- Email: [email protected]
Lethal effects of silver nanoparticles on Perkinsus marinus, a protozoan oyster parasite
Journal of Invertebrate Pathology 169 (2020) 107304
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Lethal effects of silver nanoparticles on Perkinsus marinus, a protozoan oyster parasite
T
⁎
Cecilia Bravo-Guerraa, Jorge Cáceres-Martíneza, , Rebeca Vásquez-Yeomansa, Alexey Pestryakovb, Nina Bogdanchikovac a
Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana No. 3918, CP 22860 Ensenada, Baja California, Mexico Tomsk Polytechnic University, Lenin Avenue 30, Tomsk 634050, Russia c Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, km 107 carretera Ensenada-Tijuana, CP 22860 Ensenada, Baja California, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hypnospores Perkinsus marinus Silver nanoparticles
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.
1. Introduction Diseases in aquatic organisms pose an economic risk and a management challenge for the aquaculture industry, affecting animal health and impacting production. Among these diseases, “dermo disease”, which is caused by the protozoan Perkinsus marinus, is one of the most important for the eastern oyster, Crassostrea virginica, a species cultured on the East Coast of the United States (Gullian-Klanian et al., 2008; ICES, 2011), due to the mortality it has caused in oyster populations since the 1940s (Ray, 1996). Perkinsus marinus is considered a notifiable parasite by the World Organisation for Animal Health (OIE) and has been found to infect other species such as the Cortez oyster, Crassostrea corteziensis, and the mangrove oyster, Saccostrea palmula, on the Pacific coast of northwest Mexico (Cáceres-Martínez et al., 2010; Cáceres-
⁎
Martínez et al., 2012). P. marinus can be transmitted from host to host, and all phases of its life cycle are infectious. It grows in a vegetative phase as a trophozoite, multiplying in the tissues and hemocytes of its host (Perkins, 1996) and is released into the water in feces or from the tissues of dead hosts (Bushek et al., 2002). P. marinus may develop into hypnospores, a resistant stage that remains in the environment until favorable conditions induce production of biflagellate zoospores (Perkins, 1996). Some chemotherapeutants and disinfectant compounds such as chlorine (300 ppm), UV light (> 28,000 μWs cm2), Nhalamin compounds and fresh water inactivate cultured P. marinus cells (Bushek et al., 1997; Bushek and Howell, 2000; Delaney et al., 2003). The use of bacitracin, cycloheximide and fresh water have been shown to reduce but not eliminate P. marinus in infected oysters (Calvo and Burreson, 1994; Faisal et al., 1999). Because of the ineffectiveness or
Corresponding author. E-mail address: [email protected] (J. Cáceres-Martínez).
https://doi.org/10.1016/j.jip.2019.107304 Received 23 June 2019; Received in revised form 3 December 2019; Accepted 5 December 2019 Available online 07 December 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 169 (2020) 107304
C. Bravo-Guerra, et al.
Fig. 1. Mortality of Perkinsus marinus hypnospores exposed to different silver nanoparticle treatments as a function of time. Different letters (a, b, c…n) indicate significant differences for each time period. The data are expressed as the means ± SD of three replicates per treatment. Abbreviations: Control, culture medium; PVP, 1570 μg/mL; AgNP1, 10 μg/ mL (0.009 mM Ag); AgNP2, 10 μg/ mL (0.093 mM Ag); AgNP3, 100 μg /mL (0.927 mM Ag); and AgNO3, 157 μg /mL (0.927 mM).
Fig. 2. Morphology of Perkinsus marinus hypnospores before and after exposure to AgNP. (A) Live hypnospores showing a spherical shape with well-defined cell walls (W) and granular contents. Inverted microscope 20×. (B) Dead hypnospores after exposure to 10 µg/mL AgNP for 24 h, showing an irregular structure, no cellular contents and fragmentation of the cell wall (head arrow). Inverted microscope 40x.
razor blade and incubated in Ray’s Fluid Thioglycollate Medium (RFTM), fortified with 200 U of nystatin and 200 µg of chloramphenicol per mL of medium. Tissues from each oyster were incubated separately and stored in the dark at room temperature (20 ± 2 °C) for 7 d to allow development of P. marinus hypnospores. Incubation was followed by iodine staining and microscopic examination for detection of the parasite. The hypnospores were isolated by macerating the infected tissue incubated in RFTM and passing it through a series of filters (100, 70, 40 and 20 μm, Cruz-Flores et al., 2015). The filter meshes, with hypnospores, were placed in 1.5-mL microcentrifuge tubes and washed 3 times with sterile seawater at 10,000 rpm for 5 min. The supernatant was then replaced by DME to avoid recurrent contamination that occurred in RFTM. The hypnospores were maintained, until assayed, at 28 °C in DME culture medium: Ham's F12 (1:2) supplemented with 5% inactive fetal bovine serum and antibiotics (200 μg/mL of streptomycin and 200 U/mL of penicillin G) (Gauthier and Vasta, 1995). To verify the identity of the parasite, DNA from hypnospores sampled from DME was analyzed using a polymerase chain reaction (PCR). DNA extraction was performed using a DNeasy® Blood & Tissue Kit (QIAGEN) in accordance with the manufacturer's protocol. The PCR was performed using primers that amplify the non-transcribed spacer (NTS) region of ribosomal RNA, with species-specific oligonucleotides for P. marinus (Marsh et al., 1995; Robledo et al., 1998, 1999), NTS-1F (5′- CAC TTG TAT TGT GAA GCA CCC -3′) and NTS-2R (5′- TTG GTG ACA TCT CCA AAT GAC -3′), resulting in a 307-bp product. The final volume of the PCR mixture was 25 μL, consisting of the following: 1X buffer (10 mM Tris-HCl, pH 8.5, 50 mM KCl, 1.5 mM MgCl 2), 100 μM dNTPs, 1 μM of each primer, 1.5 U of Taq DNA polymerase and 1 μg of temperate DNA (Marsh et al., 1995). The PCR was performed in an
impracticality of these treatments, alternative treatments for P. marinus are needed. Silver nanoparticles (AgNP) have been evaluated against a variety of biological entities and have been shown to be lethal to ciliated protozoans such as Tetrahymena sp. and Paramecium caudatum (Shi et al., 2013; Abe et al., 2014; Juganson et al., 2017). AgNP induces lysis of the cell membrane of Tetrahymena sp. (Fuentes-Valencia and Chávez-Sánchez, personal communication, unpublished data) and induce abnormal swimming followed by a cessation of cell locomotion in P. caudatum (Abe et al., 2014). Vázquez-Muñoz et al. (2017) used AgNP against viruses, bacteria, fungi, microalgae and human cell lines in vitro and observed mortality at silver concentrations equivalent to 10 μg/ml AgNP. The efficacy of AgNP in a saline medium is unknown; all cited studies have involved bioassays in non-saline media, where physicochemical interactions between the media and AgNP allowed their lethality to be tested. The aim of the present study is to determine the lethal effects of the Argovit™ formulation of AgNP on hypnospores of P. marinus maintained in vitro in a saline medium and to determine AgNP effects on the ultrastructure of the parasite using transmission electron microscopy (TEM).
