Lethal effects of silver nanoparticles on Perkinsus marinus, a protozoan oyster parasite.

Perkinsus marinus, a World Organisation for Animal Health (OIE) notifiable parasite, infects several species of oyster, including Crassostrea virginica and Crassostrea corteziensis. There is little information on possible treatments for this parasite, but the biocidal properties of silver nanoparticles (AgNP) suggest their potential use. The lethal effects of the Argovit™ formulation of AgNP was evaluated for the first time against hypnospores of P. marinus, a particularly resistant stage of the parasite that persists in the environment until favorable conditions occur for zoosporulation to be induced. Hypnospores were exposed to 1, 10 and 100 µg/mL of silver compounded in Argovit™ (corresponding to 0.009, 0.093 and 0.927 mM of Ag), to 157.47 µg/mL (0.927 mM) of silver nitrate (AgNO3) used as a positive control, and to polyvinylpyrrolidone (PVP, 1570 µg/mL) used as a vehicle control. Hypnospores in culture medium without treatment served as a negative control. Dose-dependence after 24 h of exposure to AgNP was observed. A concentration of 0.093 mM AgNP resulted in 50% mortality of P. marinus. Treatment with 0.927 mM of silver, as AgNP or AgNO3, was highly lethal, with greater than 90% mortality. Silver nanoparticles were implicated in the deformation of hypnospores. Transmission electron microscopy (TEM) revealed AgNP within the hypnospore wall and involved in the degradation of lipid droplets in the cytoplasm. AgNP were effective in a saline medium, suggesting the utility of detailed studies of the physicochemical interactions of AgNP under these conditions. These results suggest investigations of possible effect of Argovit™ formulation of AgNP against stages of the parasite like trophozoites and tomonts that develop in tissues or hemolymph of infected oysters as well as studies on its effects in the host and environment.

Transcriptomic Analysis Reveals the Roles of Detoxification Systems in Response to Mercury in Chromera velia.

Heavy metal pollution is an increasing global concern. Among heavy metals, mercury (Hg) is especially dangerous because of its massive release into the environment and high toxicity, especially for aquatic organisms. The molecular response mechanisms of algae to Hg exposure are mostly unknown. Here, we combine physiological, biochemical, and transcriptomic analysis to provide, for the first time, a comprehensive view on the pathways activated in Chromera velia in response to toxic levels of Hg. Production of hydrogen peroxide and superoxide anion, two reactive oxygen species (ROS), showed opposite patterns in response to Hg2+ while reactive nitrogen species (RNS) levels did not change. A deep RNA sequencing analysis generated a total of 307,738,790 high-quality reads assembled in 122,874 transcripts, representing 89,853 unigenes successfully annotated in databases. Detailed analysis of the differently expressed genes corroborates the biochemical results observed in ROS production and suggests novel putative molecular mechanisms in the algal response to Hg2+. Moreover, we indicated that important transcription factor (TF) families associated with stress responses differentially expressed in C. velia cultures under Hg stress. Our study presents the first in-depth transcriptomic analysis of C. velia, focusing on the expression of genes involved in different detoxification defense systems in response to heavy metal stress.

Effect of antiprotozoal molecules on hypnospores of Perkinsus spp. parasite.

Perkinsus protozoan parasites have been associated with high mortality of bivalves worldwide, including Brazil. The use of antiproliferative drugs to treat the Perkinsosis is an unusual prophylactic strategy. However, because of their environment impact it could be used to control parasite proliferation in closed system, such as hatchery. This study evaluated the anti-Perkinsus activity potential of synthesized and commercial compounds. Viability of hypnospores of Perkinsus spp. was assessed in vitro. Cells were incubated with three 2-amino-thiophene (6AMD, 6CN, 5CN) and one acylhydrazone derivatives (AMZ-DCL), at the concentrations of 31.25; 62.5; 125; 250 and 500 µM and one commercial chlorinated phenoxy phenol derivative, triclosan (2, 5, 10 and 20 µM), for 24-48 h. Two synthetic molecules (6CN and AMZ-DCL) caused a significant decline (38 and 39%, respectively) in hypnospores viability, at the highest concentration (500 µM), after 48 h. Triclosan was the most cytotoxic compound, causing 100% of mortality at 20 µM after 24 h and at 10 µM after 48 h. Cytotoxic effects of the compounds 6CN, AMZ-DCL, and triclosan were investigated by measuring parasite's zoosporulation, morphological changes and metabolic activities (esterase activity, production of reactive oxygen species and lipid content). Results showed that zoosporulation occurred in few cell. Triclosan caused changes in the morphology of hypnospores. The 6CN and AMZ-DCL did not alter the metabolic activities studied whilst Triclosan significantly increased the production of reactive oxygen species and changed the amount and distribution of lipids in the hypnospores. These results suggest that three compounds had potential to be used as antiprotozoal drugs, although further investigation of their mechanism of action must be enlightened.

