Getting fat and stressed: Effects of dietary intake of titanium dioxide nanoparticles in the liver of turbot Scophthalmus maximus.

The extensive use of nanomaterials, including titanium dioxide nanoparticles (TiO2 NPs), raises concerns about their persistence in ecosystems. Protecting aquatic ecosystems and ensuring healthy and safe aquaculture products requires the assessment of the potential impacts of NPs on organisms. Here, we study the effects of a sublethal concentration of citrate-coated TiO2 NPs of two different primary sizes over time in flatfish turbot, Scophthalmus maximus (Linnaeus, 1758). Bioaccumulation, histology and gene expression were assessed in the liver to address morphophysiological responses to citrate-coated TiO2 NPs. Our analyses demonstrated a variable abundance of lipid droplets (LDs) in hepatocytes dependent on TiO2 NPs size, an increase in turbot exposed to smaller TiO2 NPs and a depletion with larger TiO2 NPs. The expression patterns of genes related to oxidative and immune responses and lipid metabolism (nrf2, nfκb1, and cpt1a) were dependent on the presence of TiO2 NPs and time of exposure supporting the variance in hepatic LDs distribution over time with the different NPs. The citrate coating is proposed as the likely catalyst for such effects. Thus, our findings highlight the need to scrutinize the risks associated with exposure to NPs with distinct properties, such as primary size, coatings, and crystalline forms, in aquatic organisms.

Our current understanding of the toxicity of altered mito-ribosomal fidelity during mitochondrial protein synthesis: What can it tell us about human disease?

Altered mito-ribosomal fidelity is an important and insufficiently understood causative agent of mitochondrial dysfunction. Its pathogenic effects are particularly well-known in the case of mitochondrially induced deafness, due to the existence of the, so called, ototoxic variants at positions 847C (m.1494C) and 908A (m.1555A) of 12S mitochondrial (mt-) rRNA. It was shown long ago that the deleterious effects of these variants could remain dormant until an external stimulus triggered their pathogenicity. Yet, the link from the fidelity defect at the mito-ribosomal level to its phenotypic manifestation remained obscure. Recent work with fidelity-impaired mito-ribosomes, carrying error-prone and hyper-accurate mutations in mito-ribosomal proteins, have started to reveal the complexities of the phenotypic manifestation of mito-ribosomal fidelity defects, leading to a new understanding of mtDNA disease. While much needs to be done to arrive to a clear picture of how defects at the level of mito-ribosomal translation eventually result in the complex patterns of disease observed in patients, the current evidence indicates that altered mito-ribosome function, even at very low levels, may become highly pathogenic. The aims of this review are three-fold. First, we compare the molecular details associated with mito-ribosomal fidelity to those of general ribosomal fidelity. Second, we gather information on the cellular and organismal phenotypes associated with defective translational fidelity in order to provide the necessary grounds for an understanding of the phenotypic manifestation of defective mito-ribosomal fidelity. Finally, the results of recent experiments directly tackling mito-ribosomal fidelity are reviewed and future paths of investigation are discussed.

Structural analysis of mitochondrial rRNA gene variants identified in patients with deafness.

The last few years have witnessed dramatic advances in our understanding of the structure and function of the mammalian mito-ribosome. At the same time, the first attempts to elucidate the effects of mito-ribosomal fidelity (decoding accuracy) in disease have been made. Hence, the time is right to push an important frontier in our understanding of mitochondrial genetics, that is, the elucidation of the phenotypic effects of mtDNA variants affecting the functioning of the mito-ribosome. Here, we have assessed the structural and functional role of 93 mitochondrial (mt-) rRNA variants thought to be associated with deafness, including those located at non-conserved positions. Our analysis has used the structural description of the human mito-ribosome of the highest quality currently available, together with a new understanding of the phenotypic manifestation of mito-ribosomal-associated variants. Basically, any base change capable of inducing a fidelity phenotype may be considered non-silent. Under this light, out of 92 previously reported mt-rRNA variants thought to be associated with deafness, we found that 49 were potentially non-silent. We also dismissed a large number of reportedly pathogenic mtDNA variants, 41, as polymorphisms. These results drastically update our view on the implication of the primary sequence of mt-rRNA in the etiology of deafness and mitochondrial disease in general. Our data sheds much-needed light on the question of how mt-rRNA variants located at non-conserved positions may lead to mitochondrial disease and, most notably, provide evidence of the effect of haplotype context in the manifestation of some mt-rRNA variants.