2. Materials and methods 2.1. Perkinsus marinus To obtain hypnospores of the parasites, 30 C. corteziensis (shell height 10.5 ± 1.4 cm) were collected from Boca de Camichín in the State of Nayarit, Mexico, where P. marinus was previously reported (Cáceres-Martínez et al., 2008). Following the protocol of Ray (1966), all oysters were sacrificed, and the rectum, gill and mantle (~2 g) of each were excised. The tissues were finely cut (< 1 mm3) with a sterile 2
Journal of Invertebrate Pathology 169 (2020) 107304
C. Bravo-Guerra, et al.
in PVP at 1570 μg/mL, equivalent to the highest silver concentration of the Argovit™ AgNP. 2.3. In vitro experiment After 6 d of isolation in culture medium, the hypnospores of P. marinus were distributed among the wells of a 24-well cell culture plate (VWR®), with each well containing an average of 84 ± 3 hypnospores, 900 μL of fresh culture medium and 100 μL of the corresponding treatment solution, achieving a final volume of 1 mL. The entire procedure was performed in triplicate. Hypnospores exposed to AgNP were incubated at 28 °C for four exposure times: 1, 6, 12 and 24 h. A total of four plates were used, one for each exposure time. P. marinus hypnospores were counted directly before and after treatment, and percent mortality was calculated for each treatment and exposure time. Visualized under an inverted microscope, criteria for mortality included deformity of the hypnospores, fragmentation of the cell wall and empty appearance. 2.4. Fine structure analysis Ultrastructural analysis was performed on samples from the control treatment (culture medium) and from the AgNP treatment with a concentration of 0.093 mM and 24 h exposure. These samples were placed in a 1.5-mL microcentrifuge tube with 500 µL of isolated hypnospores and 500 µL of fresh culture medium. The resulting pellet was fixed in 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4) for 4 h at room temperature and then rinsed 3x in 0.1 M sodium cacodylate (pH 7.4) at 4 °C for approximately 1 h per rinse. Post-fixation was done using a solution of 1% osmium tetraoxide (OsO4) in a sodium cacodylate buffer for 3 h and followed by a wash with 0.1 M sodium cacodylate at 4 °C for approximately 1 h. The fixed samples were dehydrated in ethanol in a series of concentrations (20, 40, 60, 80 and twice in 100%). After dehydration, the samples were embedded in Spurr resin (low viscosity kit, PELCO®) at different concentrations of ethanol/resin (80/20, 60/40, 40/60, 20/80 and twice in 100% resin) and finally polymerized at 60 °C for 12 h. Ultracuts of 70 nm were then made with an ultramicrotome (Leica, Ultracut R) with subsequent analysis using TEM. No post-staining of the samples was done, thus allowing AgNP to be easily detected.
Fig. 3. Hypnospores of Perkinsus marinus. (A) Hypnospore from the control treatment showing the conserved round shape of the external wall (W) and lipid droplets (Li), with magnification of the image in the square to right. (B) Hypnospores exposed to 10 μg/mL of AgNP for 24 h; completely deformed, with several lipid droplets in the cytoplasm full of AgNP (indicated with arrows). See the square on the right where lipid droplets are deforming, associated with large numbers of AgNP.
2.5. Statistical analysis Hypnospore mortality was defined as the response variable, and the result was expressed as a percentage. The data were transformed and evaluated using a Kruskal-Wallis analysis of variance, comparing the effect of different concentrations of AgNP for each time period of exposure. Significant differences were determined using Fisher's Least Significant Difference (LSD) post hoc test. Treatment differences were considered statistically significant when p < 0.05 (Zar, 1974).
Apollo thermocycler (Continental Lab Products). The amplification conditions were described previously by Elandallousi et al. (2004). Briefly, they included initial denaturation at 91 °C for 3 min; 35 cycles of 91 °C for 1 min, 58 °C for 1 min 1 s, and 72 °C for 1 min 2 s; and a final extension at 72 °C for 10 min. 2.2. AgNP preparation
3. Results
Argovit™, a commercial formulation of AgNP, was provided by Dr. Nina Bogdanchikova from Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México. The Argovit™ formulation is a highly dispersed suspension of AgNP with 12 mg/mL of metallic silver stabilized in 188 mg/mL of polyvinylpyrrolidone (PVP). The size distribution and morphology of the AgNP consist of spheroidal particles with an average diameter of 35 ± 15 nm with a hydrodynamic radius of 70 nm (Juárez-Moreno et al., 2017). AgNP solutions were prepared based on the metallic silver content in the Argovit™ formulation and diluted 120× to obtain a stock solution with a concentration of 100 µg/ mL (0.927 mM of silver). Test concentrations of silver were selected based on Vázquez-Muñoz et al. (2017). For comparative purposes we prepared solutions of silver nitrate (AgNO3) at 157 μg/mL (0.927 mM)
Formation of a discharge tube by hypnospores to possibly release zoospores was rarely observed in DME culture media. The majority of hypnospores maintained their characteristic appearance. The time and concentration dependence effects on the viability of P. marinus hypnospores that were exposed to AgNP are shown in Fig. 1. The highest assayed concentration of AgNP resulted in a decrease in the viability of hypnospores after only 1 h of exposure. For this concentration of AgNP, the mortality of hypnospores increased as a function of time, reaching greater than 75% at 6 h and greater than 90% at 12 and 24 h. The effects on hypnospore viability of the other AgNP concentrations assessed were clear after 12 and 24 h of exposure. Treatment with AgNP with a concentration of 10 µg/ mL (0.093 mM) resulted in 50% mortality after 24 h of exposure. At 24 h, a clear concentration dependence 3
Journal of Invertebrate Pathology 169 (2020) 107304
C. Bravo-Guerra, et al.
Fig. 4. Transmission electron micrographs of Perkinsus marinus hypnospores. (A) The cell wall under control treatment. (B) Ultrathin section of the hypnospore wall exposed to 10 μg/ mL of AgNP for 24 h. The presence of AgNP in the wall is indicated with arrows (TEM 20,000×). The square on the right shows AgNP in the cell wall (50,000×). (C) Lipid droplets showing alterations of their structures by exposure to AgNP (arrows). (D) Close up of a lipid drop being disrupted by AgNP (arrows). Abbreviations: W, wall; Li, lipid droplet; C, cytoplasm.
