Food supplements containing mussel extracts are becoming popular in human diet, providing high levels of proteins, omega-3 polyunsatured fatty acid (PUFAs), iodine and carbohydrates [1, 2]. Besides the beneficial effects and bioactives that mussels may yield, it is vital to consider the potential harmful phycotoxins that can be present in mussel extracts and marine dietary supplements. Recently, we have detected for the first time the marine toxin 13-desmethyl spirolide C in food supplements containing green lipped mussels of Perna canaliculus at levels up to 98 µg/kg [3]. In this work, we provide new data about the presence of pinnatoxin-G (trace amounts) in the dietary supplements intended for human consumption after the analysis of the green lipped mussel powder by UPLC-MS/MS. Moreover, the status of microalgae phycotoxin contaminants is also assessed in these products and in animal dietary supplements which contained 13-desmethyl spirolide C at levels up to 39 µg/kg. The mechanism of action of spirolides and pinnatoxins is associated with the blockage of the muscarinic and nicotinic receptors (mAChR and nAChR) on the nervous system. Despite the fact that human intoxications have not been reported, it is important to identify the impact of such toxins on public health since dietary products constitute an important part of the global market.

References:

[1] C.S. Cobb, E. Ernst, Systematic review of a marine nutriceutical supplemnt in clinical trials for arthritis: The effectiveness of the New Zealand green-lipped mussel Perna canaliculus, Clin. Rheumatol., 25 (2006) 275-284.

[2] A. Treschow, L. Hodges, P. Wright, P. Wynne, N. Kalafatis, T. Macrides, Novel Anti-Inflammatory omega-3 PUFAs From the New Zealand Green-Lipped Mussel, Perna Canaliculus, Comp Biochem Physiol B Biochem Mol Biol., 147 (2007) 645-656.

[3] P. Otero, C. Vale, A. Boente-Juncal, C. C., M.C. Louzao, L. Botana, Detection of Cyclic Imine Toxins in Dietary Supplements of Green Lipped Mussels (Perna canaliculus) and in Shellfish Mytilus chilensis Toxins, 12(10) (2020) 613.

Bacillus thuringiensis is a gram-positive and spore-forming bacterium that synthesizes a wide diversity of proteins with insecticidal activity and which has demonstrated its potential and safety as a biocontrol agent for more than four decades. However, several susceptible insect species have been reported for evolving resistance, which demands screening for strains exhibiting novel insecticidal properties. In this work, we performed the genome sequence analysis and the insecticidal characterization of B. thuringiensis strain Bt-UNVM_94 isolated from Argentina. This strain produced quasi symmetric bipyramidal parasporal crystals as shown using Scanning Electron Microscopy. Its genomic sequence harbours one coding sequence showing homology to the crystal toxin Cry7Ga2 and another, with similarity to the Mpp2Aa3 (Mtx2) toxin. Known Cry7A and Cry7B are known to be active against some coleopteran and lepidopteran larvae, respectively. However, bioassays performed with spore-crystal mixtures of strain Bt-UNVM_94 exhibited dual toxicity with 50% and 91% mortality against Cydia pomonella (Lepidoptera: Tortricidae) and Anthonomus grandis (Coleoptera: Curculionidae), respectively. No toxicity was detected against the free-living nematode Panagrellus redivivus (Rhabditidae: Panagrolaimidae). This strain also showed no PCR amplification of the type I b-exotoxin thuE gene, consistent with the absence of mortality in b-exotoxin bioassays with Musca domestica (Diptera: Muscidae). Screenings of novel B. thuringiensis strains may provide toxins with novel insecticidal properties that can be used to suppress insect resistance to the most used B. thuringiensis-crops in the field.