Single-cell ICP-MS for studying the association of inorganic nanoparticles with cell lines derived from aquaculture species.

The current research deals with the use of single-cell inductively coupled plasma-mass spectrometry (scICP-MS) for the assessment of titanium dioxide nanoparticle (TiO2 NP) and silver nanoparticle (Ag NP) associations in cell lines derived from aquaculture species (sea bass, sea bream, and clams). The optimization studies have considered the avoidance of high dissolved background, multi-cell peak coincidence, and possible spectral interferences. Optimum operating conditions were found when using a dwell time of 50 µs for silver and 100 µs for titanium. The assessment of associated TiO2 NPs by scICP-MS required the use of ammonia as a reaction gas (flow rate at 0.75 mL min-1) for interference-free titanium determinations (measurements at an m/z ratio of 131 from the 48Ti(NH)(NH3)4 adduct). The influence of other parameters such as the number of washing cycles and the cell concentration on accurate determinations by scICP-MS was also fully investigated. Cell exposure trials were performed using PVP-Ag NPs (15 and 100 nm, nominal diameter) and citrate-TiO2 NPs (5, 25, and 45 nm, nominal diameter) at nominal concentrations of 10 and 50 µg mL-1 for citrate-TiO2 NPs and 5.0 and 50 µg mL-1 for PVP-Ag NPs. Results have shown that citrate-TiO2 NPs interact with the outer cell membranes, being quite low in the number of citrate-TiO2 NPs that enters the cells (the high degree of aggregation is the main factor which leads to the aggregates being in the extracellular medium). In contrast, PVP-Ag NPs have been found to enter the cells.

Proteomics reveals multiple effects of titanium dioxide and silver nanoparticles in the metabolism of turbot, Scophthalmus maximus.

Titanium dioxide (TiO2) and silver (Ag) NPs are among the most used engineered inorganic nanoparticles (NPs); however, their potential effects to marine demersal fish species, are not fully understood. Therefore, this study aimed to assess the proteomic alterations induced by sub-lethal concentrations citrate-coated 25 nm ("P25") TiO2 or polyvinylpyrrolidone (PVP) coated 15 nm Ag NPs to turbot, Scophthalmus maximus. Juvenile fish were exposed to the NPs through daily feeding for 14 days. The tested concentrations were 0, 0.75 or 1.5 mg of each NPs per kg of fish per day. The determination of NPs, Titanium and Ag levels (sp-ICP-MS/ICP-MS) and histological alterations (Transmission Electron Microscopy) supported proteomic analysis performed in the liver and kidney. Proteomic sample preparation procedure (SP3) was followed by LC-MS/MS. Label-free MS quantification methods were employed to assess differences in protein expression. Functional analysis was performed using STRING web-tool. KEGG Gene Ontology suggested terms were discussed and potential biomarkers of exposure were proposed. Overall, data shows that liver accumulated more elements than kidney, presented more histological alterations (lipid droplets counts and size) and proteomic alterations. The Differentially Expressed Proteins (DEPs) were higher in Ag NPs trial. The functional analysis revealed that both NPs caused enrichment of proteins related to generic processes (metabolic pathways). Ag NPs also affected protein synthesis and nucleic acid transcription, among other processes. Proteins related to thyroid hormone transport (Serpina7) and calcium ion binding (FAT2) were suggested as biomarkers of TiO2 NPs in liver. For Ag NPs, in kidney (and at a lower degree in liver) proteins related with metabolic activity, metabolism of exogenous substances and oxidative stress (e.g.: NADH dehydrogenase and Cytochrome P450) were suggested as potential biomarkers. Data suggests adverse effects in turbot after medium/long-term exposures and the need for additional studies to validate specific biological applications of these NPs.

Quorum sensing systems as a new target to prevent biofilm-related oral diseases.

