Akinetes of the cyanobacteriumNostoc PCC 7524: morphological changes during synchronous germination

Following dilution into fresh medium in the light, akinetes ofNostoc PCC 7524 germinated synchronously. Synchrony was maintained at a high level during the first 24 h, at which time the young filaments were composed either of three cells (with N₂ as nitrogen source) or four cells (with NO ₃ ^- or NH ₄ ⁺ ), and at a slightly lower level during the next 24 h of growth. The pattern of cell division was similar in media containing the different nitrogen sources although the timing of the major events varied. In the presence of N₂ or NO ₃ ^- , heterocysts differentiated synchronously; the first developed invariably from a terminal cell of the young filament at approximately 19 h, the second from the other terminal cell after further vegetative cell division. Heterocyst differentiation did not occur in the presence of NH ₄ ⁺ . In the absence of nitrogen (gas phase argon: CO₂) akinete germination initially followed the same pattern as that observed in N₂, this early stage probably occurring at the expense of intracellular reserve materials.During germination, a new laminated layer, similar in structure and position to that found in the heterocyst envelope, appeared in the akinete envelope. This layer was not present in the germinating akinetes of a mutant which was incapable of forming heterocysts.     

Growth and temperature‐related phenotypic plasticity in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is an invasive and potentially toxic cyanobacterium, which has recently spread worldwide, mainly because of its tolerance to a wide range of climatic conditions. C. raciborskii is able to change several traits in response to environmental changes and its morphology is also affected by these changes (especially in nutrients). We also expected temperature to affect the morphology of this cyanobacterium. We examined the growth and morphology of C. raciborskii at different temperatures and compared laboratory results to the morphology of this cyanobacterium in situ. As expected, growth rates increased with temperature. In addition, a high carrying capacity at 32°C suggests that this cyanobacterium is able to form more dense blooms at high temperatures. Fragile trichomes and low growth rates were observed at 12°C. An increase in the growth rate related to temperature resulted in a decrease in trichome length, with shorter trichomes at 32°C. The same pattern was observed in wild populations of C. raciborskii in a tropical reservoir, where shorter trichomes were observed in warmer months, when biomass was highest. This species' high ability to adapt to different environmental conditions throughout the year (i.e., nutrients, temperature) may have provided it with an additional advantage to increase its perennial blooms, mainly in tropical regions.     

Inter-annual variation in macroinvertebrate communities in a shallow forest lake in eastern Finland during 1990–2001

Cylindrospermopsis raciborskii undergoes characteristic morphological changes during its annual population development. Primary filaments (those that derive directly from the akinetes) possess the following morphological features: the trichomes have one or two acuminate ends; they are narrower (2.4–2.6 μm) tfhan older filaments, their lengths vary in a wide range (from 40 to 300 μm); some of them are straight but others are slightly coiled even in populations that later consist exclusively of straight filaments; cell walls between the cells are not or only hardly visible; the filaments have a fine granulation but no gas-vacuoles, polyphosphate- or any other contrasting bodies are seen. The secondary filaments deriving from the first division of primary filaments are similar but with only one acuminate end. This way, the number of germinating akinetes can be estimated as the number of primary filaments + 1/2 number of secondary filaments. The described morphological method to estimate inoculum size of C. raciborskii has limitations. (1) The number of akinetes that we get in this way is only a minimum number since germination is not synchronous under field conditions and probably not each akinete germinates that is present in the sediments. (2) The method is applicable only if germination occurs suddenly (relatively synchronously). This is often the case in a temperate region where germination is triggered by temperature. (3) The method cannot be used in tropical or subtropical lakes where water temperature is permanently rather high and akinetes do not develop or develop only very rarely. (4) Being an a posteriori method it cannot be used as predictive tool. Estimations for Lake Balaton are in a good agreement with results of studies with other methods (germination in laboratory cultures and direct counting) and have the advantage that they do not need time-consuming experimenting or difficult direct countings.     

Growth,trichome size and akinete production of Cylindrospermopsis raciborskii (cyanobacteria) under different temperatures: Comparison of two strains isolated from the same pond

Coiled morphotype Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju that forms a water bloom in a shallow pond in northern Taiwan exhibits a negative correlation between trichome size and temperature. To investigate how temperature influences the growth and trichome size of C. raciborskii, two C. raciborskii strains isolated from the pond in August and February were grown at three temperatures (18, 24 and 30°C). Both strains exhibited the lowest and highest specific growth rates at 18°C and 24°C, respectively, and the trichomes became the largest at 18°C. However, specific growth rates of the strain isolated in August exceeded those of the strain isolated in February, and the trichomes of the strain isolated in February were larger than those of the strain isolated in August regardless of temperature. Moreover, although both strains produced larger numbers of akinetes at higher temperatures, the strain isolated in August produced many more akinetes than did the other. These findings suggest that the two strains are not identical, leading to the conclusion that the C. raciborskii population in the pond consists of at least two ecotypes. Large trichome formation and akinete production are thought to be different types of countermeasure against cold of C. raciborskii, and the patterns of investment in developing these functions seemed to vary between the strains.     

