Free-living Forms Research Articles

18S rRNA sequences and phylogeny of platyhelminthes

The phyletic relationships, and hence the systematic position, of freshwater planarians (Tricladida, Turbellaria, Platyhelminthes) within the Turbellaria, and of Platyhelminthes within the Metazoa are uncertain. This stems partially from the shortcomings of the characters and techniques clasically used in the systematic study of these groups. It is, therefore, necessary to apply new methodologies and to propose new characters (basically proteins and nucleic acids) which offer the advantages of supplying a large amount of data and of allowing quantitative estimates. This study presents 18S rRNA partial sequences from several species belonging to the phylum Platyhelminthes, including parasitic groups and free-living forms. Sequence data were compared with a large number of animal and protist sequences using both most parsimonious and additive methods and phylogenies inferred. The main conclusions supported by the data can be summarized as follows: (1) Tricladida Paludicola are monophyletic to the rest of Platyhelminthes (100% bootstrapping). (2) The Trematode and the Neorhabdocoel do not group together. This does not support the hypothesis of the origin of trematodes from rabdocoels. (3) Platyhelminthes are monophyletic (97% of bootstrapping), being a sister group of the other Eubilateria. This supports the basal position of the Platyhelminthes in the radiation of Eubilateria, and rejects hypotheses of a secondary derivation of Platyhelminthes from the Eucoelomates.

Are Temperature and Photoperiod Necessary Cues for Encystment in the Marine Benthic Harpacticoid Copepod Heteropsyllus nunni Coull?

Heteropsyllus nunni is a marine copepod that builds a cyst and dwells within it during a period of extended diapause. The field abundance of this copepod has been monitored for 10 years, but nothing is known about the cues that induce and terminate encystment. In the laboratory, different photoperiods and temperatures were tested for their effects on encystment and excystment. The photoperiod and temperature cues tested neither induced nor inhibited encystment in H. nunni. Encystment occurred in all treatments, regardless of temperature or photoperiod, suggesting that internal genetic cues, tied to a specific ontogenetic stage, must be the central causal factor. Copepods in the hot treatments encysted and excysted more rapidly than in the cold. Many copepods in the cold treatment encysted (though later than copepods in the hotter treatments), and most were still within the cyst at the end of the 23-week experiment. There were significantly more males within the full cysts than females. A concurrent field study confirmed the known seasonal patterns in the number of encystments relative to the number of free-living forms; i.e., encystment took place in the summer.

The genomes of the family Rhizobiaceae: size, stability, and rarely cutting restriction endonucleases

The lack of high-resolution genetic or physical maps for the family Rhizobiaceae limits our understanding of this agronomically important bacterial family. On the basis of statistical analyses of DNA sequences of the Rhizobiaceae and direct evaluation by pulsed-field agarose gel electrophoresis (PFE), five restriction endonucleases with AT-rich target sites were identified as the most rarely cutting: AseI (5'-ATTAAT-3'), DraI (5'-TTTAAA-3'), SpeI (5'-ACTAGT-3'), SspI (5'-AATAAT-3'), and XbaI (5'-TCTAGA-3'). We computed the sizes of the genomes of Bradyrhizobium japonicum USDA 424 and Rhizobium meliloti 1021 by adding the sizes of DNA fragments generated by SpeI digests. The genome sizes of R. meliloti 1021 and B. japonicum USDA 424 were 5,379 +/- 282.5 kb and 6,195 +/- 192.4 kb, respectively. We also compared the organization of the genomes of free-living and bacteroid forms of B. japonicum. No differences between the PFE-resolved genomic fingerprints of free-living and mature (35 days after inoculation) bacteroids of B. japonicum USDA 123 and USDA 122 were observed. Also, B. japonicum USDA 123 genomic fingerprints were unchanged after passage through nodules and after maintenance on a rich growth medium for 100 generations. We conclude that large-scale DNA rearrangements are not seen in mature bacteroids or during free-living growth on rich growth media under laboratory conditions.

