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

Bioassays 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.

Comparative Chemical Profiling and Antimicrobial/Anticancer Evaluation of Extracts from Farmed versus Wild Agelas oroides and Sarcotragus foetidus Sponges.

Marine sponges are highly efficient in removing organic pollutants and their cultivation, adjacent to fish farms, is increasingly considered as a strategy for improving seawater quality. Moreover, these invertebrates produce a plethora of bioactive metabolites, which could translate into an extra profit for the aquaculture sector. Here, we investigated the chemical profile and bioactivity of two Mediterranean species (i.e., Agelas oroides and Sarcotragus foetidus) and we assessed whether cultivated sponges differed substantially from their wild counterparts. Metabolomic analysis of crude sponge extracts revealed species-specific chemical patterns, with A. oroides and S. foetidus dominated by alkaloids and lipids, respectively. More importantly, farmed and wild explants of each species demonstrated similar chemical fingerprints, with the majority of the metabolites showing modest differences on a sponge mass-normalized basis. Furthermore, farmed sponge extracts presented similar or slightly lower antibacterial activity against methicillin-resistant Staphylococcus aureus, compared to the extracts resulting from wild sponges. Anticancer assays against human colorectal carcinoma cells (HCT-116) revealed marginally active extracts from both wild and farmed S. foetidus populations. Our study highlights that, besides mitigating organic pollution in fish aquaculture, sponge farming can serve as a valuable resource of biomolecules, with promising potential in pharmaceutical and biomedical applications.

Uptake of aquaculture-related dissolved organic pollutants by marine sponges: Kinetics and mechanistic insights from a laboratory study.

Besides the release of organic matter from uneaten feed and fish excreta, a considerable amount of deleterious chemicals may also end up into the marine environment from intensive aquaculture. A fraction of these pollutants remains freely dissolved and pose a threat to marine life due to increased bioavailability. Given the filter-feeding ability of sponges, we investigated the capacity of four ubiquitous Mediterranean species (Agelas oroides, Axinella cannabina, Chondrosia reniformis and Sarcotragus foetidus) in removing aquaculture-related dissolved organic pollutants. These included individual chemicals belonging to antibiotics (i.e., oxytetracycline), antifouling biocides (i.e., diuron and Irgarol 1051) and polycyclic aromatic hydrocarbons (i.e., 2,6-dimethylnapththalene, phenanthrene). The uptake of pollutants was assessed in vitro by exposing small sponge explants to each chemical for a period of 8 h. Additional "cleanup" experiments were performed for complex mixtures mimicking the dissolved organic material encountered in fish farms, such as filtrates of fish feed and excreta. All sponges exhibited a pronounced preference for lipophilic pollutants and a strong positive correlation was revealed between clearance rate and substrate hydrophobicity. Our best filter-feeder (i.e., A. oroides) was able to clear 10.0 ± 1.3 mL of seawater per hour and per gram of sponge, when exposed to 2,6-dimethylnapththalene. Active pumping was found to be the predominant mechanism dictating the assimilation of dissolved pollutants in all sponge species, as it was 3-10 times faster than pollutants' passive adsorption on sponges' pinacoderm. Additionally, the uptaken pollutants were shown to be strongly retained by sponges and they were hardly released back to seawater as a result of desorption or sponge excretory mechanisms. Our study corroborates that sponges are highly efficient in uptaking dissolved organic compounds and it offers new insights into the kinetics and mechanisms ruling this process.

A Multi-Species Investigation of Sponges' Filtering Activity towards Marine Microalgae.

Chronic discharge of surplus organic matter is a typical side effect of fish aquaculture, occasionally leading to coastal eutrophication and excessive phytoplankton growth. Owing to their innate filter-feeding capacity, marine sponges could mitigate environmental impact under integrated multitrophic aquaculture (IMTA) scenarios. Herein, we investigated the clearance capacity of four ubiquitous Mediterranean sponges (Agelas oroides, Axinella cannabina, Chondrosia reniformis and Sarcotragus foetidus) against three microalgal substrates with different size/motility characteristics: the nanophytoplankton Nannochloropsis sp. (~3.2 µm, nonmotile) and Isochrysis sp. (~3.8 µm, motile), as well as the diatom Phaeodactylum tricornutum (~21.7 µm, nonmotile). In vitro cleaning experiments were conducted using sponge explants in 1 L of natural seawater and applying different microalgal cell concentrations under light/dark conditions. The investigated sponges exhibited a wide range of retention efficiencies for the different phytoplankton cells, with the lowest average values found for A. cannabina (37%) and the highest for A. oroides (70%). The latter could filter up to 14.1 mL seawater per hour and gram of sponge wet weight, by retaining 100% of Isochrysis at a density of 105 cells mL-1, under darkness. Our results highlight differences in filtering capacity among sponge species and preferences for microalgal substrates with distinct size and motility traits.

Utilization of lignocellulosic biomass towards the production of omega-3 fatty acids by the heterotrophic marine microalga Crypthecodinium cohnii.

Omega-3 fatty acids have become a commodity of high nutritional and commercial value; intensive fishing and its environmental and social cost has led researchers to seeking alternative more sustainable ways of producing them. Heterotrophic microalgae such as Crypthecodinium cohnii, a marine dinoflagellate, have the ability to utilize various substrates and accumulate high amounts of docosahexaenoic acid (DHA). In this work, a mild oxidative organosolv pretreatment of beechwood pulps was employed that allowed up to 95% of lignin removal in a single stage, thus yielding a cellulose-rich solid fraction. The enzymatic hydrolysates were evaluated for their ability to support the growth and lipid accumulation of C. cohnii in batch and fed-batch cultures; the results verified the successful microalgae growth, while DHA reached up to 43.5% of the cell's total lipids. The proposed bioprocess demonstrated the utilization of non-edible biomass towards high added value food supplements in a sustainable and efficient manner.