Domination of invasive Nostocales (Cyanoprokaryota) at 52°N latitude

Cylindrospermopsis raciborskii and Sphaerospermopsis aphanizomenoides are increasingly widespread cyanobacterial species, considered invasive. However, they have never been found to dominate the phytoplankton in the northern part of the invaded area. The aim of our study was to expand the understanding of the invasion process in Nostocales, by answering the question which environmental conditions enabled the first ever noted bloom incident of C. raciborskii and S. aphanizomenoides in the northern part of their current occurrence ranges. We examined the population dynamics of the two invasive cyanobacteria and the abiotic conditions in which their blooms developed in a reservoir at 52°N. We also examined the phytoplankton community diversity and composition before and during the invasive species blooms. As shown by redundancy analysis, the competitive advantage of S. aphanizomenoides depended strongly on high water temperature and high concentration of phosphates, and was positively related to other summer conditions (low water transparency, high ammonium nitrogen concentration). C. raciborskii biomass was not related to temperature and phosphates, but we argue that high water temperature in the first half of July enabled abundant akinete germination, and pulsed phosphorus availability synergized to bring about a later bloom. Both the invasive species co‐occurred with the native cyanobacterium Aphanizomenon gracile, but C. raciborskii bloomed after the native species domination period. The results of the study corroborate the high competitiveness of S. aphanizomenoides in hot, nutrient‐rich conditions, and the adaptation of the invaders to the environment in the invaded regions, inter alia by the overwintering mechanism of numerous akinete differentiation. Our results also contradict the assumption that the phytoplankton diversity index would decrease after colonization of freshwater bodies by invasive cyanoprokaryotes.     

FACTORS RELATED TO THE DOMINANCE OF CYLINDROSPERMOPSIS RACIBORSKII (CYANOBACTERIA) IN A SHALLOW POND IN NORTHERN TAIWAN1

Seasonal succession of phytoplankton was investigated in a shallow pond in northern Taiwan from August 2009 to January 2011, with particular reference to the dynamics of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju. The abundances of the representative species in the pond increased during high‐temperature seasons, whereas only C. raciborskii became dominant in the pond from summer to autumn in both 2009 and 2010. The high shade tolerance of C. raciborskii was likely one of the factors that enabled the cyanobacterium to grow during the summer when the transparency was low. Moreover, the heterocyst production of C. raciborskii was enhanced during summer when the concentration of dissolved inorganic nitrogen was low, implying that nitrogen fixation also played an important role in supporting the growth of C. raciborskii. Autumnal rainfall was a critical factor in the collapse of C. raciborskii blooms. C. raciborskii formed blooms with relatively small trichomes, whereas larger trichomes dominated during winter. The dependence of the trade‐off between growth rate and trichome size on temperature was assumed to be an adaptation strategy of C. raciborskii.     

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.     

Benthic growth of Gloeotrichia echinulata Cyanobacteria

Hydrobiologia - Gloeotrichia echinulata is an akinete forming cyanobacterium with both a pelagic and a benthic phase. After germination of the akinete there is a short phase of growth on the...     

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.     

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.     

MORPHOLOGICAL AND 16S rRNA GENE EVIDENCE FOR RECLASSIFICATION OF THE PARALYTIC SHELLFISH TOXIN PRODUCING APHANIZOMENON FLOS‐AQUAE LMECYA 31 AS APHANIZOMENON ISSATSCHENKOI (CYANOPHYCEAE)1

A taxonomic reevaluation of the paralytic shellfish toxin (saxitoxins) producing cyanobacterium Aphanizomenon flos‐aquae Ralfs ex Born. & Flah. LMECYA31 was done using morphology and 16S rRNA gene sequences. We found that strain LMECYA31 was incorrectly identified as Aph. flos‐aquae based on (a) lack of bundle formation in trichomes, (b) shape of terminal cells in the trichomes, (c) lower similarity (<97.5%) in the 16S rRNA gene sequences relative to those of Aph. flos‐aquae, and (d) comparison within a phylogenetic tree of 16S rRNA gene sequences. The shape of the terminal trichome cells and the shape and size of the vegetative cell, heterocyst, and akinete in strain LMECYA31 match characters of Aph. issatschenkoi (Ussachew) Proschkina‐Larvernko. 16S rRNA gene sequences and phylogenetic clusters constructed from 16S rRNA gene sequences support our conclusion that strain LMECYA31 should be Aph. issatschenkoi.     

