Molecular techniques for the early warning of toxic cyanobacteria blooms in freshwater lakes and rivers

The aim of this work was to test the efficacy of molecular techniques for detecting toxigenic cyanobacteria in environmental water samples collected from freshwater lakes, rivers and reservoirs in Portugal. Of 26 environmental samples tested, 21 were found to contain Microcystis using a genus-specific polymerase chain reaction (PCR). Another primer pair was applied to the same DNA template to test for the presence of microcystin synthetase genes. This primer pair resulted in the formation of a PCR product in 15 of the samples containing Microcystis and one sample that did not give a positive result in the Microcystis genus-specific PCR. A restriction assay using the enzyme EcoRV was then applied to show that in most cases, the gene fragment was from toxigenic strains of Microcystis and, in one above-mentioned case, from a microcystin-producing strain of Planktothrix. All environmental samples were examined microscopically to confirm the presence of cyanobacteria species. Samples were also tested for the presence of microcystins using the ELISA plate assay. There was good agreement between the results obtained with molecular techniques and those obtained from microscopy and chemical methods. The PCR techniques applied in this paper were found to be useful, particularly when the concentration of the target organism was very low compared with other organisms. This technique can be used to detect inocula for cyanobacterial populations and therefore provide a useful tool for assessing under which conditions particular species can grow into bloom populations.     

Molecular and phylogenetic characterization of potentially toxic cyanobacteria in Tunisian freshwaters

This study presents a genetic characterization of 27 potentially toxic cyanobacterial strains isolated from seven reservoirs located in the north and centre of Tunisia. These strains belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. Their toxicological potential was evaluated by molecular biology tools, which showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and saxitoxin. The majority of Microcystis isolates were able to synthesize microcystin, since they presented the six characteristic segments of the microcystin synthetase mcy cluster (mcyA, -B, -C, -D, -E and -G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight microcystin variants, including microcystin-LR. The taxonomic identification of the strains was assessed based on the variability of the 16S rRNA gene sequences. Furthermore, the 16S-23S rRNA ITS sequences of Microcystis isolates and rpoC1 sequences of Cylindrospermopsis strains were also used in the phylogenetic analysis.     

Metagenomic analysis of a freshwater toxic cyanobacteria bloom

High molecular weight (HMW) DNA prepared from a toxic freshwater cyanobacterial bloom sample was used to construct a PCR-generated 75-clone, 16S rRNA gene library and a 2850-clone bacterial artificial chromosome (BAC) library. Phylogenetic analysis of the 16S rRNA gene library demonstrated that members of eight phyla of domain Bacteria, which included Cyanobacteria, Actinobacteria, Verrucomicrobium, Bacteriodetes, Planctomycetes, Chloroflexi, Candidate Division OP10 and Alpha-, Beta- and Gammaproteobacteria, were present in the bloom community. Diversity estimates determined from 16S rRNA gene analysis and direct cell counts and morphological identification of phytoplanktons suggested that the bloom community was dominated by members of the genera Aphanizomenon and Cylindrospermopsis, phylum Cyanobacteria. BAC-end sequencing of 37 randomly selected clones and subsequent sequence analysis provided a snapshot of the total bloom community putative metabolic activities. The sequencing of the entire inserts of seven clones (clones designated 578, 67, 142, 543, 905, 1664 and 2089) selected from BAC-end sequence studies resulted in the generation of a total of 144-kb sequence data and in the identification of 130 genes for putative proteins representing at least four phyla, Proteobacteria, Actinobacteria, Bacteroidetes and Cyanobacteria. This is the first report on a snapshot analysis of a limited metagenome of a toxic cyanobacterial freshwater bloom.     