OBJECTIVE: The present study summarizes the current knowledge on the role of bacterial extracellular signaling systems, known as quorum sensing (QS), in oral biofilm formation, and on the possibility of blocking these microbial communication systems as a potential approach to prevent and treat oral infectious diseases. METHODS: A detailed literature review of the current knowledge of QS in the oral cavity was performed, using the databases MEDLINE (through PubMed) and Web of Science. RESULTS: Accumulating direct and indirect evidence indicates an important role of QS molecules in the oral microbial ecosystem. CONCLUSIONS: The mechanisms regulating gene expression through bacterial communication systems constitute a promising target to control oral biofilm formation. Although cell-to-cell communication is pivotal for biofilm formation of many pathogenic bacteria, knowledge concerning microbial interactions and signaling processes within multispecies biofilms in the oral cavity is still limited.

Depletion of a Toxoplasma porin leads to defects in mitochondrial morphology and contacts with the endoplasmic reticulum.

The voltage-dependent anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC protein, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contacts between the mitochondria and endoplasmic reticulum (ER). We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to mitochondria. Further, depletion of VDAC resulted in significant morphological changes in the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii. This article has an associated First Person interview with the first author of the paper.

Exploring the powerful phytoarsenal of white grape marc against bacteria and parasites causing significant diseases.

Natural extracts containing high polyphenolic concentration possess antibacterial, anti-parasitic and fungicidal activities. The present research characterises two extracts based on white grape marc, a winemaking by-product, describing their physicochemical features and antimicrobial capacities. The main components of these extracts are phenolic acids, flavan-3-ols and their gallates and flavonols and their glycosides. As a result of this complex composition, the extracts showed pronounced bioactivities with potential uses in agricultural, pharmaceutical and cosmetic industries. Polyphenol compounds were extracted by using hydro-organic solvent mixtures from the by-product of Albariño white wines (Galicia, NW Spain) production. The in vitro antimicrobial activity of these extracts was evaluated on Gram-positive and Gram-negative bacteria and Apicomplexan and Oomycota parasites. Microbial species investigated are causing agents of several human and animal diseases, such as foodborne illnesses (Bacillus cereus, Escherichia coli, Salmonella enterica, and Toxoplasma gondii), skin infections and/or mastitis (Staphylococcus aureus and Streptococcus uberis), malaria (Plasmodium falciparum) and plant infections as "chestnut ink" or "root rot" (Phytophthora cinnamomi). Both extracts showed activity against all the tested species, being nontoxic for the host. So, they could be used for the development of biocides to control a wide range of pathogenic agents and contribute to the enhancement of winemaking industry by-products.

Molecular Targets Implicated in the Antiparasitic and Anti-Inflammatory Activity of the Phytochemical Curcumin in Trichomoniasis.

Trichomoniasis, is the most prevalent non-viral sexually transmitted disease worldwide. Although metronidazole (MDZ) is the recommended treatment, several strains of the parasite are resistant to MDZ, and new treatments are required. Curcumin (CUR) is a polyphenol with anti-inflammatory, antioxidant and antiparasitic properties. In this study, we evaluated the effects of CUR on two biochemical targets: on proteolytic activity and hydrogenosomal metabolism in Trichomonas vaginalis. We also investigated the role of CUR on pro-inflammatory responses induced in RAW 264.7 phagocytic cells by parasite proteinases on pro-inflammatory mediators such as the nitric oxide (NO), tumor necrosis factor α (TNFα), interleukin-1beta (IL-1ß), chaperone heat shock protein 70 (Hsp70) and glucocorticoid receptor (mGR). CUR inhibited the growth of T. vaginalis trophozoites, with an IC50 value between 117 ± 7 µM and 173 ± 15 µM, depending on the culture phase. CUR increased pyruvate:ferredoxin oxidoreductase (PfoD), hydrogenosomal enzyme expression and inhibited the proteolytic activity of parasite proteinases. CUR also inhibited NO production and decreased the expression of pro-inflammatory mediators in macrophages. The findings demonstrate the potential usefulness of CUR as an antiparasitic and anti-inflammatory treatment for trichomoniasis. It could be used to control the disease and mitigate the associated immunopathogenic effects.

