A guide to the use of bioassays in exploration of natural resources

This publication is based upon work from COST Action CA18238 (Ocean4Biotech), supported by COST (European Cooperation in Science and Technology) program . Funding Information: Research of Dina Simes was funded by the Portuguese National Funds from FCT—Foundation for Science and Technology , through projects UIDB/04326/2020 , UIDP/04326/2020 and LA/P/0101/2020 and AAC n° 41/ALG/2020 - Project n° 072583 – NUTRISAFE. Funding Information: Research of Evita Strode was supported by ERDF post-doctoral research grant 1.1.1.2/16/I/001 (application No 1.1.1.2/VIAA/3/19/465). Funding Information: Susana P. Gaudêncio: This work is financed by national funds from FCT - Fundação para a Ciência e a Tecnologia , I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB . Funding Information: Research of Jerica Sabotič and Nika Janež was supported by Slovenian Research Agency ( J4- 2543 , J4-4555 , P4-0127 , P4-0432 ). Funding Information: Research of Anna Luganini and Giovanna Cristina Varese was financed by the University of Torino (Ricerca Locale) and the European Commission – NextGenerationEU , Project “Strengthening the MIRRI Italian Research Infrastructure for Sustainable Bioscience and Bioeconomy”, code n. IR0000005. Funding Information: Research of David Ezra was supported by The Chief Scientist of the Israeli Ministry of Agriculture and Rural Development (MOARD), grant number 20-02-0122 , and Copia Agro Israel. Publisher Copyright: © 2024 The AuthorsBioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purification/identification, and in later stages to evaluate their safety and efficacy. In this review, we provide a comprehensive overview of the most common bioassays used in the discovery and development of new bioactive compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial, antibiofilm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive compounds to a higher stage of development. We conclude by providing an application-oriented view focused on the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay results, as these serve as a starting point for application-based development and further testing, as well as for consideration by regulatory authorities.publishersversionpublishe

Specific Chemical and Genetic Markers Revealed a Thousands-Year Presence of Toxic Nodularia spumigena in the Baltic Sea

In the Baltic Sea, diazotrophic cyanobacteria have been present for thousands of years, over the whole brackish water phase of the ecosystem. However, our knowledge about the species composition of the cyanobacterial community is limited to the last several decades. In the current study, the presence of species-specific chemical and genetic markers in deep sediments were analyzed to increase the existing knowledge on the history of toxic Nodularia spumigena blooms in the Baltic Sea. As chemical markers, three cyclic nonribosomal peptides were applied: the hepatotoxic nodularin, which in the sea was detected solely in N. spumigena, and two anabaenopeptins (AP827 and AP883a) characteristic of two different chemotypes of this species. From the same sediment samples, DNA was isolated and the gene involved in biosynthesis of nodularin, as well as the phycocyanin intergenic spacer region (PC-IGS), were amplified. The results of chemical and genetic analyses proved for the first time the thousands-year presence of toxic N. spumigena in the Baltic Sea. They also indicated that through all this time, the same two sub-populations of the species co-existed

Morphologic, Phylogenetic and Chemical Characterization of a Brackish Colonial Picocyanobacterium (Coelosphaeriaceae) with Bioactive Properties

Despite their cosmopolitan distribution, knowledge on cyanobacteria in the family Coelosphaeriaceae is limited. In this study, a single species culture of a coelosphaeran cyanobacterium isolated from a brackish rock pool in the Baltic Sea was established. The strain was characterized by morphological features, partial 16S rRNA sequence and nonribosomal oligopeptide profile. The bioactivity of fractionated extracts against several serine proteases, as well as protein-serine/threonine phosphatases was studied. Phylogenetic analyses of the strain suggested a close relationship with Snowella litoralis, but its morphology resembled Woronichinia compacta. The controversial morphologic and phylogenetic results demonstrated remaining uncertainties regarding species division in this cyanobacteria family. Chemical analyses of the strain indicated production of nonribosomal oligopeptides. In fractionated extracts, masses and ion fragmentation spectra of seven possible anabaenopeptins were identified. Additionally, fragmentation spectra of cyanopeptolin-like peptides were collected in several of the fractions. The nonribosomal oligopeptide profile adds another potential identification criterion in future inter- and intraspecies comparisons of coelosphaeran cyanobacteria. The fractionated extracts showed significant activity against carboxypeptidase A and trypsin. Inhibition of these important metabolic enzymes might have impacts at the ecosystem level in aquatic habitats with high cyanobacteria densities