Molecular detection of potentially toxic cyanobacteria and their associated bacteria in lake water column and sediment

We investigated the molecular diversity of cyanobacteria and bacteria during a water bloom in a lake with a long history of toxic cyanobacterial blooms (Lake Kastoria, Greece). We also tested the hypothesis whether bloom-forming cyanobacteria are preserved in the lake’s sediment 2 years after the bloom. The dominant cyanobacteria during the bloom included the potentially toxin-producing Microcystis aeruginosa and several other Chroococcales forms closely related to the genus Microcystis. This suggests that the use of cyanobacterial-specific primers seems to be very informative in describing the cyanobacteria during the water blooms. The bacterial community showed high diversity, consisting mostly of singleton and doubleton phylotypes. The majority of the phylotypes were typical lake bacteria including some potential pathogens and toxin metabolising bacteria, suggesting that the dominant toxic cyanobacteria did not have any significant effect on the bacterial community structure. In the sediment, 2 years after the water bloom, no bloom-forming cyanobacteria were retrieved, suggesting that they cannot be preserved in the sediment. Similar to the water column, sediment bacterial diversity was also high, consisting mostly of yet-uncultured bacteria that are related to environments where organic matter degradation takes place.     

Detection and identification of potentially toxic cyanobacteria in Polish water bodies

The main goal of this study was to determine the distribution of potentially toxic cyanobacteria in 39 selected Polish water bodies. From the water bodies with blooms and also from those in which blooms were not visible 87 samples were investigated. For the first time samples from ponds localized in villages with high agricultural activities were included. Lakes for which microcystin concentrations had been determined before were included as a reference for the research. The detection of cyanobacteria was conducted by microscopic observation as well as by PCR amplification of the rpoC1 gene fragment. Cyanobacteria were present in 75 out of 87 samples. The presence of potentially toxic cyanobacteria was detected by amplification of the mcyB and mcyE genes, which are involved in the biosynthesis of microcystins. Both genes were detected in 7 out of 9 blooms investigated. In the case of samples collected from water bodies in which blooms were not observed, the mcyB and mcyE genes were detected in 20 out of 36. In order to identify the cyanobacteria occurring in selected reservoirs, 16S plus ITS clone libraries were constructed. The method allowed distinguishing 18 different genotypes. After sequence analysis, cyanobacteria belonging to genera Microcystis, Planktothrix, Anabaena, Pseudanabaena, Synechocystis, Synechococcus and Woronichinia were identified. Results confirmed the usefulness of the rpoC1 and mcy genes for monitoring water bodies and detection of potentially toxic cyanobacteria. Application of molecular markers allowed detecting potentially toxic cyanobacteria before the bloom was visible. This is the first comprehensive study concerning cyanobacteria present in different types of Polish water bodies performed using molecular markers.     

The taxonomic composition of macrozoobenthos in central Russian small karst lakes

The macrozoobenthos taxonomic composition in small karst lakes of Vladimir oblast is studied for the first time. In the structure of bottom communities, 149 taxa one rank below the genus have been found. Chironomid larvae and other amphibiotic animals, as well as oligochaetes, prevail. Most of them are species widely distributed in the surface waters of European Russia. The highest macrozoobenthos species diversity was recorded in neutral lakes, and the lowest diversity was in lakes with weakly acidic waters.     

淡水有害底栖蓝藻研究进展

底栖蓝藻在全球淡水水域内广泛分布,在河流、湖泊、湿地、泉水水域等均有发现。相比于浮游蓝藻,底栖蓝藻因其隐蔽性、采样困难等关注度较低。针对近十几年有害底栖蓝藻不断过量繁殖的情况,本文对有害底栖蓝藻种类、主要的环境影响因子、有害次级代谢物及监测分析方法进行了总结概括。阐述了在有害底栖蓝藻生物量集聚过程中,光照、温度、营养盐、基质、水流等主要环境因子间的交互关联,并指出藻垫小生境内有害底栖蓝藻与微生物群落间作用的重要性。分析了不同属种有害底栖蓝藻的代谢物种类及影响代谢物产出的因素,总结了目前定性和定量分析有害底栖蓝藻的方法并指出其不足,对预测、定位高水平的有害底栖蓝藻提出了建议。本文为进一步研究有害底栖蓝藻异常增殖机制和有效防控方法提供了参考和借鉴。     

Evaluation of sampling and screening techniques for tiered monitoring of toxic cyanobacteria in lakes