Immunochemical analysis of lipopolysaccharides from free-living and endosymbiotic forms of Rhizobium leguminosarum

Rhizobium leguminosarum B556 and 8002 differ only with respect to carrying symbiotic plasmids with specificity for Pisum or Phaseolus hosts, respectively. Protease-treated samples derived from free-living cultures of both strains revealed a ladder of lipopolysaccharide (LPS-1) bands after periodate-silver staining of sodium dodecyl sulfate-polyacrylamide gels. These bands were arranged as doublets. After Western (immuno-) blotting, all LPS-1 bands reacted with monoclonal antibody JIM 21, whereas monoclonal antibody MAC 57 reacted only with the upper (slower-migrating) band and monoclonal antibody MAC 114 reacted only with the lower band of each doublet pair. Preparations obtained from bacteroids of Pisum or Phaseolus nodules showed significant differences in the size distribution and antigenicity of LPS. In bacteroids from Phaseolus sp., JIM 21 and MAC 57 each stained a ladder of LPS-1 bands on sodium dodecyl sulfate-polyacrylamide gels which corresponded in mobility to the upper band of each doublet pair seen in free-living cultures. MAC 114 did not react with the LPS from Phaseolus sp.-derived bacteroids. In bacteroids from Pisum sp., only fast-migrating (lower-molecular-weight) forms of LPS-1 could be visualized on gels, but both upper and lower bands of each doublet were still present and could be stained by the appropriate monoclonal antibody, MAC 57 or MAC 114, respectively. Similarly, bacteroids from R. leguminosarum 3841, which nodulates Pisum species, differed with respect to the structure and antigenicity of their LPS-1 from bacteroids of a related strain, B625, which nodulates Phaseolus species. Physiological factors were investigated that could account for these differences between the structures of LPS-1 from free-living cultures of B556 and 8002 and that from bacteroids. The following modifications in growth conditions each tended to reduce the expression of MAC 114 antigen and enhance the expression of MAC 57 antigen: succinate rather than glucose as the carbon source; microaerobic (2.5%, vol/vol) oxygen concentrations; and acidic (pH 5 to 6) culture medium. When all three of these conditions were combined, the LPS-1 that resulted was very similar to that in bacteroids from Pisum nodules. However, it was not possible to reproduce the LPS-1 pattern observed for bacteroids from Phaseolus nodules, which maintained a ladder of LPS bands reacting with MAC 57 antibody.

Molecular biology of Leishmania

Leishmania is a trypanosomatid protozoa with a digenetic life cycle. Sandflies inject promastigotes, the free living form present in their salivary glands, into mammals where the parasite colonizes macrophages, transforming into intracellular amastigotes. The cycle is completed when during a blood meal the insect ingests infected macrophages, the amastigotes are released in the gut where they transform back into promastigotes. Leishmania has to adapt to the changing life conditions, from free-living forms in the poikilothermic insect vector to obligatory intracellular parasite in the homeothermic mammalian host. It also has to adapt to the acidic pH of the macrophage's phagolysosome where amastigotes multiply. The adaptative response of Leishmania includes morphological, physiological, and biochemical changes. Promastigotes can be grown in culture medium. Studies of changes taking place during adaptation have been facilitated by the establishment of in vitro conditions that allow the transformation of amastigotes into promastigotes and vice versa. The system is well suited for studying regulation of gene expression during adaptative differentiation. Some mechanisms of mRNA processing are unique to these protozoa: trans-splicing and RNA editing. Several genes that are differentially expressed in the two stages have been studied. No obvious cis regulatory motifs have been found in the DNA.

