Cyanobacteria and Macroinvertebrate Relationships in Freshwater Benthic Communities beyond Cytotoxicity.

Cyanobacteria are harmful algae that are monitored worldwide to prevent the effects of the toxins that they can produce. Most research efforts have focused on direct or indirect effects on human populations, with a view to gain easy accurate detection and quantification methods, mainly in planktic communities, but with increasing interest shown in benthos. However, cyanobacteria have played a fundamental role from the very beginning in both the development of our planet's biodiversity and the construction of new habitats. These organisms have colonized almost every possible planktic or benthic environment on earth, including the most extreme ones, and display a vast number of adaptations. All this explains why they are the most important or the only phototrophs in some habitats. The negative effects of cyanotoxins on macroinvertebrates have been demonstrated, but usually under conditions that are far from natural, and on forms of exposure, toxin concentration, or composition. The cohabitation of cyanobacteria with most invertebrate groups is long-standing and has probably contributed to the development of detoxification means, which would explain the survival of some species inside cyanobacteria colonies. This review focuses on benthic cyanobacteria, their capacity to produce several types of toxins, and their relationships with benthic macroinvertebrates beyond toxicity.

Identification of Marine Biotechnology Value Chains with High Potential in the Northern Mediterranean Region.

Marine (blue) biotechnology is an emerging field enabling the valorization of new products and processes with massive potential for innovation and economic growth. In the Mediterranean region, this innovation potential is not exploited as well as in other European regions due to a lack of a clear identification of the different value chains and the high fragmentation of business innovation initiatives. As a result, several opportunities to create an innovative society are being missed. To address this problem, eight Northern Mediterranean countries (Croatia, France, Greece, Italy, Montenegro, Portugal, Slovenia and Spain) established five national blue biotechnology hubs to identify and address the bottlenecks that prevent the development of marine biotechnology in the region. Following a three-step approach (1. Analysis: setting the scene; 2. Transfer: identification of promising value chains; 3. Capitalization: community creation), we identified the three value chains that are most promising for the Northern Mediterranean region: algae production for added-value compounds, integrated multi-trophic aquaculture (IMTA) and valorization aquaculture/fisheries/processing by-products, unavoidable/unwanted catches and discards. The potential for the development and the technical and non-technical skills that are necessary to advance in this exciting field were identified through several stakeholder events which provided valuable insight and feedback that should be addressed for marine biotechnology in the Northern Mediterranean region to reach its full potential.

Autofluorescence Imaging to Evaluate Red Algae Physiology.

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in full sunshine. Most rhodophytes are red, however some can appear bluish, depending on the proportion of blue and red biliproteins (phycocyanin and phycoerythrin). Different phycobiliproteins can capture light at diverse wavelengths and transmit it to chlorophyll a, which makes photosynthesis under very different light conditions possible. These pigments respond to habitat changes in light, and their autofluorescence can help to study biological processes. Using Chroothece mobilis as a model organism and the spectral lambda scan mode in a confocal microscope, the adaptation of photosynthetic pigments to different monochromatic lights was studied at the cellular level to guess the species' optimal growth conditions. The results showed that, even when the studied strain was isolated from a cave, it adapted to both dim and medium light intensities. The presented method is especially useful for studying photosynthetic organisms that do not grow or grow very slowly under laboratory conditions, which is usually the case for those living in extreme habitats.

The Green Microalga Coelastrella thermophila var. globulina (Scenedesmaceae, Chlorophyta) Isolated from an Algerian Hot Spring as a Potential Source of Fatty Acids.

Screening microalgae from extreme environments, including hot springs, is an important research topic that has lately emerged. A thermophilic green alga was isolated from a north-eastern Algerian hot spring at a temperature of 63 °C, and its fatty acid (FA) profile was explored. The strain was cultivated in BBM medium at 35 °C in a 16:8 h light/dark cycle and 75 µM photons m-2 s-1. The morphological studies combined with phylogenetic analysis revealed that the isolate was Coelastrella thermophila var. globulina Q. Wang, H. Song, X. Liu, G. Liu and Z. Hu. The monounsaturated fatty acid (MUFA) content was 51.12%. The saturated fatty acid (SFA) and polyunsaturated fatty acid (PUFA) content, respectively, accounted for 27.01% and 21.87%. The main FA was oleic acid (18:1n-9), whose value was 35.95%, followed in decreasing order by palmitic acid (16:0) with 21.45%, linoleic acid (18:2n-6) with 14.38% and α-linolenic acid (18:3n-3) with 04.22%. The FA profile exhibited high total n-6 and n-3 PUFA values (15.80% and 5.76%, respectively). Coelastrella thermophila var. globulina is particularly interesting for producing n-6 and n-3 PUFA and is likely suitable for other biotechnological purposes. This is the first time that this taxon has been reported in hot springs. Other species can be expected to be reported, which emphasises the importance of the biodiversity of extreme habitats.

