Ricardo Calado

Trends in the Discovery of New Marine Natural Products from Invertebrates over the Last Two Decades – Where and What Are We Bioprospecting?

It is acknowledged that marine invertebrates produce bioactive natural products that may be useful for developing new drugs. By exploring untapped geographical sources and/or novel groups of organisms one can maximize the search for new marine drugs to treat human diseases. The goal of this paper is to analyse the trends associated with the discovery of new marine natural products from invertebrates (NMNPI) over the last two decades. The analysis considers different taxonomical levels and geographical approaches of bioprospected species. Additionally, this research is also directed to provide new insights into less bioprospected taxa and world regions. In order to gather the information available on NMNPI, the yearly-published reviews of Marine Natural Products covering 1990–2009 were surveyed. Information on source organisms, specifically taxonomical information and collection sites, was assembled together with additional geographical information collected from the articles originally describing the new natural product. Almost 10000 NMNPI were discovered since 1990, with a pronounced increase between decades. Porifera and Cnidaria were the two dominant sources of NMNPI worldwide. The exception was polar regions where Echinodermata dominated. The majority of species that yielded the new natural products belong to only one class of each Porifera and Cnidaria phyla (Demospongiae and Anthozoa, respectively). Increased bioprospecting efforts were observed in the Pacific Ocean, particularly in Asian countries that are associated with the Japan Biodiversity Hotspot and the Kuroshio Current. Although results show comparably less NMNPI from polar regions, the number of new natural products per species is similar to that recorded for other regions. The present study provides information to future bioprospecting efforts addressing previously unexplored taxonomic groups and/or regions. We also highlight how marine invertebrates, which in some cases have no commercial value, may become highly valuable in the ongoing search for new drugs from the sea.

MARINE ORNAMENTAL DECAPODS—Popular, Pricey, And Poorly Studied

Abstract The growing demand for highly priced marine ornamental species has contributed to the endangered status of coral reefs. A list of 128 of the most heavily traded marine ornamental decapod crustacean species is tabulated. The development of commercial culture techniques, the knowledge of the larval development, and the association with vertebrate and invertebrate organisms are presented for these species. Forty-nine of the species are caridean shrimp, with the Hippolytidae family alone accounting for 15. Anomuran and brachyuran crabs are the next most traded groups (32 and 27 species, respectively), with the pricey stenopodidean shrimp, Astacidea, and Palinura lobsters being represented by a considerably lower number of species (7, 7 and 6, respectively). The main bottlenecks impairing the commercial culture of ornamental shrimp and lobsters are their long larval development and poor survival rates. The main constraint for the development of culture techniques for hermit and brachyuran crabs is their low commercial value. The ecological impacts of harvesting ornamental species are still poorly studied. Nevertheless, the collection in considerable numbers of hermit and small majid crabs (e.g., Clibanarius and Mithraculus) from tidal areas, fish cleaning shrimp (e.g., Lysmata and Stenopus), and the crown-of-thorns sea star eaters Hymenocera, is likely to have serious impacts on the ecosystem. The cooperation between researchers working on larval biology, population dynamics, ecology, aquaculture, and fisheries is essential to properly manage the collection of marine ornamental decapods.

A rearing system for the culture of ornamental decapod crustacean larvae

Abstract The design and operation of a small research scale and a mass commercial scale rearing system for the culture of marine ornamental decapod crustacean larvae are described in the present paper. Preliminary data on the culture of the Mediterranean cleaner shrimp ( Lysmata seticaudata ), peppermint shrimp ( Lysmata wurdemanni ), blue-white partner shrimp ( Periclimenes sagittifer ), sponge crab ( Cryptodromiopsis antillensis ) and green emerald crab ( Mithraculus sculptus ) are also presented. The use of these “plantonkreisel” based systems allowed the complete larval development of the above-mentioned species, inducing minimal mechanical stress while keeping an excellent water quality. Higher survival rates (up to 70% and 60% for L. seticaudata and L. wurdemanni , respectively) to the post-larval stage and a shorter larval stage duration (27 and 22 days for L. seticaudata and L. wurdemanni , respectively) were achieved, in comparison to conventional rearing systems. This culture technology may play a key role in the realisation of a commercial culture of these highly priced crustacean species and therefore the reduction of wild specimen collection.