Entomopathogenic nematodes belonging to the genus Steinernema are able to infest and kill insect hosts in association with their resident, entomopathogenic symbiont bacteria in the gram-negative genus Xenorhabdus (Enterobacteriaceae). However, only a few species of Xenorhabdus have been isolated from their hosts and their insecticidal properties reported. Here we performed the genome sequence analysis of 14 Xenorhabdus strains isolated from Steinernema nematodes in Argentina, able to kill 6^th instar Galleria mellonella (Lepidoptera) larvae. The 14 draft genome sequences encoded 110 putative insecticidal toxins (Tc, Pir and Mcf toxin homologs) plus other virulence factors with similarity to putative nematicidal toxins and chitinases. The genome sequences of strains Flor, 5, PSL, Reich, 42, Vera, M, 18, Cul, DI, 12, 38, 3 and ZM exhibited 4, 9, 2, 10, 9, 5, 7, 10, 10, 7, 3, 18, 8 and 8 putative insecticidal toxin genes, respectively. Strains 5, Reich, 42, 18, DI, 38, 3 and ZM carried their predicted insecticidal toxin genes arranged into putative pathogenicity islands whereas strains Flor, PSL, Vera, M, Cul and 12) showed them spread thorough different contigs. Multigene phylogenetic analysis and average nucleotide identity (ANI) calculations were also performed and allowed the identification of three strains (PSL, Reich and 12) that should be considered as novel Xenorhabdus genomospecies. In this work, we provide a dual insight into the diversity of the species belonging to the Xenorhabdus genus and into their predicted insecticidal toxin repertory, which is currently under investigation.

Snake venom Cysteine-Rich Secretory Proteins (svCRiSPs) are important components in the venom of many species of snakes including Viperidae and Elapidae. Although the widespread distribution of svCRiSPs in snake venoms is well known, little is known of the contribution that they make to the local pathophysiology of snakebite. This work aimed to investigate the role of svCRiSPs from the most medically significant species of North American snakes (Crotalus atrox, C. adamanteus, C. scutulatus scutulatus, C. horridus, and Agkistrodon piscivorus), focusing on the cellular and molecular mechanisms underlying vascular biology in snakebite. We evaluated the biological activities of svCRiSPs (Css-CRiSP, Catrox-CRiSP, Cada-CRiSP, Chor-CRiSP, and App-CRiSP) by using both in vitro assays on human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs) permeability and in vivo Miles assay of vascular permeability. Of all the CRiSPs tested, Css-CRiSP and App-CRiSP displayed the highest increase in acute vascular permeability compared to other crotaline CRiSPs. To elucidate the main pathway underlying the endothelial permeability induced by svCRiSPs, we initially screened the changes in protein expression and phosphorylation in HDLECs and HDBECs 30 min after treatment with Css-CRiSP and App-CRiSP using reverse phase protein arrays (RPPA). Stimulating HDBECs with Css-CRiSP and App-CRiSP enhanced caveolin-1 expression. In HDLEC cells, Css-CRiPS and App-CRiSP increased the upregulation of the expression of proteins involved in the phosphoinositide 3-kinase/Akt, Src, MAPK/JNK pathways, cell mobility, and cell adhesion molecules. These preliminary observations suggest that Css-CRiSP and App-CRiSP induce the increased endothelial monolayer permeability in HDBECs and HDLECs via different mechanisms. Knowledge gained from these studies provides insights into the molecular mechanisms that underlie the effects of svCRiSPs on vascular function and contributes to a new level of understanding of the pathophysiology of snakebite.

Food and feed are frequently contaminated by numerous regulated and emerging mycotoxins. Humans and animals are thus exposed daily to mycotoxins through the oral route making of the gut the first and the more exposed tissue. Although many studies have evaluated and demonstrated the impact of mycotoxins on the intestinal epithelial cells (IECs) and on the brain cells, surprisingly only few studies have investigated their impact on cells of the enteric nerve system (ENS). In the present work, we measured the impact of major regulated and emerging mycotoxins (17 mycotoxins in total) on the proliferation and viability of ENS cells in vitro. On the 17 mycotoxins tested, 9 were found active with anti-proliferative and cytotoxic effects observed at doses ranging from 0.19 to 16.4 µM and 0.4 to 38.4 µM, respectively. Mechanistic approaches revealed that toxicity on ENS cells was related to i) alteration of the membrane permeability, ii) ROS production and/or iii) induction of apoptosis/necrosis. Importantly, toxic doses found on ENS cells were compared to toxic doses found on IECs in order to determine if toxicity toward ENS was selective or not. Finally, toxic doses on ENS cells were compared to PMTDI and range of exposure in humans and animals in order to evaluate if ENS’s cells are a realistic target of food-associated mycotoxins and if alterations of ENS participate in the global impact of these toxins on the gut and the full organism.