4. Discussion
was observed for hypnospore viability. There was no difference in mortality between hypnospores exposed to AgNO3 for 1 h and those subjected to the negative control. A large viability decrease was observed after 6 h of exposure, similar to that found for the highest concentration of AgNP. The control treatment (culture medium) resulted in a mortality of less than 10% (Fig. 1). Observed using the inverted microscope, the hypnospores of P. marinus subjected to the negative control were spherical (55 ± 21 µm of diameter) with thick walls and granular content in their cytoplasm (Fig. 2a). The hypnospores exposed to treatments with AgNP exhibited an irregular shape, lysed walls and an empty appearance (Fig. 2b). The AgNO3 treatment completely disintegrated the structure of hypnospores, leaving no evidence of the cell. The ultrastructure of hypnospores from the control treatment exhibited spherical shapes with thick cell walls and several lipid droplets (Fig. 3a). Detailed images of hypnospore cell walls from the control group, showing a normal ultrastructure consisting of a thick wall and semispherical lipid droplets, are presented in Fig. 4a. In contrast, hypnospores exposed to treatments with AgNP showed great structural deformation, in some cases showing a kidney-shaped structure (Fig. 3b); moreover, lipid droplets located in the cell wall or within the cytoplasm were deformed, many of them containing abundant AgNP. Detailed images of hypnospores exposed to AgNP show the presence of AgNP in the cell wall and the cytoplasm (Fig. 4b). In the cytoplasm, AgNP were observed attached to the exterior of lipid droplets (Fig. 4c and d).
The Argovit™ formulation of AgNP had a clear lethal effect on P. marinus hypnospores following 24 h of exposure. Argovit™ silver nanoparticles are highly lethal to P. marinus hypnospores with doses of 100 µg/mL achieving 75% mortality following 6 h of exposure and 90% mortality at 24 h. A silver concentration ten times lower (10 µg/mL AgNP) induced 50% mortality in P. marinus hypnospores after 24 h of exposure, and maintained its lethal effect for up to 24 h in the saline medium. The same concentration of AgNP (10 µg/mL) used by Abe et al., 2014, killed 50% of P. caudatum, a freshwater protozoan. The current results were obtained in a saline medium, suggesting that saline conditions do not interfere with the lethal activity of AgNP. According to Marambio-Jones and Hoek (2010), the use of metallic nanoparticles is limited by the solubility of silver ions in biological and environmental media containing Cl-; therefore, saline medium could present practical disadvantages due to the activity of silver ions inducing the formation of stable compounds such as silver chloride (AgCl), with low solubility and rapid precipitation. Although the biochemical environment might affect AgNP dissolution (Zhang et al., 2016), Argovit™ AgNP showed activity after 24 h under experimental conditions. Coated silver nanoparticles might interfere with possible chemical reactions; however, similar results were obtained with AgNO3. Detailed studies are necessary to determine the physicochemical behavior of AgNP in PVP under saline conditions, where interaction with salt compounds undoubtedly interferes with AgNP. Several hypotheses have been raised concerning
4
Journal of Invertebrate Pathology 169 (2020) 107304
C. Bravo-Guerra, et al.
the mechanism of action of AgNP. One proposes a toxic effect related to the production of free radicals by the AgNP, which interact with the lipids of the basic structure of cell membranes by triggering the process of lipid peroxidation (Avello and Suwalsky, 2006), or due to the interaction of ionic silver with thiol groups of vital enzymes of the membrane, agglomerating on the surface or penetrating into the cell and affecting its permeability (Singh et al., 2008). It is quite possible that AgNP produces reactive oxygen species (ROS) within the cell walls and lipid drops of P. marinus hypnospores. ROS were produced on human tumor cells and plant cells exposed to the same AgNP formulation. ROS overproduction is directly associated with cell death pathways triggered in those cells (Juárez-Moreno et al., 2017; SpinosoCastillo et al., 2017). AgNP accumulation and ROS overproduction might contribute to explanations of the damage observed in lipid droplets in the cytoplasm of the parasite. The presence of AgNP within cell walls and lipid droplets suggests endocytosis and provides evidence for the proposed interaction of free radicals with lipids mentioned above. Evidence from electron microscopy provides additional insight into the mechanisms of damage to P. marinus hypnospore cell walls and cytoplasm, with AgNP apparently degrading lipid droplets, possibly the cause of parasite death and suggesting that accumulation of AgNP in the lipidic regions of parasite cells could be an important factor in the observed biological response. It is important to note that the hypnospore stage is quite different than those found in oyster tissue or hemolymph of infected oysters, thus it is necessary to determine the effect of the Argovit™ formulation of AgNP in trophozoites and tomonts that correspond to proliferative stages of the parasite in infected oysters. The data generated in this study constitute a baseline for research on the possible effect of AgNP in infected oysters and the environment.
