The interactions of ectosymbiotic Laboulbeniales (Ascomycota) fungi and their hosts are rather understudied. Rickia wasmannii Cavara is a common ant-associated Laboulbeniales species that has been reported in 17 countries of Europe, and frequently infects Myrmica scabrinodis Nylander, 1846 (Hymenoptera: Formicidae), a common ant species host, in high density. These make M. scabrinodis and R. wasmannii appropriate model organisms for studies on fungal host-ectosymbiont interactions. Aggressiveness and boldness of infected and uninfected M. scabrinodis workers from northern and eastern Hungary were studied in two laboratory-established behavioural experiments. Infected workers were significantly less aggressive and less bold (i.e. less likely to leave nest shelters) than the uninfected ones. These results suggest that R. wasmannii has considerable effects on the behaviour of M. scabrinodis. Our study brings an evidence that infection of ants with Laboulbeniales might negatively affect the workers’ behaviour. In special, the competitive abilities might be affected most by these fungi, since remaining inside and behaving submissively is not effective behaviour in the case of significant competition for resources among colonies.

behaviour, ectoparasite, Laboulbeniomycetes , fungi, infection spreading

The ascomycete order Laboulbeniales contains 141 genera and over than 2,100 species of obligatory ectosymbionts (Santamaria et al. 2017). The hosts of these fungi mainly belong to the groups Coleoptera (ca. 80%) and Diptera (ca. 10%) (Weir and Hammond 1997). Most Laboulbeniales fungi exhibit extreme host specificity, with a single host species (stenotopic) or several species in the same genus (eurytopic) (Haelewaters 2012 and references therein), but unrelated insects can also host a single Laboulbeniales species if they share the same microhabitat (De Kesel and Haelewaters 2014, Pfliegler et al. 2016). While such ectosymbiotic associations can have both positive (Konrad et al. 2015) and negative (Benjamin 1971, Gemeno et al. 2004, Nalepa and Weir 2007, Strandberg and Tucker 1974) effects on their hosts, they are sometimes considered to be neutral factors (García et al. 2010, Lapeva-Gjonova and Santamaria 2011, Whisler 1968).

To date, four ant-associated species of Laboulbeniales have been recorded in Europe: Laboulbenia formicarum Thaxt. is known in France, Portugal and Spain, parasitizing Lasius grandis Forel, 1909, L. neglectus van Loon, Boomsma & Andrasfalvy 1990 and L. niger (Linnaeus, 1758) (Espadaler and Santamaria 2012, Gómez et al. 2016). Laboulbenia camponoti S.W.T. Batra is known in Bulgaria, Spain, Austria, Romania and Italy, on C. universitatis Forel, 1890; C. pilicornis (Roger, 1859); C. sylvaticus (Olivier, 1792) and C. aethiops (Latreille, 1798) (Báthori et al. 2014, Espadaler and Santamaria 2012; Gómez et al. 2016). Rickia lenoirii Santam. has been reported in Greece, France, Hungary and Romania on Messor wasmanni Krausse, 1910 and M. structor (Latreille, 1798) (Báthori et al. 2015b, Santamaria and Espadaler 2015). Rickia wasmannii Cavara (Fig. 1) is the most widely distributed ant-associated Laboulbeniales in Europe (Santamaria and Espadaler 2015). This species has been reported from England, France, Luxemburg, Germany, the Netherlands, Switzerland, Poland, Italy, Austria, Slovenia, the Czech Republic, Slovakia, Hungary, Romania, Spain, Belgium and Bulgaria, and parasitizes nine Myrmica Latreille, 1804 species: M. gallienii Bondroit, 1920; M. rubra (Linnaeus, 1758); M. ruginodis Nylander, 1846; M. sabuleti Meinert, 1861; M. scabrinodis Nylander, 1846; M. slovaca Sadil, 1952; M. specioides Bondroit, 1918; M. spinosior Santschi, 1931 and M. vandeli Bondroit, 1920 (De Kesel et al. 2016, Haelewaters et al. 2015a, 2015b).

Figure 1. 

Myrmica scabrinodis worker with Rickia wasmannii thalli (arrows indicate some thalli).