Toxicity estimates for diuron and atrazine for the tropical marine cnidarian Exaiptasia pallida and in-hospite Symbiodinium spp. using PAM chlorophyll-a fluorometry.

Effective ecotoxicological risk assessments for herbicides in tropical marine environments are restricted by a lack of toxicity data, sensitive test methods and endpoints for relevant species, and this requires rectification. The symbiotic sea anemone Exaiptasia pallida is a suitable test species, representing the phylum Cnidaria and allowing for assessments of toxicological responses of both the animal host and in-hospite Symbiodinium spp. Pulse amplitude modulated (PAM) chlorophyll-a fluorometry is recognised as a valuable ecotoxicological tool, and here newly-developed test methods are presented using PAM fluorometry to measure herbicide effects on photosynthetic efficiency of in-hospite Symbiodinium spp. Additionally, measurements on healthy laboratory-reared E. pallida provide baseline data demonstrating the normal effective quantum yield (EQY) and the maximum electron transport rate (ETRm) for Symbiodinium spp. in the absence of herbicide stress. Concentration-dependant reductions in the EQY and ETRm occurred during diuron and atrazine exposures; a mean 48-h EC50 (effective concentration; 50%) of 8µg/L of diuron was estimated, however atrazine elicited a much lower toxicity. Twelve-day exposures to 10-200µg/L diuron showed that the greatest EQY effect occurred during the first 48h, with little subsequent change. However, longer exposures to the lowest diuron treatment (1µg/L) showed the lowest EQYs after 96h followed by recovery to control levels within 12d. Furthermore, asexual reproduction was inhibited during 12-d exposures to diuron, and 12-d EC50 values of 100 and 132µg/L were estimated to inhibit successful reproduction of pedal lacerates and juveniles by 50% respectively. This study provides much needed data contributions to species sensitivity curves for development of diuron and atrazine water quality guidelines in tropical marine environments.

Effects of cyanobacteria Synechocystis spp. in the host-parasite model Crassostrea gasar-Perkinsus marinus.

Perkinsosis is a disease caused by protozoan parasites from the Perkinsus genus. In Brazil, two species, P. beihaiensis and P. marinus, are frequently found infecting native oysters (Crassostrea gasar and C. rhizophorae) from cultured and wild populations in several states of the Northeast region. The impacts of this disease in bivalves from Brazil, as well as the interactions with environmental factors, are poorly studied. In the present work, we evaluated the in vitro effects of the cyanobacteria Synechocystis spp. on trophozoites of P. marinus and haemocytes of C. gasar. Four cyanobacteria strains isolated from the Northeast Brazilian coast were used as whole cultures (WCs) and extracellular products (ECPs). Trophozoites of P. marinus were exposed for short (4h) and long (48h and 7days, the latter only for ECPs) periods, while haemocytes were exposed for a short period (4h). Cellular and immune parameters, i.e. cell viability, cell count, reactive oxygen species production (ROS) and phagocytosis of inert (latex beads) and biological particles (zymosan and trophozoites of P. marinus) were measured by flow cytometry. The viability of P. marinus trophozoites was improved in response to WCs of Synechocystis spp., which could be a beneficial effect of the cyanobacteria providing nutrients and reducing reactive oxygen species. Long-term exposure of trophozoites to ECPs of cyanobacteria did not modify in vitro cell proliferation nor viability. In contrast, C. gasar haemocytes showed a reduction in cell viability when exposed to WCs, but not to ECPs. However, ROS production was not altered. Haemocyte ability to engulf latex particles was reduced when exposed mainly to ECPs of cyanobacteria; while neither the WCs nor the ECPs modified phagocytosis of the biological particles, zymosan and P. marinus. Our results suggest a negative effect of cyanobacteria from the Synechocystis genus on host immune cells, in contrast to a more beneficial effect on the parasite cell, which could together disrupt the balance of the host-parasite interaction and make oysters more susceptible to P. marinus as well as opportunistic infections.