Biochemical changes during the embryonic development of Norway lobster, Nephrops norvegicus

The objectives of the present study were to investigate the total and free amino acid profiles and lipid dynamics (lipid classes and fatty acids) during embryogenesis of Nephrops norvegicus, in order to understand the early larval protein and lipid requirements. There was a significant increase in total essential (EAA) and nonessential amino acid (NEAA) contents during embryonic development (P<0.05). The major EAA were arginine, histidine and leucine, while the most important NEAA were glutamic acid, aspartic acid, glycine and proline. The higher percent increase occurred in respect to NEAA (19.9%), mainly due to the significant increase of glycine (51.8%) and alanine (35.1%). The free amino acid (FAA) content, especially the free nonessential amino acids (FNEAA), also increased significantly, being the quantitatively most important glycine, proline, taurine and glutamic acid. The free essential amino acids (FEAA) also revealed significant variations and the most important were arginine, lysine and leucine. A higher percent increase was attained with FNEAA (66.8%) in comparison to FEAA (49.6%), mainly due to the significant increase of homocystine (87.7%), valine (83.5%), glutamine (82.5%) and glutamic acid (76.1%). The absorption of dissolved organic compounds from seawater can explain the increase in free and total amino acid (FAA and TAA) contents, because a significant increase in the water content during development was observed (P<0.05). A substantial decrease in all neutral lipid classes (P<0.05) was observed during embryonic development, namely diacylglycerols (DAG) (97.9% of utilization), triacylglycerols (TAG) (93.2%), sterol esters (StE) (91.3%) and monoacylglycerols (MAG) (90.4%). The quantitatively most important fatty acids were the saturates (SFA) 14:0, 16:0 and 18:0, the monounsaturates (MUFA) 16:1n-7, 18:1n-9 and 18:1n-7, and the polyunsaturates (PUFA) 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3. The unsaturated fatty acids (UFA) are used up at a higher rate (54.5% of utilization) than SFA (42.8%); within the UFA, MUFA are more consumed than PUFA (59.3% and 52.7%, respectively). In terms of the utilization of individual fatty acids, there was a preferential consumption of 20:4n-3, 22:6n-3, 18:1n-9, 22:5n-3 and 16:1n-7. It is evident that N. norvegicus depends primarily on lipid reserves of the egg during early ontogeny. On the contrary, this species tends to conserve EAA and increase NEAA and FNEAA contents during embryonic development.

Differential impacts of ocean acidification and warming on winter and summer progeny of a coastal squid (Loligo vulgaris)

Little is known about the capacity of early life stages to undergo hypercapnic and thermal acclimation under the future scenarios of ocean acidification and warming. Here, we investigated a comprehensive set of biological responses to these climate change-related variables (2°C above winter and summer average spawning temperatures and ΔpH=0.5 units) during the early ontogeny of the squid Loligo vulgaris. Embryo survival rates ranged from 92% to 96% under present-day temperature (13–17°C) and pH (8.0) scenarios. Yet, ocean acidification (pH 7.5) and summer warming (19°C) led to a significant drop in the survival rates of summer embryos (47%, P<0.05). The embryonic period was shortened by increasing temperature in both pH treatments (P<0.05). Embryo growth rates increased significantly with temperature under present-day scenarios, but there was a significant trend reversal under future summer warming conditions (P<0.05). Besides pronounced premature hatching, a higher percentage of abnormalities was found in summer embryos exposed to future warming and lower pH (P<0.05). Under the hypercapnic scenario, oxygen consumption rates decreased significantly in late embryos and newly hatched paralarvae, especially in the summer period (P<0.05). Concomitantly, there was a significant enhancement of the heat shock response (HSP70/HSC70) with warming in both pH treatments and developmental stages. Upper thermal tolerance limits were positively influenced by acclimation temperature, and such thresholds were significantly higher in late embryos than in hatchlings under present-day conditions (P<0.05). In contrast, the upper thermal tolerance limits under hypercapnia were higher in hatchlings than in embryos. Thus, we show that the stressful abiotic conditions inside the embryos capsules will be exacerbated under near-future ocean acidification and summer warming scenarios. The occurrence of prolonged embryogenesis along with lowered thermal tolerance limits under such conditions is expected to negatively affect the survival success of squid early life stages during the summer spawning period, but not winter spawning.