Chemical and Genetic Diversity of Nodularia spumigena from the Baltic Sea

Nodularia spumigena is a toxic, filamentous cyanobacterium occurring in brackish waters worldwide, yet forms extensive recurrent blooms in the Baltic Sea. N. spumigena produces several classes of non-ribosomal peptides (NRPs) that are active against several key metabolic enzymes. Previously, strains from geographically distant regions showed distinct NRP metabolic profiles. In this work, conspecific diversity in N. spumigena was studied using chemical and genetic approaches. NRP profiles were determined in 25 N. spumigena strains isolated in different years and from different locations in the Baltic Sea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genetic diversity was assessed by targeting the phycocyanin intergenic spacer and flanking regions (cpcBA-IGS). Overall, 14 spumigins, 5 aeruginosins, 2 pseudaeruginosins, 2 nodularins, 36 anabaenopeptins, and one new cyanopeptolin-like peptide were identified among the strains. Seven anabaenopeptins were new structures; one cyanopeptolin-like peptide was discovered in N. spumigena for the first time. Based on NRP profiles and cpcBA-IGS sequences, the strains were grouped into two main clusters without apparent influence of year and location, indicating persistent presence of these two subpopulations in the Baltic Sea. This study is a major step in using chemical profiling to explore conspecific diversity with a higher resolution than with a sole genetic approach

Linking environmental heterogeneity and chemo-diversity in cyanobacteria: A culture-dependent profile based analysis

Cyanobacteria are ecologically versatile microorganisms that inhabit most environments, ranging from marine systems to arid deserts. Given their ability to survive under harsh and extreme conditions, we hypothesize that cyanobacteria could produce a wide variety of compounds in specific niches. In this context, we sampled a number of different environments, from freshwater and brackish ecosystems to terrestrial and anchialine caves, spanning from the Canary Islands and Iceland to Estonia and Greece. Forty-four (44) cyanobacteria strains were analyzed with de novo peptide fragmentation in order to detect their metabolome profile; further, their antimicrobial, cytotoxic, and enzyme inhibitory activity was investigated. Both freshwater/planktic and rock-dwelling/benthic strains exhibited different types of inhibitory activities. However, cyanopeptides were only detected in freshwater strains; microcystins, anabaenopeptins, and aeruginosins congeners from Microcystis spp., Thrichormus variabilis, and Calothrix epiphytica strains. Therefore, our results indicate a high degree of unknown chemo-diversity, as we could not link the presence/absence of any known cyanopeptides and inhibitory activities from strains derived from other habitats, in contrast with freshwater cyanobacteria strains. In this work we discuss the correlation between the cyanobacteria chemo- and lifestyle diversity providing a missing study material for profile-based analysis on cyanobacteria from under-explored environments

Characterization and diversity of microcystins produced by cyanobacteria from the Curonian Lagoon (SE Baltic Sea)

Microcystins (MCs) are the most widely distributed and structurally diverse cyanotoxins that can have significant health impacts on living organisms, including humans. The identification of MC variants and their quantification is very important for toxicological assessment. Within this study, we explored the diversity of MCs and their potential producers from the Curonian Lagoon. MC profiles were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, while the potential producers were detected based on the presence of genus-specific mcyE gene sequences. Among the numerous MCs detected, one new potential MC variant with m/z 1057 was partially characterized. Moreover, two other MCs with m/z 1075 and m/z 1068 might belong to new variants with serine (Ser), rarely detected in position one of the peptides. They might also represent MC-Y(OMe)R and MC-WR, respectively. However, the application of a low-resolution MS/MS system made the unambiguous identification of the MCs impossible. Based on this example, the problems of peptide structure identification are discussed in the work. Genetic analysis revealed that potential MCs producers include Dolichospermum/Anabaena, Microcystis spp., and Planktothrix agardhii. The diversity and temporal variations in MC profiles may indicate the presence of several chemotypes of cyanobacteria in the Curonian Lagoon

Phytoplankton of the Curonian Lagoon as a new interesting source for bioactive natural products. Special impact on cyanobacterial metabolites