Exposure to cyanotoxins can pose serious human health consequences both through drinking water and recreational use of a contaminated waterway. We adapted a tiered framework proposed by the World Health Organization (WHO) for monitoring cyanobacteria populations and assessing public health risks and implemented the program in Lake Champlain. This study focused on evaluating the sampling protocols employed in this adapted monitoring program over two field seasons (2003 and 2004). Using a paired sampling design, we evaluated whether 63-μm Wisconsin net samples adequately represented whole-water conditions and whether chlorophyll a concentration could serve as a useful predictor of cyanobacteria density and microcystin concentration. We also evaluated the spatial and temporal dynamics of blooms and their implications for monitoring. Our results suggest that using threshold values of either potentially toxic cyanobacteria density counted using a rapid screening protocol or chlorophyll a concentration serve as initial indicators of potentially high levels of cyanotoxin; however, the utility of chlorophyll a is system-dependent. Whole-water samples provide more accurate estimates of population density and higher microcystin concentrations than net samples, offering a more cautionary approach to assessing risk to recreational lake users. Shoreline samples generally showed higher cyanotoxin concentrations than offshore, but restricting sampling to the shoreline may miss early warnings of bloom development.     

Fatty acid composition as a taxonomic characteristic for Microcystis and other coccoid cyanobacteria (blue-green alga) isolates

The taxonomic importance of fatty acid composition at genus and sub-genus level was evaluated by analysing the fatty acid composition of fourteen different Microcystis isolates and seven additional members of the order Chroococcales. Fatty acid composition proved to be consistent within isolates. Isolates were clustered into two major groups, namely A and B. Group B contained all the Microcystis isolates and was further divided into subgroups of varying similarity indicating the existence of different taxa. The Microcystis isolates were characterised by a high content of polyunsaturated fatty acids (27–44%) and a low content of palmitoleate. The test organisms were arranged in a scheme indicating their possible phylogenetic relationship based on fatty acid composition and other phenotypic characteristics. According to our data the toxic strains, represented by different isolates, of Microcystis appear as a distinct group. Furthermore two dubious species namely Microcystis incerta and a Synechocystis sp. could clearly be reasigned to different genera. The results demonstrated that fatty acid composition is an effective taxonomic tool in clarifying taxonomical problems of Microcystis isolates. Department of Microbiology, University of the Orange Free State     

A statistical morphological analysis and taxonomic revision of the genus Xiphophora (Fucaceae)

RICE, E. L., 1989. A statistical morphological analysis and taxonomic revision of the genus Xiphophora (Fucaceae). Xiphophora is a brown seaweed endemic to the cool temperature rocky shores of Australia and New Zealand. The two species, X. chondrophylla and X. gladiata have previously been separated by morphological criteria. However, the differences betweeen them in these characters were largely size-related, resulting in taxonomic confusion. In particular, the presence of X. gladiata in New Zealand has been disputed. In this study, a series of discriminant function analyses were performed, based on a suite of morphological characters collected from each species throughout a major portion of their geographic ranges. The results show conclusively that the present taxonomy of Xiphophora is untenable. This genus is composed of two species, but these are distributed latitudinally (either side of the 40^oS parallel). Each species contains two allopatric subspecies; one in Australia and the other in New Zealand. A formal taxonomic revision of the genus is presented.     

Rise and fall of toxic benthic freshwater cyanobacteria (Anabaena spp.) in the Eel river: Buoyancy and dispersal

Benthic cyanobacteria in rivers produce cyanotoxins and affect aquatic food webs, but knowledge of their ecology lags behind planktonic cyanobacteria. The buoyancy of benthic Anabaena spp. mats was studied to understand implications for Anabaena dispersal in the Eel River, California. Field experiments were used to investigate the effects of oxygen bubble production and dissolution on the buoyancy of Anabaena dominated benthic mats in response to light exposure. Samples of Anabaena dominated mats were harvested from the South Fork Eel River and placed in settling columns to measure floating and sinking velocities, or deployed into in situ ambient and low light treatments to measure the effect of light on flotation. Floating and sinking occurred within minutes and were driven by oxygen bubbles produced during photosynthesis, rather than intracellular changes in carbohydrates or gas vesicles. Light experiment results showed that in a natural ambient light regime, mats remained floating for at least 4 days, while in low light mats begin to sink in <24 h. Floating Anabaena samples were collected from five sites in the watershed and found to contain the cyanotoxins anatoxin-a and microcystin, with higher concentrations of anatoxin-a (median 560, max 30,693 ng/g DW) than microcystin (median 30, max 37 ng/g DW). The ability of Anabaena mats to maintain their buoyancy will markedly increase their downstream dispersal distances. Increased buoyancy also allows toxin-containing mats to collect along shorelines, increasing threats to human and animal public health.     