Health 22, 141–151. Cáceres-Martínez, J., García-Ortega, M., Vásquez-Yeomans, R., Pineda-García, T.J., Stokes, N.A., Carnegie, R.B., 2012. Natural and cultured populations of the mangrove oyster Saccostrea palmula from Sinaloa, Mexico, infected by Perkinsus marinus. J. Invertebr. Pathol. 110 (3), 321–325. https://doi.org/10.1016/j.jip.2012.03.019. Calvo, G.W., Burreson, E.M., 1994. In vitro and in vivo effects of eight chemotherapeutants on the oyster parasite Perkinsus marinus (Mackin, Owen, and Collier). J. Shellfish Res. 13, 101–107. Cruz- Flores, R., Vásquez-Yeomans, R., Cáceres-Martínez, J., 2015. A novel method for separation of Rickettsiales-like organism “Candidatus Xenohaliotis californiensis” from host abalone tissue. J. Microbiol. Methods 115, 79–82. Delaney, M., Brady, Y., Worley, S., 2003. The effectiveness of N-halamine disinfectant compounds on Perkinsus marinus, a parasite of the eastern oyster Crassostrea virginica. J. Shellfish Res. 22, 91–94. Elandalloussi, L.M., Leite, R.M., Afonso, R., Nunez, P.A., Robledo, J.A.F., Vasta, G.R., Cancela, M.L., 2004. Development of a PCR-ELISA assay for diagnosis of Perkinsus marinus and Perkinsus atlanticus infections in bivalve molluscs. Mol. Cell. Probes 18, 89–96. Faisal, M., La Peyre, J.F., Elsayed, E., Wright, D.C., 1999. Bacitracin inhibits the oyster pathogen Perkinsus marinus in vitro and in vivo. J. Aquat. Anim. Health 11, 130–138. Gauthier, J.D., Vasta, G.R., 1995. In vitro culture of the eastern oyster parasite Perkinsus marinus: optimization of the methodology. J. Invertebr. Pathol. 66, 156–168. Gullian-Klanian, M., Herrera-Silveira, J.A., Rodríguez-Canul, R., Aguirre-Macedo, L., 2008. Factors associated with the prevalence of Perkinsus marinus in Crassostrea virginica from the southern Gulf of Mexico. Dis. Aquat. Org. 79, 237–247. ICES, 2011. Dermo disease of oysters caused by Perkinsus marinus. Revised and updated by Susan E. Ford. ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish. Leaflet No. 30. 5 pp. Juárez-Moreno, K., Gonzalez, E.B., Girón-Vazquez, N., Chávez-Santoscoy, R.A., MotaMorales, J.D., Perez-Mozqueda, L.L., Garcia-Garcia, M.R., Pestryakov, A., Bogdanchikova, N., 2017. Comparison of cytotoxicity and genotoxicity effects of silver nanoparticles on human cervix and breast cancer cell lines. Hum. Exp. Toxicol. 36, 931–948. https://doi.org/10.1177/0960327116675206. Juganson, K., Mortimer, M., Ivaska, A., Pucciarelli, S., Miceli, C., Orupõldd, K., Kahru, A., 2017. Mechanisms of toxic action of silver nanoparticles in the protozoan Tetrahymena thermophila: from gene expression to phenotypic events. Environ. Pollut. 225, 481–489. Marambio Jones, C., Hoek, E., 2010. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J. Nanopart. Res. 12, 1531–1551. Marsh, A.G., Gauthier, J.D., Vasta, G.R., 1995. A semiquantitative PCR assay for assessing Perkinsus marinus infections in the eastern oyster, Crassostrea virginica. J. Parasitol. 81, 577–583. Perkins, F.O., 1996. The structure of Perkinsus marinus (Mackin, Owen and Collier, 1950) Levine, 1978 with comments on taxonomy and phylogeny of Perkinsus spp. J. Shellfish Res. 15, 67–87. Ray, S.M., 1966. A review of the culture method for detecting Dermocystidium marinum, with suggested modifications and precautions. Proc. Natl. Shellfish. Ass. 54, 55–69. Ray, S.M., 1996. Historical perspective on Perkinsus marinus disease of oysters in the Gulf of Mexico. J. Shellfish Res. 15, 9–11. Robledo, J.A.F., Gauthier, D.J., Coss, C.A., Wright, A.C., Vasta, G.R., 1998. Species-specificity and sensitivity of a PCR-based assay for Perkinsus marinus in the eastern oyster Crassostrea virginica: a comparison with the fluid thioglycollate assay. J. Parasitol. 84, 1237–1244. Robledo, J.A.F., Wright, A.C., Marsh, A.G., Vasta, G.R., 1999. Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus. J. Parasitol. 85, 650–656. Shi, J., Xu, B., Sun, X., Ma, C., Yu, C., Zhang, H., 2013. Light induced toxicity reduction of silver nanoparticles to Tetrahymena pyriformis: Effect of particle size. Aquat. Toxicol. 132, 53–60. Singh, M., Singh, S., Prasad, S., Gambhir, I.S., 2008. Nanotechnology in medicine and antibacterial effect of silver nanoparticles. Dig. J. Nanomater. Biostruct. 3, 115–122. Spinoso-Castillo, J.L., Chavez-Santoscoy, R.A., Bogdanchikova, N., Pérez-Sato, J.A., Morales-Ramos, V., Bello-Bello, J.J., 2017. Antimicrobial and hormetic effects of silver nanoparticles on in vitro regeneration of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system, Plant Cell. Tissue Org. Cult. 129, 195–207. https://doi.org/10.1007/s11240-017-1169-8. Vázquez-Muñóz, R., Borrego, B., Juárez-Moreno, K., García-García, M., Mota-Morales, J.D., Bogdanchikova, N., Huerta-Saquero, A., 2017. Toxicity of silver nanoparticles in biological systems: Does the complexity of biological systems matter? Toxicol. Lett. 276, 11–20. https://doi.org/10.1016/j.toxlet.2017.05.007. Zar, J.H., 1974. Biostatistical Analysis. Prentice Hall, Englewood Cliffs, NJ. Zhang, C., Hu, Z., Deng, B., 2016. Silver nanoparticles in aquatic environments: physiochemical behavior and antimicrobial mechanisms. Water Res. 88, 403–427.
Acknowledgments This project was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) 258607 Ciencia Básica from 2017–2019. Silver nanoparticles Argovit™ provided by Dr. Nina Bogdanchikova becomes from Tomsk Polytechnic University Competitiveness Program grant VIU-ISHBMT-65/2019. Thanks to Mc. Yanet Guerrero Rentería for her assistance in laboratory, to Mc. Juan Manuel Martínez Andrade for his service in processing TEM images and to Dr. Juan C. García-Ramos for revision of the manuscript. References Abe, T., Haneda, K., Haga, N., 2014. Silver nanoparticle cytotoxicity and antidote proteins against silver toxicity in Paramecium. Nano Biomed. 6, 35–40. Avello, M., Suwalsky, M., 2006. Radicales libres, antioxidantes naturales y mecanismos de protección. Atenea 429, 161–172. Bushek, D., Holley, R., Kell, M., 1997. Chlorine tolerance of Perkinsus marinus. J. Shellfish Res. 16, 260 (Abstract). Bushek, D., Howell, T.L., 2000. The effect of UV irradiation on Perkinsus marinus and its potential use to reduce transmission via shellfish effluents. Northeastern Regional Aquaculture Center (NRAC) Publication No. 00-008, North Dartmouth, Massachusetts, USA 5p. Bushek, D., Ford, S.E., Chintala, M.M., 2002. Comparison of in vitro-cultured and wildtype Perkinsus marinus. III. Fecal elimination and its role in transmission. Dis. Aquat. Org. 51, 217–225. Cáceres-Martínez, J., Vásquez-Yeomans, R., Padilla-Lardizábal, G., del Río-Portilla, M.A., 2008. Perkinsus marinus in pleasure oyster Crassostrea corteziensis from Nayarit, Pacific coast of México. J. Invertebr. Pathol. 99, 66–73. Cáceres-Martínez, J., Vásquez-Yeomans, R., Padilla-Lardizábal, G., 2010. Parasites of the pleasure oyster Crassostrea corteziensis cultured in Nayarit. Mexico. J. Aquat. Anim.
