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.     

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.     

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.     

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.     

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.     

Effects of Ekalux Ec-25 on akinete germination and sporulation ofPithophora kewensis

The effect of the organophosphorus insecticide Ekalux Ec-25 (quinalphos) was studied on akinete germination, and sporulation of the green algaPithophora kewensis (Cladophorales). Initiation of akinete germination was delayed by 1–3 d with 0.025–0.1% concentrations of the pesticide used. The percentage of akinete germination was found to be markedly affected by the treatment with insecticide, except with the concentration of 0.25% Ekalux Ec-25, where percent germination was observed to be higher than in the control. Generally, the time taken for the initiation of akinete formation increased and percent sporulation decreased with the increase of concentrations from 0.025 to 0.1% of Ekalux Ec-25.     

Identification of an akinete marker gene in Anabaena variabilis

Cyanobacteria that form akinetes as well as heterocysts present a rare opportunity to investigate the relationships between alternative differentiation processes and pattern formation processes in a single bacterium. Because no akinete marker gene has been identified, akinete formation has been little studied genetically. We report the first identification of an akinete marker gene.     

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.     

Freshwater algae of Southern Africa

Observations are reported on akinete formation, sheath formation and the breaking away of the rest of the trichome after akinete formation in Gloeotrichia ghosei R. N. Singh.     

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.     

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.     

Temperature-Dependent Dispersal Strategies of Aphanizomenon ovalisporum (Nostocales, Cyanobacteria): Implications for the Annual Life Cycle

Aphanizomenon ovalisporum is a planktonic nostocalean cyanobacterium with increasing research interest due to its ability to produce the potent cytotoxin cylindrospermopsin and its potential invasiveness under the global warming scenario. The present study provides novel data on the potential dispersal strategies of A. ovalisporum by analyzing the influence of temperature (10–40 °C) on akinete differentiation and cell morphometry in cultures of A. ovalisporum UAM 290 isolated from a Spanish pond. Our results confirmed a temperature-dependent akinete differentiation, with the maximum akinete production reached at 20 °C (15 % of the cells), a low basal production at 25–30 °C (<0.4 % of the cells) and no detectable production at 35 °C. Furthermore, we reported the fragmentation of A. ovalisporum filaments at temperatures of 25 °C and above. Additionally, we observed that the morphology of vegetative cells varied under different temperature scenarios. Indeed, a strong negative correlation was found between temperature and the width, length and biovolume of vegetative cells, whereas akinete dimensions remained stable along the temperature gradient. Therefore, linear regressions between temperature and the cell size parameters are herein presented aiming to facilitate the identification of A. ovalisporum in the field throughout the course of the year. This is the first study evidencing that akinete production is triggered by temperatures between 20 and 25 °C in A. ovalisporum and reporting the existence of filament fragmentation as a potential dispersal strategy of this species. The importance of these findings for understanding the annual life cycle and invasive potential of A. ovalisporum is further discussed herein.     

Characterization of akinete differentiation in the cyanobacterium Anabaena azollae

Differentiation of akinetes was investigated in the filamentous cyanobacterium Anabaena azollae Stras. In this organism all pre-existing vegetative cells are capable of developing into akinetes. Standard sporulation medium (SSM) was used to synchronously induce the formation of akinetes, while cultures in Allen and Arnon (AA/8) medium were used as controls.This paper describes the changes in photosynthetic pigments and total soluble proteins in these cultures over a 25-day period encompassing akinete differentiation. Heterocyst frequencies and nitrogenase activity were also monitored during the same period in both media. SDS-PAGE results indicated that specific proteins were synthesized in a manner correlated with akinete differentiation. The results demonstrate that in cultures undergoing akinete development, some of the photosynthetic pigments are maintained, nitrogen-fixation and heterocyst differentiation are suppressed, and the cells synthesize a variety of specific proteins.     

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.     

Cell Wall and Coat of the Developing Akinete of a Cylindrospermum Species

The mature akinete coat of a Cylindrospermum species contains laminar layers, not present in immature akinetes, and a thickened intermediate layer.     

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.     

Long-term observations on Anabaena circinalis Rabenhorst (Cyanophyta)

Field observations on Anabaena circinalis Rabenhorst over six summer seasonal appearances in two dams have shown that the frequency of occurence of heterocysts became fairly constant soon after the appearance of the species and decreased just before the end of the growing season. By contrast, akinete frequency reached a maximum, very early in the season, then decreased rapidly. Drought led to a decrease in occurrence of both Anabaena and of akinetes in several dams; this was possibly associated with an increasing concentration of NOx in the water. At Carcoar dam this akinete reduction was shown first in end-of-season figures. Drought also led to an end-of-season decrease there in the occurrence of heterocysts. Variations in morphology were noted. The coiling of the trichome, shape of akinete and relative position of heterocyst were all variable, although these characters are often assumed to be of taxonomic importance.     

Structural aspects of akinete germination in the cyanobacterium Anabaena variabilis

Electronmicroscopical investigations of light activated akinetes in different phases before outgrowth of the germinating cell showed two alterations in the akinete envelope, obviously in connection with the germination process. After induction of germination the akinetes show formation of an expanding more or less electron dense layer between the outer cell wall layer (outer membrane, L_IV) and the condensed part of the akinete coat (the transformed sheath of the vegetative cell). Between this new formed layer and the mentioned part of the akinete coat thick laminar layers are deposited which contain alternately electron dense and electron transparent strata. The expanding layer is assumed to be a mucous layer which acts as swelling body causing, after bursting of the layered shell, the expulsion of the germinating cell in the manner characteristic for Anabaena variabilis.