Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v63i2.1049Sociobiology 63(2): 851-854 (June, 2016) Antifungal Effect of Silver Nanoparticles on Rickia wasmannii Cavara (Ascomycota: Laboulbeniales) Infecting Myrmica scabrinodis Nylander (Formicidae) Ants Laboulbeniales are obligate ectoparasites of Arthropods with a peculiar biology (Weir & Beakes, 1995; Haelewaters et al., 2015). Insect-Laboulbeniales relationships have several interesting but rather understudied aspects. Recent results showed that ant parasitic Laboulbeniales fungi can influence the physiology and behavior of their hosts (Csata et al., 2014; Báthori et al., 2015a; Konrad et al., 2015), although penetration of the host cuticle was not observed (Tragust et al., 2016). Ants are optimal model organisms for studying the effects of insect parasitic microorganisms, as there is usually a high number of infected (often heavily infected) hosts available (and easily maintained) and methods for studying survival, overall health, behavioral changes, etc. have been established. Furthermore, ant-associated Laboulbeniales are also relatively easy to count using a binocular microscope (Csata et al., 2014; Báthori et al., 2015a; Markó et al., 2016). The importance of uninfected controls in such survival/ behavior oriented studies is beyond question. This may constitute Abstract Rickia wasmannii Cavara (Ascomycota: Laboulbeniales) is an ectoparasitic fungus infecting Myrmica ants. Ant-parasitic Laboulbeniales and their interactions with the hosts have been in the focus of several studies. To assess the effects of these fungi, comparison of infected and uninfected or completely treated ants are needed. So far, treating Laboulbeniales infection was only achieved with cockroaches, but not with ants. We present a simple, yet relatively long, AgNP topical treatment that reduces or eliminates Rickia infection from Myrmica scabrinodis ants without affecting their lifespan. We discuss the possibilities of the proposed treatment in the light of the biology of Rickia. Sociobiology An international journal on social insects WP Pfliegler1,2, L Tálas1, F Báthori3, A Tartally3, I Pócsi1, G Szemán-Nagy1 Article History Edited by Evandro Nascimento Silva, UEFS, Brazil Received 22 April 2016 Initial acceptance 17 May 2016 Final acceptance 17 May 2016 Publication date 15 July 2016 Keywords Antimycotic, fungal parasite, ectoparasite, AgNPs, treatment. Corresponding author András Tartally Department of Evolutionary Zoology and Human Biology University of Debrecen Egyetem tér 1., H4032 - Debrecen, Hungary E-Mail: tartally.andras@science.unideb.hu a problem as infected and uninfected specimens are not necessarily available from the same colony or even the same location (Báthori et al., 2015a). Thus, an alternative approach may be treating the hosts with an antifungal compound and thereby making comparisons between infected and uninfected insects possible. In the case of ants, it could give the possibility to have infected and uninfected individuals from the same colonies (e.g. sister or closely related workers). Control of a Laboulbenialean genus, Herpomyces Thaxt. on cockroaches was achieved using the fungicide benomyl {methyl N-[1-(butylcarbamoyl)benzimidazol-2-yl]carbamate} (Gemeno et al., 2004). Recently, this compound was found to be ineffective in treating Myrmica rubra L. ant queens infected with R. wasmannii and the antifungal treatment also negatively affected the host (Pech & Heneberg, 2015). The main target of benomyl is the polymerization of tubulin, resulting in the inhibition of cell proliferation and division (Wride et al., 2014). Rickia thalli are attached firmly to the 1 - Department of Biotechnology and Microbiology, University of Debrecen, Hungary 2 - Postdoctoral Fellowship Programme of the Hungarian Academy of Sciences (MTA), Budapest, Hungary 3 - Department of Evolutionary Zoology and