Identification of MMV Malaria Box inhibitors of Perkinsus marinus using an ATP-based bioluminescence assay.

"Dermo" disease caused by the protozoan parasite Perkinsus marinus (Perkinsozoa) is one of the main obstacles to the restoration of oyster populations in the USA. Perkinsus spp. are also a concern worldwide because there are limited approaches to intervention against the disease. Based on the phylogenetic affinity between the Perkinsozoa and Apicomplexa, we exposed Perkinsus trophozoites to the Medicines for Malaria Venture Malaria Box, an open access compound library comprised of 200 drug-like and 200 probe-like compounds that are highly active against the erythrocyte stage of Plasmodium falciparum. Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against P. marinus trophozoites. Six compounds with IC50 in the µM range were used to compare the degree of susceptibility in vitro of eight P. marinus strains from the USA and five Perkinsus species from around the world. The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across Perkinsus strains and species. Both Perkinsus marinus isolates and Perkinsus spp. presented different patterns of response to the panel of compounds tested, supporting the concept of strain/species variability. Here, we expanded the range of compounds available for inhibiting Perkinsus proliferation in vitro and characterized Perkinsus phenotypes based on their resistance to six compounds. We also discuss the implications of these findings in the context of oyster management. The Perkinsus system offers the potential for investigating the mechanism of action of the compounds of interest.

Towards molecular, physiological, and biochemical understanding of photosynthetic inhibition and oxidative stress in the toxic Alexandrium tamarense induced by a marine bacterium.

Alexandrium tamarense is a notorious harmful algal bloom species, which is associated with the largest number of paralytic shellfish poisoning cases, causing devastating economic losses and health hazards. The marine bacterium Mangrovimonas yunxiaonensis strain LY01 showed high algicidal effects on A. tamarense. A. tamarense was also susceptible to the supernatant of LY01 as revealed by algicidal activity assay, but washed bacterial cells did not show algicidal activity towards A. tamarense. In this study, we investigated the algicidal effect of the supernatant on growth, photosynthesis and the antioxidative response of A. tamarense. The results indicated that under the algicidal effect of the supernatant, the contents of cellular pigments including chlorophyll a and carotenoids were significantly decreased, and the decline of the maximum quantum yield and relative electron transport rate values suggested that photosynthetic inhibition occurred in the photosystem II system. The content of reactive oxygen species (ROS) increased after 0.5 h exposure, and the surplus ROS induced lipid peroxidation, the destruction of cellular membrane integrity and decreased cellular protein and carbohydrate contents in the algal cells. At the same time, the supernatant also induced the responses of antioxidant enzymes and non-enzymatic antioxidant. The transcription of photosynthesis- and respiration-related genes were significantly inhibited during the exposure procedure, which obstructed photosynthetic efficiency and capacity and disturbed the respiratory system, thereby increasing ROS production again. All these results elaborate clearly the entire procedure by which cellular physiological levels respond to the algicidal bacterium and may contribute to a better understanding of the bacterial control of A. tamarense.

Pallial mucus of the oyster Crassostrea virginica regulates the expression of putative virulence genes of its pathogen Perkinsus marinus.

Perkinsus marinus is a pathogen responsible for severe mortalities of the eastern oyster Crassostrea virginica along the East and Gulf coasts of the United States. When cultivated, the pathogenicity of this microorganism decreases significantly, hampering the study of its virulence factors. Recent investigations have shown a significant increase of the in vivo virulence of P. marinus exposed to oyster pallial mucus. In the current study, we investigated the effect of pallial mucus on P. marinus gene expression compared with cultures supplemented with oyster digestive extracts or with un-supplemented cultures. In parallel, parasite cells cultured under these three conditions were used to challenge oysters and to assess virulence in vivo. Perkinsus marinus mRNA sequencing was performed on an Illumina GAIIX sequencer and data were analysed using the Tuxedo RNAseq suite for mapping against the draft P. marinus genome and for differential expression analysis. Results showed that exposure of P. marinus to mucus induces significant regulation of nearly 3,600 transcripts, many of which are considered as putative virulence factors. Pallial mucus is suspected to mimic internal host conditions, thereby preparing the pathogen to overcome defense factors before invasion. This hypothesis is supported by significant regulation in several antioxidant proteins, heat shock proteins, protease inhibitors and proteasome subunits. In addition, mucus exposure induced the modulation of several genes known to affect immunity and apoptosis in vertebrates and invertebrates. Several proteases (proteolysis) and merozoite surface proteins (cell recognition) were also modulated. Overall, these results provide a baseline for targeted, in depth analysis of candidate virulence factors in P. marinus.