Effects of salinity and source of inocula on germination of Anabaena akinetes from a tidally influenced lake

1. Sedimentary akinetes (resting stages) may represent significant potential inocula for nuisance blooms of cyanobacteria. We studied the effects of salinity and sediment source on the germination and subsequent growth of Anabaena flos‐aquae akinetes from a shallow, tidally influenced lake. 2. Surface sediments collected from littoral and open‐water sites were used as inocula to culture A. flos‐aquae akinetes in four salinities (0.1, 2.2, 4.4 and 6.5) over 22 days. Akinete germination and development was followed by counting developmental stages every second day. 3. Filament growth, but not akinete germination, was inhibited by salinity and there were significantly fewer filaments at 6.5 than at 0.1 and 2.2. Cultures inoculated with littoral sediment had more akinetes, germlings and filaments than those inoculated with open‐water sediment. 4. Sediment is a potential source of inocula for Anabaena blooms in the lake, which potentially could develop solely from this source because germination and subsequent filament growth do not depend on the existence of an initial pelagic Anabaena population.     

The fine structure of akinete formation and germination in Cylindrospermum

Summary Akinete formation and germination were studied in a species of Cylindrospermum using the electron microscope. The differentiation of a vegetative cell into an akinete is characterized by cell enlargement, sheath condensation, deposition of several spore envelope layers, including a dense fibrillar layer and deposition of large cyanophycin granules. The mature akinete in addition to the multilayered envelope retains internally a large number of cyanophycin granules, a photosynthetic thylakoid system, polyhedral bodies, lipid deposits and nucleoplasmic regions. Germination of the akinete can take place in several modes differing in detail. Most frequently the spore envelope remains intact and the germling which may or may not have divided emerges through a pore at one end of the envelope. The photosynthetic thylakoid system appears to increase by the fusion of small vesicles found in the cytoplasm. Alpha-granules are numerous and cyanophycin is nearly absent in the germling.     

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.     

Varied Diazotrophies, Morphologies, and Toxicities of Genetically Similar Isolates of Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) from Northern Australia

The potentially toxic freshwater cyanobacterium Cylindrospermopsis raciborskii has become increasingly prevalent in tropical and temperate water bodies worldwide. This paper investigates the effects of different nitrogen sources (NO₃⁻, NH₄⁺, and omission of a fixed form of nitrogen) on the growth rates, morphologies, and cylindrospermopsin (CYL) concentrations (expressed as a percentage of the freeze-dried weight) of seven C. raciborskii isolates obtained from a range of water bodies in northern Australia and grown in batch culture. In general, growth rates were lowest in the absence of a fixed-nitrogen source and highest with NH₄⁺ as the nitrogen source. Conversely, the highest concentrations of CYL were recorded in cultures grown in the absence of a fixed-nitrogen source and the lowest were found in cultures supplied with NH₄⁺. Cultures supplied with NO₃⁻ were intermediate with respect to both CYL concentration and growth rate. Different nitrogen sources resulted in significant differences in the morphology of C. raciborskii trichomes. Most notable were the loss of heterocysts and the tapering of end cells in cultures supplied with NH₄⁺ and the statistically significant increase in vegetative cell length (nitrogen depleted < NO₃⁻ < NH₄⁺). The morphological changes induced by different nitrogen sources were consistent for all isolates, despite measurable differences in vegetative-cell and heterocyst dimensions among isolates. Such induced morphological variation has implications for Cylindrospermopsis taxonomy, given that distinctions between species are based on minor and overlapping differences in cell lengths and widths. The close phylogenetic association among all seven isolates was confirmed by the high level (>99.8%) of similarity of their 16S rRNA gene sequences. Another genetic technique, analysis of the HIP1 octameric-palindrome repeated sequence, showed greater heterogeneity among the isolates and appears to be a useful method for distinguishing among isolates of C. raciborskii.     