Human Biology, University of Debrecen, Hungary SHORT NOTE WP Pfliegler – Silver nanoparticles against Rickia infection on Myrmica852 host, presumably even if the cells of the thallus are dead, e.g. we observed Rickia and other Laboulbeniales thalli on decades- old museum specimens (Báthori et al., 2014, 2015b) and the fungus does not produce hyphae at all. Thus, antimycotics inhibiting cell proliferation may not be suitable to decrease the number of mature Rickia thalli on infected hosts at all. Additionally, the lack of structures penetrating the host in the case of ant-infecting Laboulbeniales fungi (Tragust et al., 2016) also probably accounts for the ineffectiveness of oral treatments administered to the host insect. On the other hand, antimicrobial compounds with cytotoxic effects may be more effective against Rickia. Silver nanoparticles (AgNPs) can interact with the cell surface causing ruptures in the cell membrane of eukaryotic cells, they are known to generate oxidative stress and disturb metabolic pathways and are also promising anti-biofilm agents (You et al., 2012; Cavalieri et al., 2014). Our aim was therefore to test whether AgNPs are able to exterminate R. wasmannii efficiently from its host ants. Myrmica scabrinodis Nylander ants infected with R. wasmannii were obtained from Rakaca (48°27’N, 20°47’E, NE Hungary) and maintained as a colony in laboratory. Workers of age groups 3 and 4 (see: Cammaerts-Tricot, 1974) were selected to avoid variation in expected lifespan. Treated (19 specimens) and water-treated control workers (18 specimens) were kept in individual plastic containers. Conditions are described in Báthori et al. (2015a). Thalli of R. wasmannii were counted using a Leica binocular microscope, using 20x to 100x magnification with hosts handled with a delicate pincer. Only thalli on the dorsal part of the heads of specimens were counted (antennae and mouthparts excluded; the mean number of thalli counted on a single ant was 26.6, SD= 12.98, for 37 specimens). Thalli were counted before the experiment and every 7 days during the four-week-long treatment. Ants were monitored and thalli counted for 2 more weeks after treatment. Thalli on dead specimens were not counted. Treatment involved submerging ants into 20, 10 or 5 ppm AgNP (Pérez et al., 2008) solution (Bay Nano, Miskolc) for 5 seconds using a pincer on a daily basis. Specimens of the control group were treated the same way with distilled water. Survival of AgNP-treated and water-treated hosts was compared by Kaplan-Meier survival analysis and its log-rank test using the MedCalc Software. As Rickia is not a mycelial fungus (Weir & Beakes, 1995), we could also assess the survival of the thalli (with a weekly resolution) using the same method (considering thalli on dead hosts as censored). Student’s 2-sample T-test (or Welch’s test depending on the equality of variance) was applied to test the significances of the differences in the proportional decrease of Rickia thalli on the two groups of ants. During our initial trial experiments, we determined the optimal concentration of AgNPs to treat M. scabrinodis workers with R. wasmannii infection. 20 to 10 ppm AgNPs proved to be unsuitable for the treatment of ants as these concentrations caused argyria and death of the host. The 5 ppm concentration showed no such effects: compared to the control (water-treated) group, the mortality of the ants was not different (log-rank test for Kaplan-Meier survival analysis, p<0.6), with only 4-4 hosts surviving the whole 6-week-long period of observations. Thus, we concluded that the AgNP treatment did not affect the lifespan of workers during the period of experiments. The low survival rate in both groups is probably associated with the negative effects of social isolation (Koto et al., 2015). It is noted that isolation was only necessary for tracking the changes on the number