Evolutionary relationships among cyanobacteria and green chloroplasts

The 16S rRNAs from 29 cyanobacteria and the cyanelle of the phytoflagellate Cyanophora paradoxa were partially sequenced by a dideoxynucleotide-terminated, primer extension method. A least-squares distance matrix analysis was used to infer phylogenetic trees that include green chloroplasts (those of euglenoids, green algae, and higher plants). The results indicate that many diverse forms of cyanobacteria diverged within a short span of evolutionary distance. Evolutionary depth within the surveyed cyanobacteria is substantially less than that separating the major eubacterial taxa, as though cyanobacterial diversification occurred significantly after the appearance of the major eubacterial groups. Three of the five taxonomic sections defined by Rippka et al. (R. Rippka, J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Stanier, J. Gen. Microbiol. 111:1-61, 1979) (sections II [pleurocapsalean], IV [heterocystous, filamentous, nonbranching], and V [heterocystous, filamentous, branching]) are phylogenetically coherent. However, the other two sections (I [unicellular] and III [nonheterocystous, filamentous]) are intermixed and hence are not natural groupings. Our results not only support the conclusion of previous workers that the cyanobacteria and green chloroplasts form a coherent phylogenetic group but also suggest that the chloroplast lineage, which includes the cyanelle of C. paradoxa, is not just a sister group to the free-living forms but rather is contained within the cyanobacterial radiation.

Isolation of monoclonal antibodies reacting with peribacteriod membranes and other components of pea root nodules containing Rhizobium leguminosarum

Plant and bacterial antigens contributing to nodule development and symbiosis in pea (Pisum sativum L.) roots were identified after isolation of a set of monoclonal antibody (McAb)-producing hybridoma lines. Rats were immunised with the peribacteriod material released by mild osmotic shock treatment from membrane-enclosed bacteroids of Rhizobium leguminosarum bv. viceae. In order to diversify the range of McAb specificities, this material was either used as immunogen directly (method 1), or after immunodepletion of a set of glycoprotein and lipopolysaccharide antigens (method 2), or after deglycosylation (method 3). After fusion and screening of cloned hybridoma lines, these three immunisation methods gave respectively 4, 2 and 1 classes of McAb with unique antigen specificities. Ultrastructural immunogold localisation studies showed four different antigens to be present on peribacteriod and plasma membranes (identified by MAC 64, 202, 206 or 209); in addition, a glycoprotein of plant origin but present in the infection-thread matrix was identified by MAC 204. Although none of the epitopes recognised by these McAb was nodule-specific, several were found to be more abundant in extracts of nodule tissue than in uninfected roots (MAC 64, 202, 204, 206). Two McAb reacted with new bacterial antigens: MAC 203 identified a bacterial antigen expressed upon infection but not in free-living cultures of Rhizobium, and MAC 115 identified a bacterial polypeptide (55 kdaltons) that was present in both free-living and bacteroid forms. There were also some McAb of broader specificity that react with antigens present in both plant and bacterial cytoplasms.

Distribution and significance of heterotrophic marine bacteria with antibacterial activity.

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.

CARBON NUTRITION AND THE REGULATION OF UPTAKE HYDROGENASE ACTIVITY IN FREE-LIVING AND SYMBIOTIC ANABAENA CYCADEAE.

Anabaena cycadeae was grown with N2 as nitrogen source, either photoautotrophically in light or with glucose as carbon source in darkness. The rate of growth was much slower in darkness but the heterocyst frequency was much the same; nitrogenase activity (on a chlorophyll basis) was about half that of light-grown cells. Light-grown organisms contained uptake hydrogenase activity but dark-grown organisms did not. The addition of glucose to light-grown organisms was followed by the disappearance of uptake hydrogenase activity over the following 48 hours and the disappearance was independent of light. Heterocyst frequency and nitrogenase activity were much less affected by glucose addition. A. cycadeae growing symbiotically in cycad roots had much higher heterocyst frequency and nitrogenase activity than the free-living form but no detectable uptake hydrogenase activity. It is suggested that the rate of supply of carbohydrate to the heterocyst controls the development of uptake hydrogenase activity and that the absence of this activity in the symbiotic cyanobacteria indicates that the organisms in the cycad roots have an ample supply of carbohydrate.