Confocal Microscopy in Ecophysiological Studies of Algae: A Door to Understanding Autofluorescence in Red Algae.

Alga in the genus Chroothece have been reported mostly from aquatic or subaerial continental environments, where they grow in extreme conditions. The strain Chroothece mobilis MAESE 20.29 was exposed to different light intensities, red and green monochromatic light, ultraviolet (UV) radiation, high nitrogen concentrations, and high salinity to assess the effect of those environmental parameters on its growth. Confocal laser scanning microscopy (CLSM) was used as an "in vivo" noninvasive single-cell method for the study. The strain seemed to prefer fairly high light intensities and showed a significant increase in allophycocyanin (APC) and chlorophyll a [photosystem I (PSI) and photosystem II (PSII)] fluorescence with 330 and 789 µM/cm2/s intensities. Green monochromatic light promoted a significant increase in the fluorescence of APC and chlorophyll a (PSI and PSII). UV-A significantly decreased phycocyanin and increased APC, while UV-A + B showed a greater decreasing effect on c-Phycocyanin but did not significantly change concentrations of APC. The increase in nitrogen concentration in the culture medium significantly and negatively affected all pigments, and no effect was observed with an increase in salinity. Our data show that CLSM represents a very powerful tool for ecological research of microalgae in small volumes and may contribute to the knowledge of phycobiliproteins in vivo behavior and the parameters for the large-scale production of these pigments.

The 6th Iberian and 2nd Ibero-American Cyanotoxin Congress CIC2019.

According to genomic data, toxin cyanobacteria production is likely as old as the group itself [...].

Are Cyanotoxins the Only Toxic Compound Potentially Present in Microalgae Supplements? Results from a Study of Ecological and Non-Ecological Products.

Food supplements with microalgae are becoming increasingly abundant and can be easily found anywhere. The most popular products are based on cyanophytes, such as Aphanizomenon flos-aquae, Arthrospira platensis and Limnospira maxima, or on chlorophytes, such as Chlorella or Haematoccus. Although they are all advertised as being very beneficial for health, these products might be harmful because they may contain cyanotoxins and other contaminants, and no information on production methods or strain origins is usually provided. While legislation on the presence of microcystins in waters for different uses is clear, toxicological analyses are not compulsory for food supplements, nor for analyzing anatoxins. Given the potential risk of eating contaminated food, cyanotoxins, heavy metals and the presence of other contaminant organisms were analyzed in 10 microalgae food supplements. Microcystin-LR and anatoxin-a were detected in three analyzed products, and in both cyanophyte- and chlorophyte-based products. The light microscope study revealed the presence of different potentially harmful microbial contaminants. The ICP (OES) analyses detected high concentrations of some heavy metals, especially Pb. The results emphasize the need to promote the better control of food products containing microalgae, and to develop standard methodologies to analyze cyanotoxins and potential toxic compounds to protect consumer health.

Are We Underestimating Benthic Cyanotoxins? Extensive Sampling Results from Spain.

Microcystins (MCs) are potent hepatotoxins, and their presence in water bodies poses a threat to wildlife and human populations. Most of the available information refers to plankton, and much less is known about microcystins in other habitats. To broaden our understanding of the presence and environmental distribution of this group of toxins, we conducted extensive sampling throughout Spain, under a range of conditions and in distinct aquatic and terrestrial habitats. More than half of the tested strains were toxic; concentrations of the hepatotoxin were low compared with planktic communities, and the number of toxic variants identified in each sample of the Spanish strains ranged from 1-3. The presence of microcystins LF and LY (MC-LF and MC-LY) in the tested samples was significant, and ranged from 21.4% to 100% of the total microcystins per strain. These strains were only detected in cyanobacteria Oscillatoriales and Nostocales. We can report, for the first time, seven new species of microcystin producers in high mountain rivers and chasmoendolithic communities. This is the first report of these species in Geitlerinema and the confirmation of Anatoxin-a in Phormidium uncinatum. Our findings show that microcystins are widespread in all habitat types, including both aerophytic and endolithic peat bogs and that it is necessary to identify all the variants of microcystins in aquatic bodies as the commonest toxins sometimes represent a very low proportion of the total.