Bottom-up versus top-down effects on ciliate community composition in four eutrophic lakes (China)

Previous studies have shown that ciliate plankton is generally controlled by food resources (e.g., algae) and predators (e.g., metazooplankton). Among lakes with similar trophic levels but different distributions of phyto- and metazooplankton, the main forces acting on ciliate assemblages may be different. We investigated the relationship between ciliate communities and bottom-up versus top-down variables based on a survey of four subtropical eutrophic lakes (China). Two of the lakes (Chaohu, Taihu) are located on the Mid-lower Yangtze Plain near sea level, and the other two (Dianchi, Xingyunhu) on the Yunnan-Kweichow Plateau at 1700 m above sea level. Blooms of cyanobacteria developed during summer in Lakes Chaohu and Taihu and throughout the year in Lakes Dianchi and Xingyunhu. Ciliate functional feeding groups differed significantly between lakes. The results of canonical correspondence analysis (CCA) and variation partitioning showed that cyanobacteria significantly influence ciliate species, whereas ‘edible’ algae (cryptophytes, diatoms) and cladocerans were the important variables in explaining the ciliate community structure of Lakes Dianchi and Xingyunhu compared with Lakes Taihu and Chaohu. Our results highlight the importance of consistent cyanobacterial blooms in shaping the ciliate community in subtropical eutrophic shallow lakes by interacting with top-down and bottom-up factors.     

Towards a phylogenetic classification of dendrocoelid freshwater planarians (Platyhelminthes): a morphological and eclectic approach

We explore and review the taxonomic distribution of morphological features that may be used as supporting apomorphies for the monophyletic status of various taxa in future, more comprehensive phylogenetic analyses of the dendrocoelid freshwater planarians and their close relatives. Characters examined are: (i) musculature of the pharynx; (ii) anatomy of the eye; (iii) distribution of testis follicles along the longitudinal axis of the body; (iv) epidermis of the body wall; (v) musculature of the ventral body wall; (vi) presence or absence of a penis papilla; (vii) position of the mouth opening; and (viii) anterior adhesive organ. A phylogenetic hypothesis, in the form of a character state tree, is developed. This hypothesis suggests that the Kenkiidae should be resurrected as a separate family, sharing a sistergroup relationship with the Dendrocoelidae. This sistergroup relationship is supported by the shared presence of an apomorphic anterior adhesive organ. Although the shape of the anterior adhesive organ, both within and between kenkiids and dendrocoelids, may differ considerably between species, they are all constructed according to the same principles. The monophyly of the Kenkiidae is supported by the presence of an apomorphic thickening of the marginal zone of the epidermis and presumably also by the presence of prepharyngeal testes. Within the Kenkiidae, the genus Macrocotyla should be considered as a junior synonym of Kenkia, which is characterized by the presence of an apomorphic ‘planariid pharynx‐type 2’. The monophyly of the Dendrocoelidae is suggested by the presence of a dendrocoelid type of pharynx. It is shown that multicellular eye cups with numerous photoreceptor cells are not restricted to the Dugesiidae and the Terricola but are also characteristic for the Dendrocoelidae; the phylogenetic implications of this character state distribution are discussed. It is pointed out that in planarians there may be a relationship between: (i) body size, the size of an eye cup and the number of its constituent pigment cells; and (ii) the size of an eye cup and the number of photoreceptor cells that it contains. Some tentative phylogenetic groups are suggested on the basis of the following presumed apomorphic characters: presence of transverse fibres in the ventral subepidermal musculature, presence of an extra longitudinal muscle layer in the outer pharynx musculature, a mouth opening that has shifted anteriad. It is suggested that the presumed monophyly of the genus Bdellocephala is supported by the apomorphic absence of a penis papilla, combined with a penial lumen provided with many plicae.     