Choline kinase II is present only in nodules that synthesize stable peribacteroid membranes

Host-cell cytoplasm from soybean plants infected with the peribacteroid membrane (PBM)-building Rhizobium japonicum strain 61-A-101 (effective, N(2)-fixing) had much higher choline kinase activity than cytoplasm from either uninfected tissue or tissue infected with the non-PBM-building (ineffective, non-N(2)-fixing) strain 61-A-24. Ion-exchange chromatography showed that both types of nodule and root tissue possessed constitutive choline kinase I activity that had a K(m) for choline of approximately 150 muM. The nodules of the effective symbiosis had another activity, choline kinase II (K(m) = 81 muM). Nondenaturing and NaDodSO(4) electrophoresis revealed no multimeric subunit structure of the two enzyme forms but did show the molecular sizes for choline kinase I, 58-59 kDa, and choline kinase II, 60 kDa. Choline kinase I and II and pI values of 8.1 and 8.5, respectively, and two-dimensional gel electrophoresis of whole cytoplasm from control and infected tissue showed a spot corresponding to choline kinase II only in the case of the effective symbiosis, whereas both tissue types had spots corresponding to choline kinase I. Choline kinase II is presumed to be encoded by the plant as neither free-living nor symbiotic (bacteroid) forms of the prokaryote showed any choline kinase activity.

Production of heterotrophic bacteria inhabiting macroscopic organic aggregates (marine snow) from surface waters1

Macroscopic detrital aggregates, known as marine snow, are a ubiquitous and abundant component of the marine pelagic zone. Descriptions of microbial communities occurring at densities 2–5 orders of magnitude higher on these particles than in the surrounding seawater have led to the suggestion that marine snow may be a site of intense heterotrophic activity. We tested this hypothesis using incorporation of [3H]thymidine into macromolecules as a measure of bacterial growth occurring on marine snow from oceanic waters in the North Atlantic and from neritic waters off southern California.Abundances of marine snow ranged from 0.1 to 4.3 aggregates liter−1. However, only 0.1–4% of the bacteria in the water occurred in association with marine snow. Mean thymidine incorporation per cell on aggregates was generally equal to or lower than that of bacteria found free‐living in the surrounding seawater, indicating that attached bacteria were not growing more rapidly than free‐living bacteria. Bacteria inhabiting aggregates were up to 25 times larger than free‐living forms. Thus, the contribution of these attached bacteria to total bacterial production in surface waters was low but occasionally significant, ranging from 3 to 26% and averaging 8 ± 7%

Microbial Ecophysiology of Whey Biomethanation: Characterization of Bacterial Trophic Populations and Prevalent Species in Continuous Culture

The organization and species composition of bacterial trophic groups associated with lactose biomethanation were investigated in a whey-processing chemostat by enumeration, isolation, and general characterization studies. The bacteria were spatially organized as free-living forms and as self-immobilized forms appearing in flocs. Three dominant bacterial trophic group populations were present (in most probable number per milliliter) whose species numbers varied with the substrate consumed: hydrolytic, 10; acetogenic, 10 to 10; and methanogenic, 10 to 10. The three prevalent species utilizing lactose were identified as Leuconostoc mesenteroides, Klebsiella oxytoca, and Clostridium butyricum. Clostridium propionicum and Desulfovibrio vulgaris were the dominant lactate-consuming, hydrogen-producing acetogenic bacteria, while D. vulgaris was the only significant ethanol-degrading species. Methanosarcina barkeri and Methanothrix soehngenii were identified as the dominant acetate-utilizing methanogens, and Methanobacterium formicicum was the prevalent hydrogen-utilizing methanogen. A microbial food chain is proposed for lactose biomethanation that comprises multiple species in three different groups, with the major hydrogen-producing acetogen being a sulfate-reducing species, D. vulgaris, which functioned in the absence of significant levels of environmental sulfate.

The significance of macroscopic aggregates (marine snow) as sites for heterotrophic bacterial production in the mesopelagic zone of the subtropical Atlantic

Macroscopic detrital aggregates (known as marine snow) harbor greatly enriched populations of bacteria and protozoans, suggesting that they may be an important site of heterotrophic activity in the deep sea. We collected flocculent marine snow larger than 3 mm in diameter in the mesopelagic zone of the subtropical Atlantic from the submersible, Johnson-Sea-Link. Although the bacteria inhabiting these aggregates incorporated as much or more [ 3H]-thymidine per bacterium than free-living forms, were up to 2 orders of magnitude more abundant on marine snow than in the surrounding seawater, and were twice as large as unattached bacteria, the marine snow itself was rare, ranging from 0.5 to 4 aggregates m −3. Thus the contribution of bacteria inhabiting flocculent marine snow to total bacterial production in the mesopelagic zone was insignificant, ranging from 0.01 to 0.39% over depths of 130 to 650 m.

