenerative life support systems, and can be used as a model organism for understanding microbial survival in space. Akinetes are resting-state cells of cyanobacteria that are produced by certain genera of heterocystous cyanobacteria to survive extreme environmental conditions. Although they are similar in nature to endospores, there have been no investigations into the survival of akinetes in extraterrestrial environments. The aim of this work was to examine the survival of akinetes from Anabaena cylindrica in simulated extraterrestrial conditions and in Low Earth Orbit (LEO). Akinetes were dried onto limestone rocks and sent into LEO for 10 days on the ESA Biopan VI. In ground-based experiments, the rocks were exposed to periods of desiccation, vacuum (0.7 × 10⁻³ kPa), temperature extremes (−80 to 80°C), Mars conditions (−27°C, 0.8 kPa, CO₂) and UV radiation (325–400 nm). A proportion of the akinete population was able to survive a period of 10 days in LEO and 28 days in Mars simulated conditions, when the rocks were not subjected to UV radiation. Furthermore, the akinetes were able to survive 28 days of exposure to desiccation and low temperature with high viability remaining. Yet long periods of vacuum and high temperature were lethal to the akinetes. This work shows that akinetes are extreme-tolerating states of cyanobacteria that have a practical use in space applications and yield new insight into the survival of microbial resting-state cells in space conditions.     

2-Methylhopanoids are maximally produced in akinetes of Nostoc punctiforme: geobiological implications

2-Methylhopanes, molecular fossils of 2-methylbacteriohopanepolyol (2-MeBHP) lipids, have been proposed as biomarkers for cyanobacteria, and by extension, oxygenic photosynthesis. However, the robustness of this interpretation is unclear, as 2-methylhopanoids occur in organisms besides cyanobacteria and their physiological functions are unknown. As a first step toward understanding the role of 2-MeBHP in cyanobacteria, we examined the expression and intercellular localization of hopanoids in the three cell types of Nostoc punctiforme : vegetative cells, akinetes, and heterocysts. Cultures in which N. punctiforme had differentiated into akinetes contained approximately 10-fold higher concentrations of 2-methylhopanoids than did cultures that contained only vegetative cells. In contrast, 2-methylhopanoids were only present at very low concentrations in heterocysts. Hopanoid production initially increased threefold in cells starved of nitrogen but returned to levels consistent with vegetative cells within 2 weeks. Vegetative and akinete cell types were separated into cytoplasmic, thylakoid, and outer membrane fractions; the increase in hopanoid expression observed in akinetes was due to a 34-fold enrichment of hopanoid content in their outer membrane relative to vegetative cells. Akinetes formed in response either to low light or phosphorus limitation, exhibited the same 2-methylhopanoid localization and concentration, demonstrating that 2-methylhopanoids are associated with the akinete cell type per se . Because akinetes are resting cells that are not photosynthetically active, 2-methylhopanoids cannot be functionally linked to oxygenic photosynthesis in N.   punctiforme .     

AKINETE FORMATION IN PLANKTONIC ANABAENA SPP. (CYANOBACTEFUA) BY TREATMENT WITH LOW TEMPERATURE1

The effects of temperature, light intensity and nutrient depletion on akinete formation in seven strains of planktonic Anabaena spp.: A. mucosa TAC426; A. crassa TAC436; A. spiroides TAC443 and TAC444; A. flosaquae TAC446; and A. ucrainica TAC448 and TAC449 were examined. A Marked Pfft of temperature on akinete formation was observed at 40 μmol photons·m^?2·sec^?1 and nutrient-sufficient conditions. At 20° C, akinetes did not develop in A. mucosa TAC426, A. crassa TAC436, A. spiroides TAC443, A. flos-aquae TAC446, or A. ucrainica TAC449 but were formed at frequencies of a little over 11% (ratio of filaments with akinetes to total filaments) in A. spiroides TAC444 and A. ucrainica TAC448. None of the strains fmd akinetes or heterocysts at 30° C and 35° C. At lower temperature (10° C and 15° C), akinetes developed in all the strains at maximum frequencies of 13.4–77.4% during the late exponential phase or late exponential to stationary phases of growth. With only one exception, low light or nutrient deletion did not lead to the induction of akinete diferentiation at 20° C. Only akinete formation in A. flosaquae TAC446 was induced by nitrogen deletion with a frequency of 12.1%, similar to that induced by low temperature, but the initiation of akinete formation in the strain was delayed compared to treatment with low temperature. These results show that temperature was the most important environmental factor triggering akinete formation in these species. In A. crassa TAC436 and A. spiroides TAC443 and TAC444, akinetes developed during the late exponential growth phase even though heterocysts were formed at a 100% frequency (ratio of filaments with heterocysts to total filaments) throughout the entire growth phase. In A. mucosa TAC426, A. flos-aquae TAC446, and A. ucrainica TAC448 and TAC449, there was a positive correlation between heterocyst and akinete formation, suggesting that the presence of a heterocyst may play a role in akinete formation.