Raphidiopsis mediterranea Skuja represents non-heterocytous life-cycle stages of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju in Lake Kastoria (Greece), its type locality: Evidence by morphological and phylogenetic analysis

The taxonomical relationship of Cylindrospermopsis raciborskii and Raphidiopsis mediterranea was studied by morphological and 16S rRNA gene diversity analyses of natural populations from Lake Kastoria, Greece. Samples were obtained during a bloom (23,830 trichomes mL⁻¹) in August 2003. A high diversity of apical cell, trichome, heterocyte and akinete morphology, trichome fragmentation and reproduction was observed. Trichomes were grouped into three dominant morphotypes: the typical and the non-heterocytous morphotype of C. raciborskii and the typical morphotype of R. mediterranea. A morphometric comparison of the dominant morphotypes showed significant differences in mean values of cell and trichome sizes despite the high overlap in the range of the respective size values. Additionally, two new morphotypes representing developmental stages of the species are described while a new mode of reproduction involving a structurally distinct reproductive cell is described for the first time in planktic Nostocales. A putative life-cycle, common for C. raciborskii and R. mediterranea is proposed revealing that trichome reproduction of R. mediterranea gives rise both to R. mediterranea and C. raciborskii non-heterocytous morphotypes. The phylogenetic analysis of partial 16S rRNA gene (ca. 920 bp) of the co-existing Cylindrospermopsis and Raphidiopsis morphotypes revealed only one phylotype which showed 99.54% similarity to R. mediterranea HB2 (China) and 99.19% similarity to C. raciborskii form 1 (Australia). We propose that all morphotypes comprised stages of the life cycle of C. raciborkii whereas R. mediterranea from Lake Kastoria (its type locality) represents non-heterocytous stages of Cylindrospermopsis complex life cycle.     

PULSES OF PHOSPHATE PROMOTE DOMINANCE OF THE TOXIC CYANOPHYTE CYLINDROSPERMOPSIS RACIBORSKII IN A SUBTROPICAL WATER RESERVOIR1

The role of dissolved inorganic phosphorus (DIP) in promoting dominance of the toxic nitrogen (N)‐fixing cyanobacterium Cylindrospermopsis raciborskii (Wo?osz.) Seenayya et Subba Raju was examined in a subtropical water reservoir, Lake Samsonvale (=North Pine reservoir). A novel in situ bioassay approach, using dialysis tubing rather than bottles or bags, was used to determine the change in C. raciborskii dominance with daily additions of DIP. A statistically significant increase in dominance of C. raciborskii was observed when DIP was added at two concentrations (0.32 μM and 16 μM) in a daily pulse over a 4 d period in three separate experiments in the summer of 2006/2007. There was an increase in both C. raciborskii cell concentrations and biovolume in two DIP treatments, but not in the ammoniacal N + DIP treatment. In addition, overall phytoplankton cell concentrations increased with DIP addition, indicating that Lake Samsonvale was DIP limited at the time of experiments. Given the bioassay response, it is likely that dominance of C. raciborskii could increase in Lake Samsonvale with periodic injections of DIP such as inflow events.     

Factors regulating recruitment from the sediment to the water column in the bloom-forming cyanobacterium Gloeotrichia echinulata

1. The influence of light, temperature, sediment mixing and sediment origin (water depth) on the recruitment of the cyanobacterium Gloeotrichia echinulata was examined in the laboratory. 2. Light and temperature were the most important factors initiating germination in G. echinulata. 3. The extent of germination (recruited biovolume) was mainly regulated by temperature and sediment mixing. Furthermore, sediment mixing significantly enhanced the frequency of observed heterocysts and colonies. 4. Despite the fact that the deep and shallow sediments contained a similar number of akinete colonies, the highest recruitment occurred from shallow sediments, indicating that akinetes from shallow sediments have a higher viability than those from deeper parts of the lake. 5. Our results support the hypothesis that shallow sediments are more important than profundal sediments for the recruitment of G. echinulata to the pelagic zone.     

Effects of thermal acclimation and photoacclimation on lipophilic pigments in an invasive and a native cyanobacterium of temperate regions

The freshwater cyanobacterium Cylindrospermopsis raciborskii spreads from tropical to temperate regions worldwide. This entails acclimation to varied light and temperature conditions. We studied the thermal and light acclimation of the photosynthetic machinery of C. raciborskii by monitoring alteration of the chlorophyll a and carotenoid content in German strains of C. raciborskii, in African and Australian strains of C. raciborskii, and in German strains of Aphanizomenon gracile, a native cyanobacterium belonging to the same order (Nostocales). Our results showed that temperate and tropical C. raciborskii strains did not differ in pigment acclimation to light and temperature. In contrast, the ratio of photoprotective carotenoids (namely the carotenoid glycoside 4-hydroxymyxol glycoside [aphanizophyll]) to chlorophyll a increased significantly more in C. raciborskii in comparison with A. gracile (1) with decreasing temperatures from 20 to 10°C and a moderate light intensity of 80?µmol photons m^?2?s^?1 and (2) with increasing light intensities at a suboptimal temperature of 15°C, compared to 20°C. We conclude that below 20°C photoinhibition is avoided by greater photoprotection in the invasive species C. raciborskii compared to the native species A. gracile.     

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.