Effects of global change factors on fatty acids and mycosporine-like amino acid production in Chroothece richteriana (Rhodophyta).

Under natural conditions, Chroothece richteriana synthesizes a fairly high proportion of fatty acids. However, nothing is known about how environmental changes affect their production, or about the production of protective compounds, when colonies develop under full sunshine with high levels of UV radiation. In this study, wild colonies of C. richteriana were subjected to increasing temperature, conductivity, ammonium concentrations and photosynthetically active radiation (PAR), and UV radiations to assess the potential changes in lipid composition and mycosporine-like amino acids (MAAs) concentration. The PERMANOVA analysis detected no differences for the whole fatty acid profile among treatments, but the percentages of α-linolenic acid and total polyunsaturated fatty acids increased at the lowest assayed temperature. The percentages of linoleic and α-linolenic acids increased with lowering temperature. γ-linolenic and arachidonic acids decreased with increasing conductivity, and a high arachidonic acid concentration was related with increased conductivity. The samples exposed to UVB radiation showed higher percentages of eicosapentaenoic acid and total monounsaturated fatty acids, at the expense of saturated fatty acids. MAAs accumulation increased but not significantly at the lowest conductivity, and also with the highest PAR and UVR exposure, while ammonium and temperature had no effect. The observed changes are probably related with adaptations of both membrane fluidity to low temperature, and metabolism to protect cells against UV radiation damage. The results suggest the potential to change lipid composition and MAAs concentration in response to environmental stressful conditions due to climate change, and highlight the interest of the species in future research about the biotechnological production of both compound types.

Microcystins and cyanophyte extracts inhibit or promote the photosynthesis of fluvial algae. Ecological and management implications.

The ecological influence of cyanotoxins on aquatic biota remains unclear despite the numerous published references on toxicological and sanitary problems related with cyanophyte proliferation. The effects of microcystins and cyanophyte extracts on the photosynthesis of the algae that belong to two taxonomic groups, Rhodophyta and Bacillariophyta, were studied in an attempt to elucidate their role in the intraspecific competence and physiognomy of fluvial communities. The data showed that both cyanobacteria extracts and pure microcystin-LR affected the photosynthetic activity of all the tested organisms, diatoms (Fistulifera pelliculosa, Gomphonema parvulum, Nitzschia frustulum and Stephanodiscus minutulus) and red algae (Chroothece richteriana) at environmentally relevant concentrations. Effects varied with strains and time, and promoted or inhibited photosynthesis. The microcystins and the other compounds present in cyanobacteria extracts may explain the competence effects observed in nature, especially in calcareous environments where they predominate, and after disturbing events like heavy rains or floods, which may destroy cyanophyte mats and release toxic or inhibitory compounds in a seasonal scale pattern.

Decoding cyanobacterial phylogeny and molecular evolution using an evonumeric approach.

Forty-one heterocystous cyanobacteria strains representing 12 cyanobacterial genera collected from all across India were assessed in phylogenetic and evolutionary perspectives. The structural gene 16S ribosomal RNA (rRNA) and the functional genes nifD and psbA were selected as molecular chronometers in this study. The phylogenetic analyses demonstrated the monophyly of heterocystous cyanobacteria with significant intermixing, along with establishing the polyphyly of Stigonematales, strongly supporting the need for re-amendments in cyanobacterial taxonomy and systematics. Molecular trends obtained did not clearly reflect the phenotypic affiliations, thus advocating for genetic characterizations using more molecular markers. Large-scale evonumeric extrapolations of gene sequence data of all the three molecular markers was performed to assess the evolutionary pace of heterocystous cyanobacteria on the basis of nucleotide diversity, recombination frequencies, and the DNA divergence between the sampled taxa. The obtained results tilted the evolutionary pace in favor of the less complex Nostocales thus indicating that possibly the simple non-branched forms are more flexible and adaptive for evolutionary diversifications as compared to the more complex and branched ones. This study hence represents a unique blend of molecular phylogeny with evogenomic sequence analyses for understanding the genetic diversity, phylogeny, and evolutionary pace within the heterocystous cyanobacteria.

The freshwater alga Chroothece richteriana (Rhodophyta) as a potential source of lipids.