Protein Import into the Endosymbiotic Organelles of Apicomplexan Parasites.

: The organelles of endosymbiotic origin, plastids, and mitochondria, evolved through the serial acquisition of endosymbionts by a host cell. These events were accompanied by gene transfer from the symbionts to the host, resulting in most of the organellar proteins being encoded in the cell nuclear genome and trafficked into the organelle via a series of translocation complexes. Much of what is known about organelle protein translocation mechanisms is based on studies performed in common model organisms; e.g., yeast and humans or Arabidopsis. However, studies performed in divergent organisms are gradually accumulating. These studies provide insights into universally conserved traits, while discovering traits that are specific to organisms or clades. Apicomplexan parasites feature two organelles of endosymbiotic origin: a secondary plastid named the apicoplast and a mitochondrion. In the context of the diseases caused by apicomplexan parasites, the essential roles and divergent features of both organelles make them prime targets for drug discovery. This potential and the amenability of the apicomplexan Toxoplasma gondii to genetic manipulation motivated research about the mechanisms controlling both organelles' biogenesis. Here we provide an overview of what is known about apicomplexan organelle protein import. We focus on work done mainly in T. gondii and provide a comparison to model organisms.

Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment.

Presence of an isoform of H+-pyrophosphatase located in the alveolar sacs of a scuticociliate parasite of turbot: physiological consequences.

H+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2 storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasite Philasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that the P. dicentrarchi H+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance in P. dicentrarchi.

Enzymes Involved in Pyrophosphate and Calcium Metabolism as Targets for Anti-scuticociliate Chemotherapy.

Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca(2+) homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the in vitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+-PPases (H+-PPases). CQ and ART inhibited the in vitro growth of ciliates with IC50 values of respectively 74 ± 9 µM and 80 ± 8 µM. CQ inhibits the H+ translocation (with an IC50 of 13.4 ± 0.2 µM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+-PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca(2+) induced by ATP, indicating an effect on the Ca(2+) -ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca(2+) metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis.

Presence of a plant-like proton-translocating pyrophosphatase in a scuticociliate parasite and its role as a possible drug target.

The proton-translocating inorganic pyrophosphatases (H(+)-PPases) are primary electrogenic H(+) pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H(+)-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61.7 kDa and an isoelectric point of 5.0. Several motifs characteristic of plant vacuolar H(+)-PPases (V-H(+)-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V-H(+)-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K(+)-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V-H(+)-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.

Alternative oxidase inhibitors as antiparasitic agents against scuticociliatosis.

Philasterides dicentrarchi causes a severe disease in turbot, and at present there are no drugs available to treat infected fish. We have previously demonstrated that, in addition to the classical respiratory pathway, P. dicentrarchi possesses an alternative mitochondrial respiratory pathway that is cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive. In this study, we found that during the initial phase of growth in normoxia, ciliate respiration is sensitive to the natural polyphenol resveratrol (RESV) and to Antimycin A (AMA). However, under hypoxic conditions, the parasite utilizes AMA-insensitive respiration, which is completely inhibited by RESV and by the antioxidant propyl gallate (PG), an alternative oxidase (AOX) inhibitor. PG caused significantly dose-dependent inhibition of the in vitro growth of the parasite under normoxia and hypoxia and an over-expression of heat shock proteins of the Hsp70 subfamily. RESV and PG may affect the protective role of the AOX against mitochondrial oxidative stress, leading to an impaired mitochondrial membrane potential and mitochondrial dysfunction, which the parasite attempts to neutralize by increasing the expression of Hsp70. In view of the antiparasitic effects induced by AOX inhibitors and the absence of AOX in their host, this enzyme constitutes a potential target for the development of new drugs against scuticociliatosis.

Evidence of an alternative oxidase pathway for mitochondrial respiration in the scuticociliate Philasterides dicentrarchi.