of thalli on each specimens. Treatment to ants may be applied without isolation in further experiments. The effect of the 4-week-long daily AgNP treatment resulted in the decrease in the number of thalli on the ants significantly more effectively than water treatment (number of thalli on ants in the group treated only with water also decreased in most cases). Significance was p<0.05 for the first week of treatment and p<0.01 for the second to fourth week. After 4 weeks of AgNP treatment, the mean decrease of thalli number was 82.99 % (SD=18.13) and all thalli disappeared from 4 of the surviving 10 AgNP treated ants (40 %) (Fig 1c). None of the control water-treated ants lost more than 75 % of thalli during this period and the mean decrease was 39.68 % (SD=26.62) for the surviving 9 control ants. Post treatment observation after 2 weeks also confirmed the significant (p<0.05 and p<0.1) difference between the two groups’ loss of thalli, although the number of surviving ants was low. Fig 1a summarizes the observations on the decrease in thalli number on the two ant groups. The results of the analysis and the comparison of the two survival curves using Kaplan- Meier survival analysis for the thalli of Rickia confirms that the treatment was effective with high statistical support (p<0.0001), backed by a high number (n=983) of tracked thalli (Fig 1b). Our results offer a possible treatment of Laboulbeniales infection of ants that could be exploited in the study their host- parasite interactions. Infected and post-treatment uninfected ants from the same colony could be used for experiments, allowing better comparisons to assess the effect of Rickia on behavior (especially social behavior between infected/uninfected sister ants), mortality, etc. As far as we know, our method is the first topical treatment of an insect for a fungal parasite and also the first effective treatment for Laboulbeniales on ants. We also tried to apply the treatment to Blatta lateralis L. cockroaches infected with Herpomyces stylopygae Speg., but the number of thalli quickly increased in both the treated and control groups (results not shown). This may be related to the fact that Hesperomyces spp. penetrate the host’s cuticle (Richards & Smith, 1956), while R. wasmannii was recently shown to be only attached to the surface of the ant cuticle (Tragust et al., 2016), but this question requires further study. Studying other host-Laboulbeniales pairs was not possible due to the rarity of these fungi and the usually small number of thalli on other insects (e.g. beetles). Thus, topical treatment is so far only effective for ants. Sociobiology 63(2): 851-854 (June, 2016) 853 Acknowledgements AT and FB was supported by the ‘AntLab’ Marie Curie Career Integration Grant (of AT), part the 7th European Community Framework Programme. AT was supported by a ‘Bolyai János’ scholarship of the Hungarian Academy of Sciences (MTA). References Báthori, F., Pfliegler, W. P. & Tartally, A. (2014). First records of the Myrmecophilous Fungus Laboulbenia camponoti Batra (Ascomycota: Laboulbeniales) from the Carpathian Basin. Sociobiology, 61: 338-340. doi: 10.13102/sociobiology.v61i3.338-340 Báthori, F., Csata, E. & Tartally, A. (2015a). Rickia wasmannii Increases the Need for Water in Myrmica scabrinodis (Ascomycota: Laboulbeniales; Hymenoptera: Formicidae). Journal of Invertebrate Pathology, 126: 78-82. doi:10.1016/j. jip.2015.01.005. Báthori, F., Pfliegler, W. P. & Tartally, A. (2015b). First Records Of The Recently Described Ectoparasitic Rickia lenoirii Santam. (Ascomycota: Laboulbeniales) In The Carpathian Basin. Sociobiology, 62: 620-622. doi: 10.13102/sociobiology. v62i4.901. Cammaerts-Tricot, M.C. (1974). Production and Perception of Attractive Pheromones by Differently Aged Workers of Myrmica rubra (Hymenoptera Formicidae). Insectes Sociaux, 21: 235-247. doi: 