During an ecological study of Chroothece (Rhodophyta) in a small river in a semi-arid region of south-east Spain it became clear that most of these cells had a high lipid content. This suggested potential uses in biotechnology, which has been investigated further. The colonies, which occur in full sunlight, are typically orange-brown. Most, perhaps all, the yellow-orange colour is associated with their high carotenoid content, with the carotenoid to chlorophyll ratio up to 2.7. The polyunsaturated fatty acyl composition of the glycerides was 35.3% of the dry weight. This consisted mainly of omega-3 (5.9%) and omega-6 (29.4%) fats. The relatively high proportion of docosahexaenoyl (1.78%), eicosapentaenoyl (14.15%), arachidonoyl (0.92%) and γ-linolenoyl (0.78%) suggests use for medical and dietary purposes. All cells have a high phycocyanin content whilst phycoerythrin is absent. The alga has a wide distribution globally and hence provides scope for selecting strains with optimum properties.

Environmental gradients and macroalgae in Mediterranean marshes: the case of Pego-Oliva marsh (East Iberian Peninsula).

Although Mediterranean marshes have historically suffered high anthropogenic pressure, they have maintained their remarkable biodiversity. They are severely threatened but remain comparatively unexplored systems from the algological point of view. For example, most of the indexes proposed for monitoring ecological quality are based on diatoms and very few have explored the use of macroalgae. The Pego-Oliva marsh is located in the east of the Iberian Peninsula close to the Mediterranean coast with warm annual temperature and fairly high precipitation. The aims of this study were to ascertain the ecological variables that explained macroalgal distribution in the Pego-Oliva marsh and to assess their indicator value. Macroalgal biodiversity was seen to be high (50 taxa) despite the high nitrogen concentration of the marsh. All the environmental variables studied had a broad range of variation throughout the marsh, especially conductivity (500-12290 µS/cm), temperature (14.3-31.7 °C), nitrate (9.493-64.113 mg/L) and ammonium (0.004-0.814 mg/L). A clear gradient of conductivity and dissolved oxygen was observed from fresh to saltwater. Batrachospermum arcuatum, Calothrix parietina, Chaetophora tuberculosa, Draparnaldia mutabilis, Hildenbrandia angolensis and Leptolyngbya angustissima were seen to act as indicators of low conductivity and dissolved inorganic nitrogen, and high dissolved oxygen, while Calothrix pulvinata, Ulva intestinalis, Homoeothrix violacea, Phormidium tergestinum and Thorea violacea were indicators of high conductivity and low dissolved nitrogen habitats. Cladophora glomerata, Compsopogon coeruleus, Polysiphonia subtilissima and Ulva flexuosa are the most widespread species and have a broad ecological range. Irrigation ditches have high ammonium and low dissolved oxygen concentrations and host infrequently reported species like Kumanoa mahlacensis. The data presented confirm the usefulness of macroalgae for the ecological monitoring of marshes, while increasing our knowledge of the distribution and ecological range of some species. However, more experimental work is needed to know the tolerance range of species living in dynamic systems like Mediterranean marshes.

Effects of living cyanobacteria, cyanobacterial extracts and pure microcystins on growth and ultrastructure of microalgae and bacteria.

In this study, we demonstrate the inhibitory effect of both cyanobacterial extracts and pure microcystins on the growth of microalgae and bacteria. This inhibitory effect was more persistent in pure microcystins than in the extracts, which lost their properties eight days after exposure. In addition, the effects on bacteria were longerlasting than those on microalgae. The microalgae exposed to both extracts and cultures of microcystin producing species showed morphological and ultrastructural alterations, even in cases where there was no clear effect on growth. The implications for colonisation and benthic communities structure and development are discussed in the context of biomonitoring.

Intracellular and dissolved microcystin in reservoirs of the river Segura basin, Murcia, SE Spain.

The seasonal sampling of irrigation or drinking water reservoirs in the province of Murcia (SE Spain) in the hydrological year 2000-2001 revealed the presence of intracellular microcystins in the benthic cyanophyte communities throughout the year. The total microcystin levels, as measured by HPLC, were relatively high but lower than others published for planktonic communities of the European continent or certain African countries. The diversity of forms was also very high and comparable to those found for other European countries. The concentrations of microcystins dissolved in the water were always below limits recommended by the WHO for drinking water and, during most of the year, undetectable by immunological methods. We discuss the need for setting up a control network for detecting benthic cyanobacteria growth to prevent long-term undesirable effects in the human population in small towns (through drinking water or the consumption of vegetables) and in wild animals. In the particular case of the Iberian Peninsula, a joint strategy between Portugal and Spain is recommendable.