Lipophilic marine toxins in molluscs constitute an important threat to human health and high number of intoxications occur every year. These toxins restrict the progress of aquaculture, which is one of the fastest growing food sectors in the world. The region of Galicia (Spain), Chile and South East Pacific are commercially important producers of edible bivalve mollusc, however they have been subjected to recurring cases of shellfish farm closures in the last decade. This work aimed to study the lipophilic toxic profile of commercial shellfish (including emerging toxins) from these locations in order to establish a potential risk when ingested. For this, a total of 41 samples of Galician mussels (Mytilus galloprovincialis), 6 samples of mussels from Chile (Mytilus chilensis) and 6 samples of other shellfish from South East Pacific (Tawerea gayi and Meretrix lyrata ) were purchased in local markets and analysed by ultra-high-performance liquid chromatography system coupled to mass spectrometry (UPLC–MS/MS). Chromatograms from Mytilus galloprovincialis showed the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), pectenotoxin-2 (PTX-2), azaspiracid-2 (AZA-2) and the emerging toxins 13-desmethyl spirolide C (SPX-13) and pinnatoxin-G (PnTX-G). Data showed that OA group toxins are the main risk in Galician mollusks, which was detected in 38 samples (93%) at levels close to the legislated limit, followed by SPX-13 that was detected in 19 samples (46%) in quantities of up to 28.9 μg/kg. Analysis from PTX-2, AZA-2, and PnTX-G showed lower amounts, all below 3 μg/kg. Results also showed the presence of the emerging PnTX-G in mussels Mytilus chilensis at levels up to 5.2 μg/kg and AZA-2 and PTX-2 in clams Tawera gayi up to 4.33μg/kg and 10.88μg/kg, respectively. Despite no potential risk through mussel ingestion was found for the emerging toxins (SPX-13 and PnTX-G), there is a need for robust methodologies that can detect a wide range of known or emerging toxins in different matrix due to the geographical expansion of marine toxins.

Natural inhibitors are ubiquitous components, existing in a variety of organisms. Kunitz-type inhibitors (KTIs) are a group of proteins that bear remarkable homology to the Bovine Pancreatic Trypsin Inhibitor (BPTI) and have been extensively isolated and reported in snake venoms. The KTIs exhibit a wide variety of biological activities, including inhibition of various proteases, interference with hemostasis, inflammation, activation or blockade of acid-sensing ion channels (ASICs), and blockade of ion channels; showing their extensive functional diversity and biomedically relevant pharmacological properties. The king cobra (Ophiophagus hannah) is the longest venomous snake in the world, endemic from India through Southeast Asia. Members of the KTI family have been isolated and identify as either a weak chymotrypsin inhibitor or a trypsin/chymotrypsin inhibitor. This study aims to isolate and further characterize the pharmacological properties of a KTI from the Malaysian king cobra venom. The inhibitory effect on serine protease activity was determined using chromogenic substrates. The whole venom was partially purified employing size exclusion HPLC, and the isolated fractions were identified with N-terminal sequencing. The fractions (20 µg) were then incubated with plasmin (0.26 U) at 37 °C, at different times, and tested for inhibitory activities on its biological substrates. The trypsin activity towards its chromogenic substrate was 65% inhibited by the whole venom. After size exclusion chromatography, 13 fractions were isolated and were tested for inhibition of plasmin activity. The most remarkable effect was obtained with fraction 10, where a complete inhibition of the fibrinolytic activity of plasmin on fibrin plates was seen. Likewise, it partially inhibited the fibrino(geno)lytic activity of plasmin. These characterization studies will elucidate the biomedically relevant pharmacological properties intrinsic to a KTI from king cobra venom, which may lead to their potential use as therapeutic drugs and analytical tools for biomedical applications.

Beauvericin (BEA) and its analogs are non-ribosomal cyclodepsipeptide mycotoxins produced by a wide range of fungal species, including saprotrophs, plant, and insect pathogens, particularly belonging to Fusarium, Trichoderma, Beauveria, and Isaria genera. Most beauvericin analogs were described among Beauveria and Isaria genera as “unnatural” beauvericins by adding amino acid precursors to the growing media. The aim of the study was to find BEAs naturally-synthesized by Fusarium species and tentatively determine their structures using mass spectrometry. Moreover, because of the unknown ability to produce beauvericin by Trichoderma fungi, we carried out the quantitative analysis using UPLC-MS. We also analyzed the polymorphism of the BEAS gene by sequencing partial BEAS regions from Trichoderma and Fusarium species.