5
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Lethal effects of silver nanoparticles on Perkinsus marinus, a protozoan oyster parasite
T
⁎
Cecilia Bravo-Guerraa, Jorge Cáceres-Martíneza, , Rebeca Vásquez-Yeomansa, Alexey Pestryakovb, Nina Bogdanchikovac a
Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana No. 3918, CP 22860 Ensenada, Baja California, Mexico Tomsk Polytechnic University, Lenin Avenue 30, Tomsk 634050, Russia c Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, km 107 carretera Ensenada-Tijuana, CP 22860 Ensenada, Baja California, Mexico b
A R T I C LE I N FO
A B S T R A C T
Keywords: Hypnospores Perkinsus marinus Silver nanoparticles
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.
1. Introduction Diseases in aquatic organisms pose an economic risk and a management challenge for the aquaculture industry, affecting animal health and impacting production. Among these diseases, “dermo disease”, which is caused by the protozoan Perkinsus marinus, is one of the most important for the eastern oyster, Crassostrea virginica, a species cultured on the East Coast of the United States (Gullian-Klanian et al., 2008; ICES, 2011), due to the mortality it has caused in oyster populations since the 1940s (Ray, 1996). Perkinsus marinus is considered a notifiable parasite by the World Organisation for Animal Health (OIE) and has been found to infect other species such as the Cortez oyster, Crassostrea corteziensis, and the mangrove oyster, Saccostrea palmula, on the Pacific coast of northwest Mexico (Cáceres-Martínez et al., 2010; Cáceres-
⁎
Martínez et al., 2012). P. marinus can be transmitted from host to host, and all phases of its life cycle are infectious. It grows in a vegetative phase as a trophozoite, multiplying in the tissues and hemocytes of its host (Perkins, 1996) and is released into the water in feces or from the tissues of dead hosts (Bushek et al., 2002). P. marinus may develop into hypnospores, a resistant stage that remains in the environment until favorable conditions induce production of biflagellate zoospores (Perkins, 1996). Some chemotherapeutants and disinfectant compounds such as chlorine (300 ppm), UV light (> 28,000 μWs cm2), Nhalamin compounds and fresh water inactivate cultured P. marinus cells (Bushek et al., 1997; Bushek and Howell, 2000; Delaney et al., 2003). The use of bacitracin, cycloheximide and fresh water have been shown to reduce but not eliminate P. marinus in infected oysters (Calvo and Burreson, 1994; Faisal et al., 1999). Because of the ineffectiveness or
Corresponding author. E-mail address: [email protected] (J. Cáceres-Martínez).
https://doi.org/10.1016/j.jip.2019.107304 Received 23 June 2019; Received in revised form 3 December 2019; Accepted 5 December 2019 Available online 07 December 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 169 (2020) 107304
C. Bravo-Guerra, et al.
Fig. 1. Mortality of Perkinsus marinus hypnospores exposed to different silver nanoparticle treatments as a function of time. Different letters (a, b, c…n) indicate significant differences for each time period. The data are expressed as the means ± SD of three replicates per treatment. Abbreviations: Control, culture medium; PVP, 1570 μg/mL; AgNP1, 10 μg/ mL (0.009 mM Ag); AgNP2, 10 μg/ mL (0.093 mM Ag); AgNP3, 100 μg /mL (0.927 mM Ag); and AgNO3, 157 μg /mL (0.927 mM).
Fig. 2. Morphology of Perkinsus marinus hypnospores before and after exposure to AgNP. (A) Live hypnospores showing a spherical shape with well-defined cell walls (W) and granular contents. Inverted microscope 20×. (B) Dead hypnospores after exposure to 10 µg/mL AgNP for 24 h, showing an irregular structure, no cellular contents and fragmentation of the cell wall (head arrow). Inverted microscope 40x.
razor blade and incubated in Ray’s Fluid Thioglycollate Medium (RFTM), fortified with 200 U of nystatin and 200 µg of chloramphenicol per mL of medium. Tissues from each oyster were incubated separately and stored in the dark at room temperature (20 ± 2 °C) for 7 d to allow development of P. marinus hypnospores. Incubation was followed by iodine staining and microscopic examination for detection of the parasite. The hypnospores were isolated by macerating the infected tissue incubated in RFTM and passing it through a series of filters (100, 70, 40 and 20 μm, Cruz-Flores et al., 2015). The filter meshes, with hypnospores, were placed in 1.5-mL microcentrifuge tubes and washed 3 times with sterile seawater at 10,000 rpm for 5 min. The supernatant was then replaced by DME to avoid recurrent contamination that occurred in RFTM. The hypnospores were maintained, until assayed, at 28 °C in DME culture medium: Ham's F12 (1:2) supplemented with 5% inactive fetal bovine serum and antibiotics (200 μg/mL of streptomycin and 200 U/mL of penicillin G) (Gauthier and Vasta, 1995). To verify the identity of the parasite, DNA from hypnospores sampled from DME was analyzed using a polymerase chain reaction (PCR). DNA extraction was performed using a DNeasy® Blood & Tissue Kit (QIAGEN) in accordance with the manufacturer's protocol. The PCR was performed using primers that amplify the non-transcribed spacer (NTS) region of ribosomal RNA, with species-specific oligonucleotides for P. marinus (Marsh et al., 1995; Robledo et al., 1998, 1999), NTS-1F (5′- CAC TTG TAT TGT GAA GCA CCC -3′) and NTS-2R (5′- TTG GTG ACA TCT CCA AAT GAC -3′), resulting in a 307-bp product. The final volume of the PCR mixture was 25 μL, consisting of the following: 1X buffer (10 mM Tris-HCl, pH 8.5, 50 mM KCl, 1.5 mM MgCl 2), 100 μM dNTPs, 1 μM of each primer, 1.5 U of Taq DNA polymerase and 1 μg of temperate DNA (Marsh et al., 1995). The PCR was performed in an
impracticality of these treatments, alternative treatments for P. marinus are needed. Silver nanoparticles (AgNP) have been evaluated against a variety of biological entities and have been shown to be lethal to ciliated protozoans such as Tetrahymena sp. and Paramecium caudatum (Shi et al., 2013; Abe et al., 2014; Juganson et al., 2017). AgNP induces lysis of the cell membrane of Tetrahymena sp. (Fuentes-Valencia and Chávez-Sánchez, personal communication, unpublished data) and induce abnormal swimming followed by a cessation of cell locomotion in P. caudatum (Abe et al., 2014). Vázquez-Muñoz et al. (2017) used AgNP against viruses, bacteria, fungi, microalgae and human cell lines in vitro and observed mortality at silver concentrations equivalent to 10 μg/ml AgNP. The efficacy of AgNP in a saline medium is unknown; all cited studies have involved bioassays in non-saline media, where physicochemical interactions between the media and AgNP allowed their lethality to be tested. The aim of the present study is to determine the lethal effects of the Argovit™ formulation of AgNP on hypnospores of P. marinus maintained in vitro in a saline medium and to determine AgNP effects on the ultrastructure of the parasite using transmission electron microscopy (TEM).