A combined morphological and molecular phylogenetic analysis of Parthenocissus (Vitaceae) and taxonomic implications

Parthenocissus (the Virginia creeper genus, Vitaceae) consists of 13 species and shows a disjunct distribution between Asia and North America. We investigated the inflorescence structure, calyx morphology, appendages on the inner side of petals, leaf epidermis, pollen and seed characters throughout the genus. A combined phylogenetic analysis with 27 morphological and 4137 molecular characters was conducted and the result was largely congruent with that of the previous molecular work, but with higher resolution. The combined analysis identified two clades corresponding to the Asian and North American taxa. Parthenocissus feddei was resolved as closely related to the clade containing P. cuspidifera, P. heterophylla and P. semicordata. The four species share synapomorphies of having conspicuously raised veinlets, an obscurely five‐ (to eight‐) lobed calyx, appendages on the inner side of petals covering the entire length of anthers and foveolate pollen exine ornamentation. Within the Old World clade, the pentafoliolate species were weakly supported as more closely related to species with both simple and trifoliolate leaves. Furthermore, the ancestral states of tendril apices, inflorescence structure, appendages on the inner side of petals and exine ornamentation of pollen grains were reconstructed on the molecular strict consensus tree. The appendages on the inner side of petals and exine ornamentation of pollen grains were suggested to be important characters in the taxonomy of Parthenocissus, especially for species with trifoliolate leaves. Finally, the previous classifications of Parthenocissus were evaluated within the phylogenetic framework. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, ?? , ??–??.     

A taxonomic position of Armenian endemic freshwater snails of the genus Shadinia Akramowski, 1976 (Caenogastropoda: Hydrobiidae): combining morphological and molecular evidence

According to analyses of anatomical data and nucleotide sequences of three genes (16S, 18S rDNA and CO 1 mtDNA), the Armenian endemic genus Shadinia Akramowski, 1976 Akramowski, N.N. (1976) Fauna of Armenia SSR. Mollusca. Zoological Institute Academy of Science, Yerevan, Armenian SSR. [Google Scholar] should be considered as a separate subfamily, Shadiniinae of Hydrobiidae Stimpson, 1865. A combination of genetic resemblance and similarity in conchology, morphology of radula, male and female anatomy (except some details) assures us of the close relationship of Shadinia and the genera Persipyrgula and Intermaria recently described from riverine waters of Iran. We provisionally assign these two genera to the new subfamily, which also includes the genus Nicolaia, established in 2015 for small hydrobiids from the Kasach River (Armenia). The latter genus has not yet been studied genetically, but morphological evidence leads us to consider it as compatible with Persipyrgula and Intermaria. All three genera differ significantly from the genus Shadinia in only one respect: females of these snails have one seminal receptacle, while those of Shadinia have two. The subfamily Shadiniinae is a sister group to Pyrgulinae; the former differs morphologically from the latter in possessing a pigmented nervous system and odontophore, presence of basal cusps on the central tooth of the radula and a black pigmented coiled oviduct without the widening that characterises most of the Pyrgulinae.

http://zoobank.org/urn:lsid:zoobank.org:pub:3009CB4E-EE5B-44BE-B8E4-497B5CB3BE5D     

Molecular and morphological phylogenetic analysis of Brachiaria and Urochloa (Poaceae)