Bioprospecting of Marine Invertebrates for New Natural Products — A Chemical and Zoogeographical Perspective

Bioprospecting for new marine natural products (NPs) has increased significantly over the last decades, leading to an unprecedented discovery of new molecules. Marine invertebrates have been the most important source of these NPs, with researchers commonly targeting particular taxonomic groups, marine regions and/or molecules from specific chemical groups. The present review focuses on new NPs identified from marine invertebrates between 2000 and 2009, and performs a detailed analysis on: (1) the chemical groups of these NPs; (2) the association of particular chemical groups to specific marine invertebrate taxa; and (3) the yielding of molecules from the same chemical group from organisms occurring in a particular geographic region. Our survey revealed an increasing number of new terpenoids being discovered between 2000 and 2009, contrasting with the decreasing trend in the discovery of new alkaloids and aliphatic molecules. Overall, no particular association was identified between marine invertebrate taxa and chemical groups of new NPs. Nonetheless, it is worth noting that most NPs recorded from cnidarians and mollusks were terpenoids, while most NPs identified in echinoderms were aliphatic compounds or carbohydrates. The geographical trends observed in our study do not support the idea of particular chemical groups of new NPs being associated with marine invertebrates from any specific geographical region, as NPs from different chemical groups were commonly distributed worldwide.

Seafood traceability: current needs, available tools, and biotechnological challenges for origin certification

Market globalization and recurring food safety alerts have resulted in a growing consumer awareness of the need for food traceability. This is particularly relevant for seafood due to its perishable nature and importance as a key protein source for the population of the world. Here, we provide an overview of the current needs for seafood origin traceability, along with the limitations and challenges for its implementation. We focus on geochemical, biochemical, and molecular tools and how they should be optimized to be implemented globally and to address our societal needs. We suggest that seafood traceability is key to enforcing food safety regulations and fisheries control, combat fraud, and fulfill present and future expectations of conscientious producers, consumers, and authorities.

Phage therapy as an approach to prevent Vibrio anguillarum infections in fish larvae production.

Fish larvae in aquaculture have high mortality rates due to pathogenic bacteria, especially the Vibrio species, and ineffective prophylactic strategies. Vaccination is not feasible in larvae and antibiotics have reduced efficacy against multidrug resistant bacteria. A novel approach to controlling Vibrio infections in aquaculture is needed. The potential of phage therapy to combat vibriosis in fish larvae production has not yet been examined. We describe the isolation and characterization of two bacteriophages capable of infecting pathogenic Vibrio and their application to prevent bacterial infection in fish larvae. Two groups of zebrafish larvae were infected with V. anguillarum (∼106 CFU mL−1) and one was later treated with a phage lysate (∼108 PFU mL−1). A third group was only added with phages. A fourth group received neither bacteria nor phages (fish control). Larvae mortality, after 72 h, in the infected and treated group was similar to normal levels and significantly lower than that of the infected but not treated group, indicating that phage treatment was effective. Thus, directly supplying phages to the culture water could be an effective and inexpensive approach toward reducing the negative impact of vibriosis in larviculture.