2. Materials and methods 2.1. Perkinsus marinus To obtain hypnospores of the parasites, 30 C. corteziensis (shell height 10.5 ± 1.4 cm) were collected from Boca de Camichín in the State of Nayarit, Mexico, where P. marinus was previously reported (Cáceres-Martínez et al., 2008). Following the protocol of Ray (1966), all oysters were sacrificed, and the rectum, gill and mantle (~2 g) of each were excised. The tissues were finely cut (< 1 mm3) with a sterile 2
Journal of Invertebrate Pathology 169 (2020) 107304
C. Bravo-Guerra, et al.
in PVP at 1570 μg/mL, equivalent to the highest silver concentration of the Argovit™ AgNP. 2.3. In vitro experiment After 6 d of isolation in culture medium, the hypnospores of P. marinus were distributed among the wells of a 24-well cell culture plate (VWR®), with each well containing an average of 84 ± 3 hypnospores, 900 μL of fresh culture medium and 100 μL of the corresponding treatment solution, achieving a final volume of 1 mL. The entire procedure was performed in triplicate. Hypnospores exposed to AgNP were incubated at 28 °C for four exposure times: 1, 6, 12 and 24 h. A total of four plates were used, one for each exposure time. P. marinus hypnospores were counted directly before and after treatment, and percent mortality was calculated for each treatment and exposure time. Visualized under an inverted microscope, criteria for mortality included deformity of the hypnospores, fragmentation of the cell wall and empty appearance. 2.4. Fine structure analysis Ultrastructural analysis was performed on samples from the control treatment (culture medium) and from the AgNP treatment with a concentration of 0.093 mM and 24 h exposure. These samples were placed in a 1.5-mL microcentrifuge tube with 500 µL of isolated hypnospores and 500 µL of fresh culture medium. The resulting pellet was fixed in 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4) for 4 h at room temperature and then rinsed 3x in 0.1 M sodium cacodylate (pH 7.4) at 4 °C for approximately 1 h per rinse. Post-fixation was done using a solution of 1% osmium tetraoxide (OsO4) in a sodium cacodylate buffer for 3 h and followed by a wash with 0.1 M sodium cacodylate at 4 °C for approximately 1 h. The fixed samples were dehydrated in ethanol in a series of concentrations (20, 40, 60, 80 and twice in 100%). After dehydration, the samples were embedded in Spurr resin (low viscosity kit, PELCO®) at different concentrations of ethanol/resin (80/20, 60/40, 40/60, 20/80 and twice in 100% resin) and finally polymerized at 60 °C for 12 h. Ultracuts of 70 nm were then made with an ultramicrotome (Leica, Ultracut R) with subsequent analysis using TEM. No post-staining of the samples was done, thus allowing AgNP to be easily detected.
Fig. 3. Hypnospores of Perkinsus marinus. (A) Hypnospore from the control treatment showing the conserved round shape of the external wall (W) and lipid droplets (Li), with magnification of the image in the square to right. (B) Hypnospores exposed to 10 μg/mL of AgNP for 24 h; completely deformed, with several lipid droplets in the cytoplasm full of AgNP (indicated with arrows). See the square on the right where lipid droplets are deforming, associated with large numbers of AgNP.
2.5. Statistical analysis Hypnospore mortality was defined as the response variable, and the result was expressed as a percentage. The data were transformed and evaluated using a Kruskal-Wallis analysis of variance, comparing the effect of different concentrations of AgNP for each time period of exposure. Significant differences were determined using Fisher's Least Significant Difference (LSD) post hoc test. Treatment differences were considered statistically significant when p < 0.05 (Zar, 1974).
Apollo thermocycler (Continental Lab Products). The amplification conditions were described previously by Elandallousi et al. (2004). Briefly, they included initial denaturation at 91 °C for 3 min; 35 cycles of 91 °C for 1 min, 58 °C for 1 min 1 s, and 72 °C for 1 min 2 s; and a final extension at 72 °C for 10 min. 2.2. AgNP preparation
3. Results
Argovit™, a commercial formulation of AgNP, was provided by Dr. Nina Bogdanchikova from Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México. The Argovit™ formulation is a highly dispersed suspension of AgNP with 12 mg/mL of metallic silver stabilized in 188 mg/mL of polyvinylpyrrolidone (PVP). The size distribution and morphology of the AgNP consist of spheroidal particles with an average diameter of 35 ± 15 nm with a hydrodynamic radius of 70 nm (Juárez-Moreno et al., 2017). AgNP solutions were prepared based on the metallic silver content in the Argovit™ formulation and diluted 120× to obtain a stock solution with a concentration of 100 µg/ mL (0.927 mM of silver). Test concentrations of silver were selected based on Vázquez-Muñoz et al. (2017). For comparative purposes we prepared solutions of silver nitrate (AgNO3) at 157 μg/mL (0.927 mM)
Formation of a discharge tube by hypnospores to possibly release zoospores was rarely observed in DME culture media. The majority of hypnospores maintained their characteristic appearance. The time and concentration dependence effects on the viability of P. marinus hypnospores that were exposed to AgNP are shown in Fig. 1. The highest assayed concentration of AgNP resulted in a decrease in the viability of hypnospores after only 1 h of exposure. For this concentration of AgNP, the mortality of hypnospores increased as a function of time, reaching greater than 75% at 6 h and greater than 90% at 12 and 24 h. The effects on hypnospore viability of the other AgNP concentrations assessed were clear after 12 and 24 h of exposure. Treatment with AgNP with a concentration of 10 µg/ mL (0.093 mM) resulted in 50% mortality after 24 h of exposure. At 24 h, a clear concentration dependence 3
Journal of Invertebrate Pathology 169 (2020) 107304
C. Bravo-Guerra, et al.
Fig. 4. Transmission electron micrographs of Perkinsus marinus hypnospores. (A) The cell wall under control treatment. (B) Ultrathin section of the hypnospore wall exposed to 10 μg/ mL of AgNP for 24 h. The presence of AgNP in the wall is indicated with arrows (TEM 20,000×). The square on the right shows AgNP in the cell wall (50,000×). (C) Lipid droplets showing alterations of their structures by exposure to AgNP (arrows). (D) Close up of a lipid drop being disrupted by AgNP (arrows). Abbreviations: W, wall; Li, lipid droplet; C, cytoplasm.