Ant species within the genus Myrmica can be found in several different kinds of habitats across European temperate zones, including meadows, steppes, woodlands, forests, and mountainous regions. Colonies of these ants can be either monogynous or polygynous and contain up to a few thousand workers (Radchenko and Elmes 2010, Czechowski et al. 2012). Myrmica ants can be hosts to many parasitic organisms from several taxonomic groups including ecto- and endoparasitic fungi and other microbial pathogens, such as: R. wasmannii; Beauveria bassiana (Bals.-Criv.) Vuill.; Isaria fumosorosea Wize; Metarhizium anisopliae (Metschn.) Sorokin; Hirsutella stibelliformis var. myrmicarum H.C. Evans & Groden; H. subramanianii var. myrmicarum H.C. Evans & J.F. Bischof; Paraisaria myrmicarum H.C. Evans, Groden & J.F. Bischof; Ophiocordyceps myrmicarum D.R. Simmons & Groden (Radchenko and Elmes 2010, Simmons et al. 2015, Witek et al. 2014). In particular, M. scabrinodis is common in Europe (Czechowski et al. 2012, Radchenko and Elmes 2010, Seifert 2007), frequently parasitized by R. wasmannii (Espadaler and Santamaria 2012) and other (social) parasites (Tartally 2008, Witek et al. 2014). Myrmica scabrinodis is a mesothermo- and mesohygrophilic species of different kinds of humid habitats, which is tolerant of soil moisture but avoids expressly xerothermal habitats. The abundance of both R. wasmannii and M. scabrinodis make them appropriate species for the study of host-Laboulbeniales interactions, as corroborated by a number of recent studies (Báthori et al. 2015a, Csata et al. 2014, Haelewaters et al. 2015a, 2015b, Markó et al. 2016, Witek et al. 2014).

Previous work has shown that different ant-fungal interactions can influence the behaviour of hosts in several ways (e.g., increased grooming and nest cleaning, secretion of antibiotics, pathogen avoidance, dispersal of infected individuals, and the relocation of an entire colony) (e.g., Csata et al. 2014, Roy et al. 2006, Oi and Pereira 1993). Laboulbeniales fungi are often present on the ant body with high thallus densities and the results of recent studies suggest that they could have both positive and negative effects on their hosts and are able to influence their behaviour (Báthori et al. 2015a, Csata et al. 2014, Konrad et al. 2015, Pech and Heneberg 2015). For example, Csata et al. (2014) showed that individuals of M. scabrinodis infected by R. wasmanni had significantly reduced lifespans under laboratory conditions; heavily infected individuals died significantly faster from starvation and spent more time consuming water than their more lightly parasitized counterparts. Starvation survival of L. neglectus workers infected by L. formicarum was also significantly decreased (Konrad et al. 2015), while host survival due to Metarhizium exposure increased. Ant workers with high thallus densities exhibited significantly longer periods of self-grooming as well as elevated expression of immune genes (Konrad et al. 2015).

To date, it has not been studied whether the boldness and aggressive behaviour differ between infected and uninfected M. scabrinodis workers. The behavioural characteristics of different ant workers could be relevant, for example, in terms of competition between infected and uninfected colonies. In this study, we evaluate the boldness and aggressive behaviour of uninfected M. scabrinodis workers versus their heavily infected counterparts.

On the basis of our experimental results, R. wasmannii seems to have a considerable effect on the behaviour of M. scabrinodis workers with regards to boldness and interactions with uninfected workers. Since infection by the fungus appears to be permanent (Haelewaters et al. 2015c, AT and FB pers. observ. on long-term reared laboratorial colonies), this could lead to persistent differences in the ants’ behaviour in a given colony (De Kesel et al. 2016). It is worth noting that when a colony is infected, the vast majority of its workers are also infected (Markó et al. 2016; AT and FB, pers. observ.), and the infection therefore could have effects not only on the individual but also on the colonial level. Thus, infection by R. wasmannii possibly has long-term effects on the performance of infected individuals and colonies in natural habitats. It should be noted here that we had the possibility to work with populations with either infected or uninfected colonies from slightly different sites (see Methods for details). Thus, it would be important to follow similar experiments on infected and uninfected colonies collected from the same site, if such sites would be recorded in the future to confirm that our results are not affected by habitat-driven differences of maternal colonies, although it is here unlikely. It should be also emphasized that so far all results about negative and positive interactions were reached in the course of laboratory experiments, so additional experiments are needed to conduct with the ant hosts in nature.