The presence of an alternative oxidase (AOX) in the mitochondria of the scuticociliate P. dicentrarchi was investigated. The mitochondrial oxygen consumption was measured in the presence of KCN, an inhibitor of cytochrome pathway (CP) respiration and salicylhydroxamic acid (SHAM), a specific inhibitor of alternative pathway (AP) respiration. AOX expression was monitored by western blotting with an AOX polyclonal antibody. The results showed that P. dicentrarchi possesses a branched mitochondrial electron transport chain with both cyanide-sensitive and -insensitive oxygen consumption. Mitochondrial respiration was partially inhibited by cyanide and completely inhibited by the combination of cyanide and SHAM, which is direct evidence for the existence of an AP in this ciliate. SHAM significantly inhibited in vitro growth of trophozoites both under normoxic and hypoxic conditions. AOX is a 42kD monomeric protein inducible by hypoxic conditions in experimental infections and by CP inhibitors such as cyanide and antimycin A, or by AP inhibitors such as SHAM. CP respiration was greatly stimulated during the exponential growth phase, while AP respiration increased during the stationary phase, in which AOX expression is induced. As the host does not possess AOX, and because during infection P. dicentrarchi respires via AP, it may be possible to develop inhibitors targeting the AP as a novel anti-scuticociliate therapy.

Hydrogenosome metabolism is the key target for antiparasitic activity of resveratrol against Trichomonas vaginalis.

Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 µM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 µM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 µM). At doses of 50 to 100 µM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs.

Effect of resveratrol on oxygen consumption by Philasterides dicentrarchi, a scuticociliate parasite of turbot.

The phytoalexin resveratrol (RESV) displays antiparasitic activity against Philasterides dicentrarchi, a scuticociliate pathogen of turbot, and causes oxidative stress, inhibition of antioxidant enzyme activity and morphological alterations in the parasite mitochondria. In this study, we analysed the mitochondrial biology of P. dicentrarchi and assessed the effect of RESV on mitochondrial metabolism. We found that RESV caused dose-dependent inhibition of mitochondrial electron transport and O2 consumption in ciliates permeabilized with digitonin. Although the RESV molecule has a high capacity for antiradical and antioxidant activity, it induced a high level of pro-oxidant activity against the ciliate, thus causing a significant increase in intracellular ROS production. The increased ROS production was accompanied by mitochondrial collapse and dysfunction of mitochondrial membrane potential (ΔΨm) and by a significant increase in intracellular Ca⁺² levels. RESV inhibited parasite growth in a similar way to antimycin A, an inhibitor of mitochondrial electron transport and ROS generator. The findings confirm the mitochondria as a target in the potential development of effective antiparasitic treatments.

Evaluation of some physical and chemical treatments for inactivating microsporidian spores isolated from fish.

Microsporidia are a large diverse group of intracellular parasites now considered as fungi. They are particularly prevalent in fish and are recognized as important opportunistic parasites in humans. Although the mode of transmission of microsporidia has not been fully clarified, the consumption and manipulation of infected fish may be a risk factor for humans. Comparative analysis of rDNA sequence revealed that the microsporidians used in the present study had 99-100% identity with anglerfish microsporidians of the genus Spraguea and very low identity with microsporidians that infect humans. Microsporidian spores were exposed to different physical and chemical treatments: freezing at -20°C for 24-78 h, heating at 60°C for 5-15 min, microwaving at 700 W, 2.45 GHz for 15-60s, and treatment with ethanol at concentrations of between 1 and 70% for 15 min. The viability of the spores after each treatment was evaluated by two methods: a) haemocytometer counts, measuring the extrusion of the polar filament in control and treated spores, and b) a fluorometric method, testing the membrane integrity by propidium iodide exclusion. The results of both methods were concordant. Spores were inactivated by freezing at -20°C for more than 48 h, by heating to 60°C for 10 min and by microwaving at 750 W, for 20s. Exposure to 70% ethanol for 15 min also inactivated microsporidian spores. The results suggest that both freezing and heating are effective treatments for destroying microsporidian spores in European white anglerfish, and that 70% ethanol could be used by fish processors to disinfect their hands and the utensils used in processing fish. The fluorometric method can be used as an alternative to haemocytometer counts in disinfection studies aimed at establishing strategies for inactivating and reducing the viability and the potential infectivity of microsporidians present in fish or in the environment.