Mg 2+ adenosine triphosphatase from cell envelopes of free-living and bacteroid forms of Rhizobium lupini strain NZP2257

A procedure for the purification of Mg 2+ adenosine triphosphatase (EC 3.6.1.3) from free-living and bacteroid forms of Rhizobium lupini NZP2257 is described. The enzyme was released from cell envelopes using Triton X-100 and purified by gel filtration on Ultrogel AcA 22, followed by preparative gel electrophoresis on agarose. The purified ATPase had a molecular weight of about 355,000, as determined from sedimentation coefficients on sucrose gradients. Kinetic analysis of activity of the enzyme from free-living R. lupini showed it to be typical of F 1-type Mg 2+ ATPases from bacteria. Mg stimulated activity at pH 7.0, although, when present as the free ion, Mg caused non-competitive inhibition ( K I = 1.5 mM). Maximum activity with ATP occurred over a broad pH range from 6.0 to 10.5. ATP, GTP, and UTP, and, to a much lesser degree, CTP and ADP, were hydrolyzed by the enzyme. Hydrolysis of glucose 6-phosphate was not observed. The K m for ATP at pH 7.0 was 0.67 and for GTP 1.4 m m. ATPase activity was inhibited by ADP, and competitive with ATP ( K I = 0.18 mM ). Azide also caused inhibition but fluoride and DCCD had no effect. Native and sodium dodecyl sulfate-gel electrophoretic analysis revealed no obvious differences between ATPases from free-living and bacteroid forms of R. lupini.

Succinate transport by free-living forms of Rhizobium japonicum.

We have demonstrated that the transport of succinate into the cells of Rhizobium japonicum strains USDA 110 and USDA 217 is severely inhibited by cyanide, azide, and 2,4-dinitrophenol, but not by arsenate. These results suggest an active mechanism of transport that is dependent on an energized membrane, but does not directly utilize ATP. The apparent Km for succinate was 3.8 microM for strain USDA 110 and 1.8 microM for strain USDA 217; maximal transport velocities were 1.5 and 3.3 nmol of succinate per min per mg of protein, respectively. The expression of the succinate uptake activity was inducible rather than constitutive, with succinate and structurally related compounds being the most effective inducers. The mechanism showed some specificity for succinate and similar organic acids; fumarate and L-malate were classical competitive inhibitors of the system. In general, the best competing compounds were also the best carbon substrates for induction of succinate uptake activity. EDTA inhibited the transport of succinate, implying a role for divalent cations in the system. When various divalent cations were used to reconstitute EDTA-inhibited activity, Ca2+ was most effective, followed by Mg2+, which restored activity at about half the efficiency of Ca2+. Growth media that were supplemented with increased Ca2+ concentration supported more rapid growth with succinate as the carbon substrate, and cells from such media showed higher specific activities of succinate transport.

The life history and morphology of free-living Pilayella littoralis (L.) Kjellm. (Ectocarpaceae, Ectocarpales) in Nahant Bay, Massachusetts

Abstract A previously unreported free-living form of Pilayella is described. During all months of the year, drifting clouds of this plant may carpet up to half of the sandy bottom and beaches of Nahant Bay, a 15 km2 rectangular embayment on the north coast of Massachusetts. Four features distinguish the atypical free-living form from the typical attached form of this species: (1) a ball shape and a concomitant general lack of polarity in the development of the central axes; (2) a polystichous condition present throughout much of the central axes; (3) a dearth of reproductive organs; and (4) a peak summer-time abundance of hundreds of tonnes (live weight). Plant reproduction and growth appear to result from ball fragmentation and subsequent regeneration solely by vegetative cell division. Fragmentation results from seasonal senescence of the filamentous axes and from infection by the marine fungus Eurychasma dicksonii. The Pilayella drift community is characterized by its virtual monospecificity by weight,...