10.1007/BF02226916. Cavalieri, F., Tortora, M., Stringaro, A., Colone, M. & Baldassarri L. (2014). Nanomedicines for Antimicrobial Interventions. The Journal of Hospital Infection, 88: 183-190. doi: 10.1016/j.jhin.2014.09.009. Csata, E., Erős, K. & Markó, B. (2014). Effects of the Ectoparasitic Fungus Rickia wasmannii on its Ant Host Myrmica scabrinodis: changes in host mortality and behavior. Insectes Sociaux, 61: 247-252. doi: 10.1007/s00040-014-0349-3 Fig 1a. Decrease in the number of thalli (in percentage) on AgNP-treated and control (water-treated) ants during the 4 weeks of treatment and the 2 weeks of post-treatment tracking. Number of hosts in each group on bars, error bars show SD. b. Survival of thalli in the two groups. c. Example of a completely treated host ant on 1st and 28th day of AgNP treatment. WP Pfliegler – Silver nanoparticles against Rickia infection on Myrmica854 Gemeno, C., Zurek, L. & Schal, C. (2004). Control of Herpomyces spp. (Ascomycetes: Laboulbeniales) Infection in the Wood Cockroach, Parcoblatta lata (Dictyoptera: Blattodea: Blattellidae), with Benomyl. Journal of Invertebrate Pathology, 85: 132-135. doi: 10.1016/j.jip.2004.01.005. Haelewaters, D., Zhao, S.Y., De Kesel, A., Handlin, R.E., Royer, I.R., Farrell, B.D. & Pfister, D.H. (2015). Laboul- beniales (Ascomycota) of the Boston Harbor Islands I: Species Parasitizing Coccinellidae and Staphylinidae, with Comments on Typification. Northeastern Naturalist, 22: 459-477. doi: 10.1656/045.022.0304. Konrad, M., Grasse, A. V., Tragust, S. & Cremer, S. (2015). Anti-pathogen Protection Versus Survival Costs Mediated by an Ectosymbiont in an Ant Host. Proceedings of the Royal Society B: Biological Sciences, 282: 20141976. doi: 10.1098/ rspb.2014.1976. Koto, A., Mersch, D., Hollis, B. & Keller, L. (2015). Social Isolation Causes Mortality by Disrupting Energy Homeostasis in Ants. Behavioral Ecology and Sociobiology, 69: 583-591. doi: 10.1007/s00265-014-1869-6. Markó, B., Csata, E., Erős, K., Német, E., Czekes, Z. & Rózsa, L. (2016). Distribution of the Myrmecoparasitic Fungus Rickia wasmannii (Ascomycota: Laboulbeniales) Across Colonies, Individuals, and Body Parts of Myrmica scabrinodis. Journal of Invertebrate Pathology, 136: 74-80. doi: 10.1016/j.jip.2016.03.008. Pech, P. & Heneberg, P. (2015). Benomyl Treatment Decreases Fecundity of Ant Queens. Journal of Invertebrate Pathology, 130: 61-63. doi:10.1016/j.jip.2015.06.012 Pérez, M. A., Moiraghi, R., Coronado, E.A. & Macagno, V. A. (2008). Hydroquinone Synthesis of Silver Nanoparticles: A Simple Model Reaction To Understand the Factors That Determine Their Nucleation and Growth. Crystal Growth and Design, 8: 1377-1383. doi: 10.1021/cg7009644. Richards, A.G. & Smith, M. N. (1956). Infection of Cockroa- ches with Herpomyces (Laboulbeniales) II. Histology and Histopathology. Annals of the Entomological Society of America, 49: 85-93. doi: 10.1093/aesa/49.1.85. Tragust, S., Tartally, A., Espalader, X. & Billen, J. (2016). Histopathology of Laboulbeniales (Ascomycota: Laboulbeniales): Ectoparasitic Fungi on Ants (Hymenoptera: Formicidae). Myrmecological News, 23: 81-89. Weir, A. & Beakes, G. (1995). An Introduction to the Laboul- beniales: A Fascinating Group of Entomogenous fungi. Mycologist, 9: 6-10. doi: 10.1016/S0269-915X(09)80238-3. Wride D. A., Pourmand, N., Bray, W. M., Kosarchuk, J. J., Nisam, S. C., Quan, T. K., Berkeley, R. F., Katzman, S., Hartzog, G. A., Dobkin, C. E. & Scott Lokey, R. (2014). Confirmation of the Cellular Targets of Benomyl and Rapamycin Using Next-generation Sequencing of Resistant Mutants in S. cerevisiae. Molecular bioSystems, 10: 3179-3187. doi: 10.1039/c4mb00146j. You, C., Han, C., Wang, X., Zheng, Y., Li, Q., Hu, X. & Sun, H. (2012). The Progress of Silver Nanoparticles in the Antibacterial Mechanism, Clinical Application and Cytotoxicity. Molecular Biology Reports, 39: 9193-201. doi: 10.1007/s11033-012-1792-8.