We propose the following hypotheses regarding the increased latencies in time of leaving shelter in the case of infected workers: (1) they may have been in poorer condition and physically weak or impaired, e.g. the chances of survival of infected ants were lower under starvation (Konrad et al. 2015) and lab conditions (Csata et al. 2014); and/or (2) the fungus induced the host to remain in the shelter tube for a longer period of time. If the latter were true, this would mean that R. wasmannii would induce behavioural changes. It should be noted that Csata et al. (2017) did not found any differences in the locomotion of infected worker specimens compared with the uninfected ones. Thus, the infected ants did not left the tube later because of locomotion but rather because of other behavioural changes (i.e. boldness). The mechanisms causing infected ants to stay longer in the shelter could be of importance, and should be the subject of future study.

On the other hand, R. wasmannii, as an ectosymbiotic fungus, could be expected to be capable of spreading not only within but also between ant colonies. If increased worker activity (outside of the nest) would increase the spreading success of the fungus, one may expect infected ants to exhibit a higher degree of out-of-nest activity (Bos et al. 2012, sensu Hughes et al. 2011). Where a host forages more outside and fights, this would have a positive effect on the spread of the fungus compared to the behaviour of a submissive host who stays inside the nest. Furthermore, local competitive disadvantages that influence infected colonies (see: Báthori et al. 2015a, Csata et al. 2014) in a given habitat might be reduced by spreading the infection to local rival colonies. However, our results show that the infected M. scabrinodis workers do not behave in these ways. Thus, the intercolonial spread of R. wasmannii caused by individual foragers might be rare and occasional. It would need further research to check whether the fungus rather spreads by queens or by whole colony buddings, a phenomenon that is well-known in Myrmica ants (Radchenko and Elmes 2010). Spreading mainly by colony budding would suggest an extreme host specialisation of the fungus on the host ant colonies. Such an extreme host colony specialization is recorded in the case of at least one ant-parasitic species [the social parasite Microdon mutabilis (Linnaeus, 1758) hoverfly (Diptera: Syrphidae); see Schönrogge et al. 2006], but since currently no information is available on the genetic relationships among different R. wasmannii populations, it is impossible to determine whether or not fungus lineages are similarly adapted genetically to the different host ant colonies (and thus infecting genetically distant host colonies would not be effective). Furthermore, we cannot exclude the possibility that the spreading of the fungus requires the specific microhabitat provided by the ant nest [compare to the ecological specificity of R. wasmannii, (Pfliegler et al. 2016)], e.g. because the ascospores of Rickia ejected from mature thalli are extremely vulnerable to desiccation outside the ant nest microhabitat. Such hypothetical constraints on the spreading of the infection could actually act in favour of the observed behavioural effects on the host, or at least may account for the lack of negative selection regarding less active workers. In this sense, the poorer general condition of the infected host that results in less active behaviour would mean an advantage for the fungus.

For a long time, there was no knowledge of the effects of ant-associated Laboulbeniales fungi on the behaviour of their hosts, but recent research (Báthori et al. 2015a, Csata et al. 2014, Konrad et al. 2015) has begun to fill this gap. These studies revealed behavioural changes in the ant hosts, such as increased water consumption (Báthori et al. 2015a) and intensified grooming (Csata et al. 2014). Based on our current results, infection might negatively affect also the workers’ behaviour linked to competitive abilities, since remaining inside and behaving submissively is not effective when there is significant competition for resources.

We would like to express our sincere thanks to Enikő Csata, Viven Márku, Anna Ágnes Somogyi, and Kitti Barta for the assistance they provided in the field in the course of our laboratory work, to Gareth Dyke, Danny Haelewaters, Petr Klimes, Walter P. Pfliegler and Enikő Tóth for providing valuable comments on our manuscript, and to Walter P. Pfliegler for the photo used for Fig. 1. This research was supported by the ‘AntLab’ Marie Curie Career Integration Grant (to AT), part of the 7^th European Community Framework Programme. AT was supported by a ‘Bolyai János’ scholarship of the Hungarian Academy of Sciences (MTA).