An immunological comparison of nitrogenase proteins of fast and slow growing rhizobia

The enzyme complex nitrogenase, which catalyzes nitrogen reduction consists of two component proteins [ 1,2]: component I, a molybdenum and iron containing protein comprising two types of subunits and component II, a dimer iron-sulfur protein with identical subunits. The actual nitrogen reduction takes place on component I and this component is called dinitrogenase; component II which supplies component I with electrons is called dinitrogenase reductase [3]. The nitrogenase complex shows a remarkable structural similarity [2] among widely divergent organisms. The genus Rhizobium can be divided into two groups, viz. slow and fast-growing species [4,5]. These two groups differ in several characteristics. The fastgrowing rhizobia have a generation time of 2-4 h, are acid producers, have laterally arranged flagella and do not reduce nitrogen in the free-living form [4,5]. The slow-growing rhizobia have a generation time of 6-8 h, are alkali producers, have polar or subpolar flagella and reduce nitrogen in free-living and symbiotic form. Furthermore there are differences in the metabolic pathways and DNA base ratios. The results of Ruvkun and Ausubel [6] on the sequence homology between the nifgenes of a fast and a slow-growing Rhizobium pointed to a rather restricted homology. This result implies that a substantial difference may exist between nitrogenase proteins of fast and slow-growing rhizobia. In this paper we report on the immunological comparison of component I proteins of fast and slowgrowing rhizobia by the use of radioimmunoassays. We have used this technique because comparative immunological analyses of proteins from different organisms have been useful in asserting taxonomic relationships [7,8] and only small amounts of proteins are required in this procedure.

Mechanisms of survival of nematode parasites with emphasis on hypobiosis

Mechanisms involved in parasitic nematode survival must be considered with reference to their host and environmental interrelationships since these interrelationhips ultimately influence any parasite adaptations aimed at survival. The most important of the potential environmental constraints are climatic, particularly temperature and humidity, and these can drastically influence larval development and survival. One of the major host factors influencing successful parasite survival is the availability of suitable (susceptible) new hosts to the infective stages of the parasite at the appropriate time for transmission to be achieved. Other host factors that influence parasite survival are those that affect the entrance, establishment and reproduction of the parasite within its new host; mainly problems of acclimatization to a parasitic way of life as well as the countering or adaptation to a variety of host resistance factors, both molecular and cellular, by the parasite. Finally in order for life cycles to be completed, the parasite must evolve means whereby its larval forms can leave the host so that eventual transmission to a new host can be accomplished. In this paper a number of adaptations which enable the parasite to overcome these constraints are discussed. These include such things as larval resistance to environmental effects, the utilization of intermediate hosts or vectors for transmission, seasonally-increased fecundity rates, anti-host immunity stratagems and hypobiosis. This latter phenomenon, hypobiosis or prolonged but temporarily arrested larval development, represents one of the most useful of life cycle adaptations to ensure parasite adaption enables the parasite to synchronize its life cycle to changing environmental or survival and appears to be widespread among parasitic nematodes. Among its benefits, this host conditions. It can thus be of major importance in ensuring survival of the parasite during periods of environmental adversity when conditions for transmission are poor and survival of free-living forms may be minimal. It also enables the parasite to have available large numbers of infective forms at points in the host reproductive cycle which coincide with the production of the susceptible neonates thereby greatly facilitating transmission. Additionally, with certain species of nematodes, occurring as it does at times of the year when the numbers of infective stages may be high while host resources may be limited, oscillations in parasite biomass can be avoided. It thus serves as a highly adaptive mechanism for regulating populations of adult worms, lessening stress on the host and favoring parasite survival as a result.

Encystment Discovered in a Marine Copepod

It has been believed that marine crustacea do not encyst as adults. The benthic copepod Heteropsyllus nunni has been found to encyst in intertidal sands in the summer; free-living forms are abundant in the winter. A unique platelike structure on the cephalothorax is thought to be the site of cyst formation.

Pigment distribution and nitrogen fixation inAnabaena azollae

The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity. In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N2-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla). Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O2 evolution and the absence of photo-fixation of CO2 in the cyanobionts. Pigment composition and N2-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility. It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.