Peroxides with antiplasmodial activity inhibit proliferation of Perkinsus olseni, the causative agent of Perkinsosis in bivalves.

Perkinsus olseni, the causative agent of Perkinsosis, can drastically affect the survival of target marine mollusks, with dramatic economic consequences for aquaculture. P. olseni is a member of the Alveolata group, which also comprises parasites that are highly relevant for medical and veterinary sciences such as Plasmodium falciparum and Toxoplasma. P. olseni shares several unique metabolic pathways with those pathological parasites but is not toxic to humans. In this work, six antimalarially active peroxides, derived from the natural product artemisinin or synthetic trioxolanes, were synthesized and tested on P. olseni proliferation and survival. All peroxides tested revealed an inhibitory effect on P. olseni proliferation at micromolar concentrations. The relevance of the peroxide functionality on toxicity and the effect of Fe(II)-intracellular concentration on activity were also evaluated. Results demonstrated that the peroxide functionality is the toxofore and intracellular iron concentration also proved to be a crucial co-factor on the activation of peroxides in P. olseni. These data points to a mechanism of bioactivation in P. olseni sharing similarities with the one proposed in P. falciparum parasites. Preliminary studies on bioaccumulation were conducted using fluorescent-labeled peroxides. Results show that synthetic trioxolanes tend to accumulate on a vacuole while the labeled artemisinin accumulates in the cytoplasm. Preliminary experiments on differential genes expression associated to Fe(II) transport protein (Nramp) and calcium transport protein (ATP6/SERCA) were also conducted by qPCR. Results point to a fourfold increase in expression of both genes upon exposure to trioxolanes and approximately twofold upon exposure to artemisinin derivatives. Data obtained in this investigation is relevant for better understanding of the biology of Perkinsus and may also be important in the development of new strategies for Perkinsosis prevention and control.

Stress-related responses in Alexandrium tamarense cells exposed to environmental changes.

Organisms tend to be sensitive to drastic changes in environmental conditions. For unicellular microorganisms, variations in physico-chemical conditions are particularly challenging and may result in acclimation, entrance into quiescence, or death through necrotic or autocatalytic pathways. This study focuses on the thecate dinoflagellate Alexandrium tamarense. Cellular responses to oxidative, thermal, and nutrient stress were characterized using stress indicators, such as pigment content, efficiency of photosystem II or production of reactive oxygen species (ROS), as well as hallmarks of apoptosis including activity of caspase-like enzymes and expression of a metacaspase gene homolog. The formation of temporary cysts, a survival strategy of short-term quiescence, was also monitored. Cellular responses appeared to depend on multifactorial influences where type and intensity of stimulus as well as position in cell cycle may act in combination. Sequences of events observed implicate ROS production as a key determinant of stress-related pathways, playing potential roles in intracellular signaling, formation of temporary cysts, or cellular damage. Variations observed in caspase-like activities and metacaspase gene expression did not appear to be associated with programmed cell death pathways; our results suggest a wider range of functions for these proteases in phytoplankton cells, including roles in survival pathways and cell cycle progression.

Quantification of toxic effects of the herbicide metolachlor on marine microalgae Ditylum brightwellii (Bacillariophyceae), Prorocentrum minimum (Dinophyceae), and Tetraselmis suecica (Chlorophyceae).

Toxic effects of the herbicide metolachlor (MC) were evaluated for three marine microalgae, Tetraselmis suecica (chlorophyte), Ditylum brightwellii (diatom), and Prorocentrum minimum (dinoflagellate). MC showed a significant reduction in cell counts and chlorophyll a levels. Median effective concentration (EC50) was calculated based on chlorophyll a levels after a 72-h MC exposure. EC50 values for T. suecica, D. brightwellii, and P. minimum were 21.3, 0.423, and 0.07 mg/L, respectively. These values showed that the dinoflagellate was most sensitive when exposed to the herbicide, at a concentration comparable to freshwater algae, suggesting its potential as an appropriate model organism for ecotoxicity assessments in marine environments.

Recent developments in rationally designed multitarget antiprotozoan agents.