The taxonomic relationships of Brachiaria and Urochloa have been questioned based on previous morphological studies. In this paper, we reconsider the phylogenetic relationships of these genera using 22 species of Brachiaria and Urochloa and six species of Paniceae as out-groups. The ITS1, 5.8S, and ITS2 region (internal transcribed spacer) of nuclear ribosomal DNA and eight morphological characters of the inflorescence were compiled into a data matrix. The cladistic analyses suggest that Urochloa-Brachiaria as a complex is paraphyletic with Eriochloa and Melinis. Species of all these genera share molecular synapomorphies and belong to the same monophyletic groups. The results confirm the continuous gradation between those genera previously found in several morphological studies. Therefore, the following eight new combinations are made: Urochloa bovonei (Chiov.) A.M. Torres & C.M. Morton, Urochloa dura (Stapf) A.M. Torres & C.M. Morton, Urochloa dura var. dura (Stapf) A.M. Torres & C.M. Morton, Urochloa dura var. pilosa (J.G. Anderson) A.M. Torres & C.M. Morton, Urochloa lachnantha (Hochst.) A.M. Torres & C.M. Morton, Urochloa leersioides (Hochst.) A.M. Torres & C.M. Morton, Urochloa nigropedata (Munro ex Ficalho & Hiern) A.M. Torres & C.M. Morton, and Urochloa subulifolia (Mez) A.M. Torres & C.M. Morton.     

Factors influencing taxonomic composition and abundance of macrozoobenthos in extralittoral zone of shallow eutrophic lakes

Zoobenthos is an essential part of shallow lake ecosystems, exerting a considerable impact upon their functioning. We studied 13 eutrophic, shallow, polymictic lakes from Northern Poland to find out which environmental factors influence taxonomic composition, abundance and biodiversity of their zoobenthos. The Canonical Correspondence Analysis allowed to distinguish three lake types: (1) macrophyte-dominated lakes, with high plant cover and well illuminated bottom, inhabited by abundant, diverse benthic taxa; (2) deeper phytoplankton-dominated lakes, with shaded bottom, high sediment oxygen demand (SOD) and rather sparse zobenthos community, dominated by Chironomus and Chaoborus larvae; (3) shallower phytoplankton-dominated lakes, with intermediate amount of light at the bottom and lower SOD values and comparatively diverse zoobenthos, but with lower number of taxa than in the first group. Apart from plant presence, distinguishing between macrophyte-dominated lakes and the other types, the most important variable in the CCA was amount of light reaching the bottom. Probably the impact of light on the bottom fauna was indirect: light stimulated development of macrophytes or phytobenthos (depending on its intensity) and thus improved food and oxygen conditions. Zoobenthos was also affected by oxygen conditions (mainly SOD), presumably by short-time oxygen depletions occurring in the deep phytoplankton-dominated lakes and preventing survival of some benthic taxa.     

Depth profiles of cyanobacterial hepatotoxins (microcystins) in three Turkish freshwater lakes

The Turkish freshwater lakes, Sapanca, Iznik and Taskisi (Calticak) have been enriched with nutrients from agriculture and domestic sources for many years. A major bloom of cyanobacteria (blue-green algae) in Lake Sapanca was recorded in May 1997, closely followed by a fish kill. Investigations were subsequently made on the cyanobacteria and water quality of the lakes, including analysis for cyanobacterial hepatotoxins (microcystins) in the filtered particulate fraction. Samples, taken from the beginning of May to end of August 1998, were analysed for microcystins by high–performance liquid chromatography with photodiode array detection (HPLC-PDA), protein phosphatase inhibition assay (PPIA) and an enzyme-linked immunosorbent assay (ELISA). No microcystins were detected in the water column in Lake Sapanca above 10 m, but toxins were found in filtered cyanobacterial samples from 20 m depth at a concentration of 3.65 μg l^?1 microcystin–LR equivalents. Ninety percent of the microcystin pool detected in L. Sapanca was found between depths of 15 and 25 m. The principal microcystin detected by HPLC-PDA was similar to microcystin–RR. Two unidentified microcystin variants were found in Lake Taskisi surface samples at a concentration of 2.43 μg l^?1 microcystin–LR equivalents in the filtered cyanobacterial cell fraction. Although 10 water samples (10 × 5 l) were taken from Lake Iznik (surface to 20 m, 5 m intervals), no microcystins were detected by HPLC-PDA (limit of detection 10 ng). The depth at which microcystins were detected in L. Sapanca coincided with the draw-off depth for the drinking water supply for the city of Sakarya