The bioprospecting of marine and brackish water systems has increased during the last decades. In this respect, microalgae, including cyanobacteria, and their metabolites are one of the most widely explored resources. Most of the bioactive compounds are isolated from ex situ cultures of microorganisms; however, analysis of field samples could also supply valuable information about the metabolic and biotechnological potential of microalgae communities. In this work, the activity of phytoplankton samples from the Curonian Lagoon was studied. The samples were active against antibiotic resistant clinical and environmental bacterial strains as well as against serine proteases and T47D human breast adenocarcinoma cells. No significant effect was found on Daphnia magna. In addition, using LC-MS/MS, we documented the diversity of metabolites present in field samples. A list of 117 detected cyanopeptides was presented. Cyanopeptolins constituted the largest class of cyanopeptides. As complex bloom samples were analyzed, no link between the observed activity and a specific sample component can be established. However, the results of the study showed a biotechnological potential of natural products from the Curonian Lagoon

Blooms of toxic cyanobacterium Nodularia spumigena in Norwegian fjords during Holocene warm periods

In paleoecological studies, molecular markers are being used increasingly often to reconstruct community structures, environmental conditions and ecosystem changes. In this work, nodularin, anabaenopeptins and selected DNA sequences were applied as Nodularia spumigena markers to reconstruct the history of the cyanobacterium in the Norwegian fjords. For the purpose of this study, three sediment cores collected in Oslofjorden, Trondheimsfjorden and Balsfjorden were analyzed. The lack of nodularin in most recent sediments is consistent with the fact that only one report on the sporadic occurrence and low amounts of the cyanobacterium in Norwegian Fjords in 1976 has been published. However, analyses of species-specific chemical markers in deep sediments showed that thousands of years ago, N. spumigena constituted an important component of the phytoplankton community. The content of the markers in the cores indicated that the biomass of the cyanobacterium increased during the warmer Holocene periods. The analyses of genetic markers were less conclusive; they showed the occurrence of microcystin/nodularin producing cyanobacteria of Nostocales order, but they did not allow for the identification of the organisms at a species level

Effect of Increased Temperature on Native and Alien Nuisance Cyanobacteria from Temperate Lakes: An Experimental Approach

In response to global warming, an increase in cyanobacterial blooms is expected. In this work, the response of two native species of Planktothrix agardhii and Aphanizomenon gracile, as well as the response of two species alien to Europe—Chrysosporum bergii and Sphaerospermopsis aphanizomenoides—to gradual temperature increase was tested. The northernmost point of alien species distribution in the European continent was recorded. The tested strains of native species were favoured at 20⁻28 °C. Alien species acted differently along temperature gradient and their growth rate was higher than native species. Temperature range of optimal growth rate for S. aphanizomenoides was similar to native species, while C. bergii was favoured at 26⁻30 °C but sensitive at 18⁻20 °C. Under all tested temperatures, non-toxic strains of the native cyanobacteria species prevailed over the toxic ones. In P. agardhii, the decrease in concentration of microcystins and other oligopeptides with the increasing temperature was related to higher growth rate. However, changes in saxitoxin concentration in A. gracile under different temperatures were not detected. Accommodating climate change perspectives, the current work showed a high necessity of further studies of temperature effect on distribution and toxicity of both native and alien cyanobacterial species

First report of saxitoxins and anatoxin-a production by cyanobacteria from Lithuanian lakes

This study describes the first report on saxitoxins (STXs) and anatoxin-a (ANTX-a) production by cyanobacteria in Lithuanian lakes. Seasonal fluctuation patterns for neurotoxins were determined in samples collected from eutrophic lakes during July–September. Strains of Aphanizomenon gracile, A. flos-aquae, Cuspidothrix issatschenkoi, Sphaerospermopsis aphanizomenoides and Anabaenopsis cf. elenkinii were also isolated from the lakes and screened for the occurrence of neurotoxic STXs, ANTX-a and for the cytotoxin cylindrospermopsin (CYN). The highest concentration of STX (up to 1.06 µg l–1 in Lake Jieznas) was detected during July‒August and coincided with a predominance of A. gracile in the phytoplankton. Of the isolated strains, only A. gracile was confirmed to be a producer of STXs by chemical (LC-MS/MS) and genetic (sxtA) methods. Sequencing of the phycocyanin intergenic spacer and flanking regions (cpcBA) revealed that A. gracile formed a genetically homogenous group. Only trace amounts of ANTX-a were detected in field samples (up to 0.31 µg l–1 in Lake Širvys). CYN was detected in neither the bloom samples nor in the isolated strains