We screened five fungal cultures from the Fusarium genus cultivated on rice grain for the presence of the new natural beauvericins. The peptide sequence data of beauvericin analogs were established using MS/MS experiments as well as amino acid and hydroxy acid analysis following acid hydrolysis. Ten cyclodepsipeptide analogs described earlier were tentatively identified in the extract. In addition, two so far undescribed tyrosine-containing beauvericin analogs were tentatively identified in the cultures. Moreover, a quantitative analysis of beauvericin was performed using a UPLC-MS in eleven Trichoderma and six Fusarium rice cultures. The phylogenic analyses of beauvericin synthase (BEAS) divergence were performed on the basis of sequenced PCR-amplified fragments from Trichoderma and Fusarium fungi and partial reference genes from the GenBank database (representing Beauveria, Fusarium, and Trichoderma genera).

This study demonstrates the high variability of naturally-produced new types of beauvericins, such as tyrosine-containing analogs in Fusarium fungi. It also shows that fungi belonging to the Trichoderma genus possess the ability to produce beauvericin.

The aim of the present study was to evaluate the impact of picking frequency and dryers materials on fungal infection and aflatoxins content in cashew nuts produced in Côte d’Ivoire. Some cashew nuts collected after a delay of 2, 3 and 7 days on the ground, were sun-dried on racks-table, tarpaulin and cemented area to a moisture content of 8%. After 6 months of storage, 18 samples (3kg of each) of cashew nuts were collected and the fungal infection and aflatoxins content were evaluated using standard methods. Our results revealed that the rate of fungal infection evolved in keeping with the frequency of nuts picking. Thus, the rate of fungal infection was 5.7% at 2 days of frequency of nuts picking, 22.7% at 3 days and 54.6% at 7 days respectively. Proportionally, the loss rates were 1.85, 4.73 and 11.03%. The dryers had no significant effect on the infection and loss rates with corresponding values ranging from 24.49 to 31.45% and 5.41 to 10.32% respectively. A total of 12 genera and 148 fungal species were isolated and identified. The genus Aspergillus represented by Aspergillus niger (71.78%), Aspergillus flavus (4.29%), Aspergillus fumigatus (2.45%) and Aspergillus sp (1.84%) was the most preponderant. Although aflatoxin levels were marginal, they can reach 0.34 µg/kg with the 7-days pickup time. This aflatoxin level is related to the presence of aflatoxin B1 (0.29 µg/kg). The drying supports namely tarpaulins or cemented areas seemed to influence the level of aflatoxins secretion. Taken together, our results suggested that the frequency of cashew nuts picking was a critical control point in value chain for both fungal infection and aflatoxins contamination. In certain way, the racks-table seemed to be suitable drying support to avoid aflatoxins contamination.

The quality and safety of raw materials and food products are inextricably linked. Table eggs are subject to special monitoring due to microbial hazards. So far bacterial hazards have mostly been monitored. However, the latest reports have pointed to a threat that has not been considered for table eggs. Microfungi can grow on the surface of eggshells and penetrate into the egg content. Therefore, it is necessary to improve the microbiological state of the eggshell surface, which will guarantee the safety of egg consumption and slow down spoilage. The aim of the study was to examine how the sanitation of eggs with a hydrogen peroxide preparation containing silver ions affected the dynamics of growth of microfungi and the biosynthesis of mycotoxins during egg storage. The research results showed that H₂O₂ with silver ions was effective against microfungi and simultaneously limited the biosynthesis of mycotoxins. The egg sanitation treatment with a solution of hydrogen peroxide and silver ions reduced the count of microfungi, which stopped growing after one week of storage. The effectiveness of much lower concentrations of the preparation against these fungi may have been caused by the content of silver ions. There was a smaller decrease in the Haugh unit value in eggs sanitised with hydrogen peroxide and silver ions in the final period of storage. This means that the eggs lost freshness less dynamically. The research results showed that the treatment of eggs with the H₂O₂ preparation with silver ions slowed down their spoilage processes and effectively reduced their content of microfungi and mycotoxins.