Molecular analysis of bacterial communities and detection of potential pathogens in a recirculating aquaculture system for Scophthalmus maximus and Solea senegalensis.

The present study combined a DGGE and barcoded 16S rRNA pyrosequencing approach to assess bacterial composition in the water of a recirculating aquaculture system (RAS) with a shallow raceway system (SRS) for turbot (Scophthalmus maximus) and sole (Solea senegalensis). Barcoded pyrosequencing results were also used to determine the potential pathogen load in the RAS studied. Samples were collected from the water supply pipeline (Sup), fish production tanks (Pro), sedimentation filter (Sed), biofilter tank (Bio), and protein skimmer (Ozo; also used as an ozone reaction chamber) of twin RAS operating in parallel (one for each fish species). Our results revealed pronounced differences in bacterial community composition between turbot and sole RAS, suggesting that in the systems studied there is a strong species-specific effect on water bacterial communities. Proteobacteria was the most abundant phylum in the water supply and all RAS compartments. Other important taxonomic groups included the phylum Bacteriodetes. The saltwater supplied displayed a markedly lower richness and appeared to have very little influence on bacterial composition. The following potentially pathogenic species were detected: Photobacterium damselae in turbot (all compartments), Tenacibaculum discolor in turbot and sole (all compartments), Tenacibaculum soleae in turbot (all compartments) and sole (Pro, Sed and Bio), and Serratia marcescens in turbot (Sup, Sed, Bio and Ozo) and sole (only Sed) RAS. Despite the presence of these pathogens, no symptomatic fish were observed. Although we were able to identify potential pathogens, this approach should be employed with caution when monitoring aquaculture systems, as the required phylogenetic resolution for reliable identification of pathogens may not always be possible to achieve when employing 16S rRNA gene fragments.

Applicability of photodynamic antimicrobial chemotherapy as an alternative to inactivate fish pathogenic bacteria in aquaculture systems

Aquaculture activities are increasing worldwide, stimulated by the progressive reduction of natural fish stocks in the oceans. However, these activities also suffer heavy production and financial losses resulting from fish infections caused by microbial pathogens, including multidrug resistant bacteria. Therefore, strategies to control fish infections are urgently needed, in order to make aquaculture industry more sustainable. Antimicrobial photodynamic therapy (aPDT) has emerged as an alternative to treat diseases and prevent the development of antibiotic resistance by pathogenic bacteria. The aim of this work was to evaluate the applicability of aPDT to inactivate pathogenic fish bacteria. To reach this objective a cationic porphyrin Tri-Py(+)-Me-PF was tested against nine pathogenic bacteria isolated from a semi-intensive aquaculture system and against the cultivable bacteria of the aquaculture system. The ecological impact of aPDT in the aquatic environment was also tested on the natural bacterial community, using the overall bacterial community structure and the cultivable bacteria as indicators. Photodynamic inactivation of bacterial isolates and of cultivable bacteria was assessed counting the number of colonies. The impact of aPDT in the overall bacterial community structure of the aquaculture water was evaluated by denaturing gel gradient electrophoresis (DGGE). The results showed that, in the presence of Tri-Py(+)-Me-PF, the growth of bacterial isolates was inhibited, resulting in a decrease of ≈7-8 log after 60-270 min of irradiation. Cultivable bacteria were also considerably affected, showing decreases up to the detection limit (≈2 log decrease on cell survival), but the inactivation rate varied significantly with the sampling period. The DGGE fingerprint analyses revealed changes in the bacterial community structure caused by the combination of aPDT and light. The results indicate that aPDT can be regarded as a new approach to control fish infections in aquaculture systems, but it is clearly more difficult to inactivate the complex natural bacterial communities of aquaculture waters than pure cultures of bacteria isolated from aquaculture systems. Considering the use of aPDT to inactivate pathogenic microbial community of aquaculture systems the monitoring of microorganisms is needed in order to select the most effective conditions.