4. Discussion
was observed for hypnospore viability. There was no difference in mortality between hypnospores exposed to AgNO3 for 1 h and those subjected to the negative control. A large viability decrease was observed after 6 h of exposure, similar to that found for the highest concentration of AgNP. The control treatment (culture medium) resulted in a mortality of less than 10% (Fig. 1). Observed using the inverted microscope, the hypnospores of P. marinus subjected to the negative control were spherical (55 ± 21 µm of diameter) with thick walls and granular content in their cytoplasm (Fig. 2a). The hypnospores exposed to treatments with AgNP exhibited an irregular shape, lysed walls and an empty appearance (Fig. 2b). The AgNO3 treatment completely disintegrated the structure of hypnospores, leaving no evidence of the cell. The ultrastructure of hypnospores from the control treatment exhibited spherical shapes with thick cell walls and several lipid droplets (Fig. 3a). Detailed images of hypnospore cell walls from the control group, showing a normal ultrastructure consisting of a thick wall and semispherical lipid droplets, are presented in Fig. 4a. In contrast, hypnospores exposed to treatments with AgNP showed great structural deformation, in some cases showing a kidney-shaped structure (Fig. 3b); moreover, lipid droplets located in the cell wall or within the cytoplasm were deformed, many of them containing abundant AgNP. Detailed images of hypnospores exposed to AgNP show the presence of AgNP in the cell wall and the cytoplasm (Fig. 4b). In the cytoplasm, AgNP were observed attached to the exterior of lipid droplets (Fig. 4c and d).
The Argovit™ formulation of AgNP had a clear lethal effect on P. marinus hypnospores following 24 h of exposure. Argovit™ silver nanoparticles are highly lethal to P. marinus hypnospores with doses of 100 µg/mL achieving 75% mortality following 6 h of exposure and 90% mortality at 24 h. A silver concentration ten times lower (10 µg/mL AgNP) induced 50% mortality in P. marinus hypnospores after 24 h of exposure, and maintained its lethal effect for up to 24 h in the saline medium. The same concentration of AgNP (10 µg/mL) used by Abe et al., 2014, killed 50% of P. caudatum, a freshwater protozoan. The current results were obtained in a saline medium, suggesting that saline conditions do not interfere with the lethal activity of AgNP. According to Marambio-Jones and Hoek (2010), the use of metallic nanoparticles is limited by the solubility of silver ions in biological and environmental media containing Cl-; therefore, saline medium could present practical disadvantages due to the activity of silver ions inducing the formation of stable compounds such as silver chloride (AgCl), with low solubility and rapid precipitation. Although the biochemical environment might affect AgNP dissolution (Zhang et al., 2016), Argovit™ AgNP showed activity after 24 h under experimental conditions. Coated silver nanoparticles might interfere with possible chemical reactions; however, similar results were obtained with AgNO3. Detailed studies are necessary to determine the physicochemical behavior of AgNP in PVP under saline conditions, where interaction with salt compounds undoubtedly interferes with AgNP. Several hypotheses have been raised concerning
4
Journal of Invertebrate Pathology 169 (2020) 107304
C. Bravo-Guerra, et al.
the mechanism of action of AgNP. One proposes a toxic effect related to the production of free radicals by the AgNP, which interact with the lipids of the basic structure of cell membranes by triggering the process of lipid peroxidation (Avello and Suwalsky, 2006), or due to the interaction of ionic silver with thiol groups of vital enzymes of the membrane, agglomerating on the surface or penetrating into the cell and affecting its permeability (Singh et al., 2008). It is quite possible that AgNP produces reactive oxygen species (ROS) within the cell walls and lipid drops of P. marinus hypnospores. ROS were produced on human tumor cells and plant cells exposed to the same AgNP formulation. ROS overproduction is directly associated with cell death pathways triggered in those cells (Juárez-Moreno et al., 2017; SpinosoCastillo et al., 2017). AgNP accumulation and ROS overproduction might contribute to explanations of the damage observed in lipid droplets in the cytoplasm of the parasite. The presence of AgNP within cell walls and lipid droplets suggests endocytosis and provides evidence for the proposed interaction of free radicals with lipids mentioned above. Evidence from electron microscopy provides additional insight into the mechanisms of damage to P. marinus hypnospore cell walls and cytoplasm, with AgNP apparently degrading lipid droplets, possibly the cause of parasite death and suggesting that accumulation of AgNP in the lipidic regions of parasite cells could be an important factor in the observed biological response. It is important to note that the hypnospore stage is quite different than those found in oyster tissue or hemolymph of infected oysters, thus it is necessary to determine the effect of the Argovit™ formulation of AgNP in trophozoites and tomonts that correspond to proliferative stages of the parasite in infected oysters. The data generated in this study constitute a baseline for research on the possible effect of AgNP in infected oysters and the environment.
