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Freshwater mussels belonging to the genus Nodularia (Family Unionidae) are known to be widely distributed in East Asia. Although phylogenetic and population genetic studies have been performed for these species, there still remain unresolved questions in their taxonomic status and biogeographic distribution pathways. Here, the nucleotide sequences of CO1 and 16S rRNA were newly determined from 86 N. douglasiae and 83 N. breviconcha individuals collected on the Korean Peninsula. Based on these data, we revealed the following results: (1) N. douglasiae can be divided into the three genetic clades of A (only found in Korean Peninsula), B (widely distributed in East Asia), and C (only found in the west of China and Russia), (2) the clade A is not an independent species but a concrete member of N. douglasiae given the lack of genetic differences between the clades A and B, and (3) N. breviconcha is not a subspecies of N. douglasiae but an independent species apart from N. douglasiae. In addition, we suggested the plausible scenarios of biogeographic distribution events and demographic history of Nodularia species.

A polyphasic study of a benthic Nodularia isolate (LEGE06071) from an Atlantic environment, specifically salt pans, was performed. LEGE06071 resembled both type strains of Nodularia sphaerocarpa and Nodularia harveyana, while ACOI00729 (purchased isolate) was identified as N. sphaerocarpa. The length and width of vegetative cells varied from 3.10 to 3.15 microm and from 3.71 to 4.25 microm, respectively, while heterocyts were 3.91-4.89 microm long and 4.20-4.74 microm wide. None of the isolates had aerotopes. The sequencing of the 16S rRNA gene from the two Nodularia isolates indicated that they belonged neither to Nodularia spumigena nor N. harveyana. Nodularin and other cyanotoxin synthesis-associated genes could not be detected, nor could nodularin production be detected by ELISA. However, MALDI-TOF analysis of LEGE06071 revealed the presence of other compounds, namely, glycolipids. Hence, toxicological screenings against Artemia nauplii, Escherichia coli and Salmonella typhimurium were performed. Toxic effects could only be observed against Artemia, with 48 h-LC(50) values for the aqueous and crude extract of methanol of 53.21 mg ml(-1) and 17.81 mg ml(-1), respectively. This study presents the first evidence of a non-nodularin-producing Nodularia isolate in Atlantic salt pan ecosystems and its potential ecotoxicity against Artemia sp., (Copyright 2009 Elsevier Masson SAS. All rights reserved.)

Cyanobacterial blooms in the Baltic Sea are a common phenomenon and are formed by the heterocystous, filamentous species Nodularia spumigena. The toxicity of these blooms is attributed to the hepatotoxin nodularin, produced by N. spumigena. Little is known regarding the regulatory mechanisms or environmental signaling that control nodularin production. Here we report the characterization of the transcriptional expression pattern of the nodularin synthetase gene cluster (nda) during phosphate depletion, and nitrogen supplementation. Real-time PCR analysis of these genes revealed that while cells continuously expressed the nda cluster, the expression of all nda genes increased when cells were subjected to phosphate depletion, and decreased in the presence of ammonium. In contrast to the shifts in expression, the intracellular and extracellular nodularin concentrations did not vary significantly during the treatments.

In this article we summarize the current knowledge of Baltic Sea cyanobacteria, focusing on diversity, toxicity, and nitrogen fixation in the filamentous heterocystous taxa. We also review the recent results of our microbial diversity studies in planktonic and benthic habitats in the Baltic Sea. Based on molecular analyses, we have improved the understanding of cyanobacterial population structure by assessing genetic diversity within species that are morphologically inseparable. Moreover, we have studied microbial functions such as toxin production and nitrogen fixation in situ under different environmental conditions. Phosphorus limitation of bloom-forming, nitrogen-fixing cyanobacteria was clearly verified, emphasizing the importance of continuous efforts to reduce this element in the Baltic Sea. We have designed a rapid and reliable detection method for the toxic cyanobacterium Nodularia spumigena, which can be used to study bloom formation of this important toxin producer.

The community structure of the bacteria associated with Nodularia spumigena (Mertens) cyanobacterial aggregates in the Baltic Sea was studied with temperature gradient gel electrophoresis (TGGE), using a 16S rRNA gene fragment as a target. Various developmental stages of the aggregates and free-floating cyanobacterial filaments were sampled to reveal possible changes in associated microbial community structure during development and senescence of the aggregates. The microbial community structures of all samples differed, and the communities of young and decaying aggregates were separated by cluster analysis of the TGGE fingerprint data. Sequencing of the TGGE fragments indicated the presence of bacteria from the alpha-, beta-, and gamma-proteobacterial groups, as well as members of Cytophaga-Flexibacter-Bacteroides lineages and gram-positive Actinobacteria spp. The majority of the Nodularia-associated sequences were not closely related to previously reported 16S rDNA sequences from the Baltic Sea or any other environment. The structure of the bacterial assemblage reflects the environmental changes associated with the succession and decay of the cyanobacterial aggregates. In addition, the sequence data suggest that the N. spumigena (Mertens) blooms in the Baltic Sea may host thus far uncharacterized bacterial species.

Diversity and ecological features of cyanobacteria of the genus Nodularia from benthic, periphytic and soil habitats are less well known than those of Nodularia from planktonic habitats. Novel benthic Nodularia strains were isolated from the Baltic Sea and their morphology, the presence of gas vacuoles, nodularin production, gliding, 16S rRNA gene sequences, rpoB, rbcLX and ndaF genes, and gvpA-IGS regions were examined, as well as short tandemly repeated repetitive sequence fingerprints. Strains were identified as Nodularia spumigena, Nodularia sphaerocarpa or Nodularia harveyana on the basis of the size and shape of the different types of cells and the presence or absence of gas vacuoles. The planktonic strains of N. spumigena mostly had gas vacuoles and produced nodularin, whereas the benthic strains of N. sphaerocarpa and N. harveyana lacked gas vacuoles and did not produce nodularin (except for strain PCC 7804). The benthic strains were also able to glide on surfaces. In the genetic analyses, the planktonic N. spumigena and benthic N. sphaerocarpa formed monophyletic clusters, but the clusters were very closely related. Benthic strains determined as N. harveyana formed the most diverse and distant group of strains. In addition to phylogenetic analyses, the lack of the gvpA-IGS region and ndaF in N. sphaerocarpa and N. harveyana distinguished these species from the planktonic N. spumigena. Therefore, ndaF can be considered as a potential diagnostic tool for detecting and quantifying Baltic Sea bloom-forming, nodularin-producing N. spumigena strains. The data confirm that only one morphologically and genetically distinct planktonic species of Nodularia, N. spumigena, and at least two benthic species, N. sphaerocarpa and N. harveyana, exist in the Baltic Sea.