Protozoan infections are the leading cause of morbidity and mortality among parasitic infections of humans, accounting for approximately 800 thousand mortalities and a loss of more than 30 million disability-adjusted life years annually. The major protozoan infections of humans, namely malaria, Chagas disease, human African trypanosomiasis, and leishmaniasis, are primarily centered in the tropics, with a reach into some subtropical regions of the world. Though globally massive in their impact, these diseases mostly afflict the least economically endowed and geographically marginalized populations in low-income countries. As such, there is no sufficient market incentive for industrial business-driven antiprotozoal drug discovery due to poor marketing prospects and low returns on investment. Consequently, the pharmacopoeia for majority of these diseases, composed mainly of agents with poor efficacy and unsatisfactory safety profiles, has essentially remained unchanged for decades, creating a compelling need for more efficacious and better tolerated medicines. The policy makers and the scientific community are seeking effective ways to meet this need. So far, two approaches have emerged promising in this regard: combination chemotherapy and drug repositioning. Molecular hybridization has been cited as a potential third approach that could be used to deliver new antiprotozoal chemical entities. In this review article, recent applications of this novel strategy in antimalarial, antichagasic, antitrypanosomal, and antileishmanial drug discovery research and development over the last five years will be presented and discussed.

Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species.

Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species.

Toxicological effects of cypermethrin to marine phytoplankton in a co-culture system under laboratory conditions.

The growth of three marine phytoplankton species Skeletonema costatum, Scrippsiella trochoidea and Chattonella marina and the response of the antioxidant defense system have been investigated on exposure to commercial cypermethrin for 96 h and 32 days in a co-culture system. Growth of the three species was generally comparable over 96 h with an inoculation of 1:3:6.5 (C. marina:S. trochoidea:S. costatum), with stimulation at 5 µg l(-1) and inhibition under higher concentrations (50, 100 µg l(-1)). However, when inoculating at ratios of 1:1:1 during a 32 day test, S. costatum became the most sensitive species and was significantly inhibited in all test groups under the dual stresses of cypermethrin and interspecies competition. The growth of C. marina was significantly inhibited at the concentrations higher than 5 µg l(-1), while the growth of S. trochoidea was significantly promoted at low concentrations. Superoxide dismutase (SOD) activities significantly increased during 6-12 h exposure periods in test treatments at low concentrations, and enhanced in the control as well due to interspecies competition. The lipid peroxidation product malondialdehyde was enhanced at high concentrations, but did not increase in control and low concentration cultures with high SOD activities, indicating that algal cells activated the antioxidant enzymes promptly to protect the cells from lipid membrane damage. Results from this study suggested that cypermethrin pollution in maricultural sea waters might lead to a shift in phytoplankton community structure from diatom to harmful dinoflagellate species, and thus potentially stimulatory for harmful algal blooms.

Development of an in vitro assay to examine intracellular survival of Perkinsus marinus trophozoites upon phagocytosis by oyster (Crassostrea virginica and Crassostrea ariakensis) hemocytes.

Perkinsus marinus is a facultative intracellular parasite that causes "Dermo" disease in the eastern oyster Crassostrea virginica. Although hemocytes from healthy oysters rapidly phagocytize P. marinus trophozoites, they fail to efficiently kill them. Instead, trophozoites survive and proliferate, eventually overwhelming the host. Because Chesapeake Bay oyster populations have been reduced to unprecedented levels, the introduction of the Suminoe oyster, Crassostrea ariakensis (synonymous C. rivularis), has recently been proposed. Although this species is refractory to developing Dermo disease, it can be infected by Perkinsus spp. and, thus, the mechanistic basis of its disease resistance remains intriguing. To examine whether the resistance to develop Dermo is due to a high capacity of C ariakensis hemocytes to kill internalized P. marinus, we developed an in vitro assay to compare intracellular survival and proliferation of P. marinus in C. virginica and C ariakensis hemocytes. Our results revealed that P. marinus cultured trophozoites have a similar capacity for in vitro survival within hemocytes from both oyster species, suggesting that the resistance of C. ariakensis to develop Dermo disease is most likely due to reduced parasite pathogenicity for the latter oyster species, rather than to infectivity. Together with the currently available P. marinus genome, EST sequences, and the transfection methodology we recently developed, this assay should significantly contribute to a rigorous identification of the P. marinus genes responsible for its intrahemocytic survival.