Health 22, 141–151. Cáceres-Martínez, J., García-Ortega, M., Vásquez-Yeomans, R., Pineda-García, T.J., Stokes, N.A., Carnegie, R.B., 2012. Natural and cultured populations of the mangrove oyster Saccostrea palmula from Sinaloa, Mexico, infected by Perkinsus marinus. J. Invertebr. Pathol. 110 (3), 321–325. https://doi.org/10.1016/j.jip.2012.03.019. Calvo, G.W., Burreson, E.M., 1994. In vitro and in vivo effects of eight chemotherapeutants on the oyster parasite Perkinsus marinus (Mackin, Owen, and Collier). J. Shellfish Res. 13, 101–107. Cruz- Flores, R., Vásquez-Yeomans, R., Cáceres-Martínez, J., 2015. A novel method for separation of Rickettsiales-like organism “Candidatus Xenohaliotis californiensis” from host abalone tissue. J. Microbiol. Methods 115, 79–82. Delaney, M., Brady, Y., Worley, S., 2003. The effectiveness of N-halamine disinfectant compounds on Perkinsus marinus, a parasite of the eastern oyster Crassostrea virginica. J. Shellfish Res. 22, 91–94. Elandalloussi, L.M., Leite, R.M., Afonso, R., Nunez, P.A., Robledo, J.A.F., Vasta, G.R., Cancela, M.L., 2004. Development of a PCR-ELISA assay for diagnosis of Perkinsus marinus and Perkinsus atlanticus infections in bivalve molluscs. Mol. Cell. Probes 18, 89–96. Faisal, M., La Peyre, J.F., Elsayed, E., Wright, D.C., 1999. Bacitracin inhibits the oyster pathogen Perkinsus marinus in vitro and in vivo. J. Aquat. Anim. Health 11, 130–138. Gauthier, J.D., Vasta, G.R., 1995. In vitro culture of the eastern oyster parasite Perkinsus marinus: optimization of the methodology. J. Invertebr. Pathol. 66, 156–168. Gullian-Klanian, M., Herrera-Silveira, J.A., Rodríguez-Canul, R., Aguirre-Macedo, L., 2008. Factors associated with the prevalence of Perkinsus marinus in Crassostrea virginica from the southern Gulf of Mexico. Dis. Aquat. Org. 79, 237–247. ICES, 2011. Dermo disease of oysters caused by Perkinsus marinus. Revised and updated by Susan E. Ford. ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish. Leaflet No. 30. 5 pp. Juárez-Moreno, K., Gonzalez, E.B., Girón-Vazquez, N., Chávez-Santoscoy, R.A., MotaMorales, J.D., Perez-Mozqueda, L.L., Garcia-Garcia, M.R., Pestryakov, A., Bogdanchikova, N., 2017. Comparison of cytotoxicity and genotoxicity effects of silver nanoparticles on human cervix and breast cancer cell lines. Hum. Exp. Toxicol. 36, 931–948. https://doi.org/10.1177/0960327116675206. Juganson, K., Mortimer, M., Ivaska, A., Pucciarelli, S., Miceli, C., Orupõldd, K., Kahru, A., 2017. Mechanisms of toxic action of silver nanoparticles in the protozoan Tetrahymena thermophila: from gene expression to phenotypic events. Environ. Pollut. 225, 481–489. Marambio Jones, C., Hoek, E., 2010. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J. Nanopart. Res. 12, 1531–1551. Marsh, A.G., Gauthier, J.D., Vasta, G.R., 1995. A semiquantitative PCR assay for assessing Perkinsus marinus infections in the eastern oyster, Crassostrea virginica. J. Parasitol. 81, 577–583. Perkins, F.O., 1996. The structure of Perkinsus marinus (Mackin, Owen and Collier, 1950) Levine, 1978 with comments on taxonomy and phylogeny of Perkinsus spp. J. Shellfish Res. 15, 67–87. Ray, S.M., 1966. A review of the culture method for detecting Dermocystidium marinum, with suggested modifications and precautions. Proc. Natl. Shellfish. Ass. 54, 55–69. Ray, S.M., 1996. Historical perspective on Perkinsus marinus disease of oysters in the Gulf of Mexico. J. Shellfish Res. 15, 9–11. Robledo, J.A.F., Gauthier, D.J., Coss, C.A., Wright, A.C., Vasta, G.R., 1998. Species-specificity and sensitivity of a PCR-based assay for Perkinsus marinus in the eastern oyster Crassostrea virginica: a comparison with the fluid thioglycollate assay. J. Parasitol. 84, 1237–1244. Robledo, J.A.F., Wright, A.C., Marsh, A.G., Vasta, G.R., 1999. Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus. J. Parasitol. 85, 650–656. Shi, J., Xu, B., Sun, X., Ma, C., Yu, C., Zhang, H., 2013. Light induced toxicity reduction of silver nanoparticles to Tetrahymena pyriformis: Effect of particle size. Aquat. Toxicol. 132, 53–60. Singh, M., Singh, S., Prasad, S., Gambhir, I.S., 2008. Nanotechnology in medicine and antibacterial effect of silver nanoparticles. Dig. J. Nanomater. Biostruct. 3, 115–122. Spinoso-Castillo, J.L., Chavez-Santoscoy, R.A., Bogdanchikova, N., Pérez-Sato, J.A., Morales-Ramos, V., Bello-Bello, J.J., 2017. Antimicrobial and hormetic effects of silver nanoparticles on in vitro regeneration of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system, Plant Cell. Tissue Org. Cult. 129, 195–207. https://doi.org/10.1007/s11240-017-1169-8. Vázquez-Muñóz, R., Borrego, B., Juárez-Moreno, K., García-García, M., Mota-Morales, J.D., Bogdanchikova, N., Huerta-Saquero, A., 2017. Toxicity of silver nanoparticles in biological systems: Does the complexity of biological systems matter? Toxicol. Lett. 276, 11–20. https://doi.org/10.1016/j.toxlet.2017.05.007. Zar, J.H., 1974. Biostatistical Analysis. Prentice Hall, Englewood Cliffs, NJ. Zhang, C., Hu, Z., Deng, B., 2016. Silver nanoparticles in aquatic environments: physiochemical behavior and antimicrobial mechanisms. Water Res. 88, 403–427.
Acknowledgments This project was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) 258607 Ciencia Básica from 2017–2019. Silver nanoparticles Argovit™ provided by Dr. Nina Bogdanchikova becomes from Tomsk Polytechnic University Competitiveness Program grant VIU-ISHBMT-65/2019. Thanks to Mc. Yanet Guerrero Rentería for her assistance in laboratory, to Mc. Juan Manuel Martínez Andrade for his service in processing TEM images and to Dr. Juan C. García-Ramos for revision of the manuscript. References Abe, T., Haneda, K., Haga, N., 2014. Silver nanoparticle cytotoxicity and antidote proteins against silver toxicity in Paramecium. Nano Biomed. 6, 35–40. Avello, M., Suwalsky, M., 2006. Radicales libres, antioxidantes naturales y mecanismos de protección. Atenea 429, 161–172. Bushek, D., Holley, R., Kell, M., 1997. Chlorine tolerance of Perkinsus marinus. J. Shellfish Res. 16, 260 (Abstract). Bushek, D., Howell, T.L., 2000. The effect of UV irradiation on Perkinsus marinus and its potential use to reduce transmission via shellfish effluents. Northeastern Regional Aquaculture Center (NRAC) Publication No. 00-008, North Dartmouth, Massachusetts, USA 5p. Bushek, D., Ford, S.E., Chintala, M.M., 2002. Comparison of in vitro-cultured and wildtype Perkinsus marinus. III. Fecal elimination and its role in transmission. Dis. Aquat. Org. 51, 217–225. Cáceres-Martínez, J., Vásquez-Yeomans, R., Padilla-Lardizábal, G., del Río-Portilla, M.A., 2008. Perkinsus marinus in pleasure oyster Crassostrea corteziensis from Nayarit, Pacific coast of México. J. Invertebr. Pathol. 99, 66–73. Cáceres-Martínez, J., Vásquez-Yeomans, R., Padilla-Lardizábal, G., 2010. Parasites of the pleasure oyster Crassostrea corteziensis cultured in Nayarit. Mexico. J. Aquat. Anim.
5