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JHR 58: 41-52 (2017) JOURNAL OF *0erviewed opevaccersjoural
eee hn ore (f-) Hymenoptera

http://jhr.pensoft.net The Inerational Society of Hymenoptersts. RESEARCH

The effect of Rickia wasmannii (Ascomycota,
Laboulbeniales) on the aggression and boldness of
Myrmica scabrinodis (Hymenoptera, Formicidae)

Ferenc Bathori', Zoltan Radai', Andras Tartally'

| Department of Evolutionary Zoology and Human Biology, University of Debrecen, Egyetem Tér 1, H-4032
Debrecen, Hungary

Corresponding author: Ferenc Bathori (bathori.ferenc@science.unideb.hu)

Academic editor: P Klimes | Received 14 April 2017 | Accepted 9 August 2017 | Published 31 August 2017
Attp://zoobank. org/B1D19B45-F40C-4EFC-B659-B634BBF640D2

Citation: Bathori E Radai Z, Tartally A (2017) The effect of Rickia wasmannii (Ascomycota, Laboulbeniales) on the
ageression and boldness of Myrmica scabrinodis (Hymenoptera, Formicidae). Journal of Hymenoptera Research 58:

41-52. https://doi.org/10.3897/jhr.58.13253

Abstract

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 Rk. 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.

Keywords

behaviour, ectoparasite, Laboulbeniomycetes, fungi, infection spreading

Copyright Ferenc Bathori et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

42 Ferenc Bathori et al. / Journal of Hymenoptera Research 58: 41-52 (2017)

Introduction

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 spe-
cies (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
(Garcia 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, Gémez et al. 2016). Laboul-
benia 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) (Bathori et al. 2014, Espadaler and Santamaria 2012;
Goémez et al. 2016). Rickia lenoirii Santam. has been reported in Greece, France, Hun-
gary and Romania on Messor wasmanni Krausse, 1910 and /. structor (Latreille, 1798)
(Bathori 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 Re-
public, 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).

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 micro-
bial pathogens, such as: R. wasmannii; Beauveria bassiana (Bals.-Criv.) Vuill.; Lsaria
fumosorosea Wize; Metarhizium anisopliae (Metschn.) Sorokin; Hirsutella stibelliformis
var. myrmicarum H.C. Evans & Groden; H. subramanianii var. myrmicarum H.C. Ev-
ans & J.E Bischof; Paraisaria myrmicarum H.C. Evans, Groden & J.F. Bischof; Ophio-
cordyceps myrmicarum D.R. Simmons & Groden (Radchenko and Elmes 2010, Sim-
mons et al. 2015, Witek et al. 2014). In particular, M. scabrinodis is common in Europe

The effect of Rickia wasmannii (Ascomycota, Laboulbeniales) on the aggression.... 43

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

(Czechowski et al. 2012, Radchenko and Elmes 2010, Seifert 2007), frequently para-
sitized by R. wasmannii (Espadaler and Santamaria 2012) and other (social) parasites
(Tartally 2008, Witek et al. 2014). Myrmica scabrinodis is a mesothermo- and mesohy-
grophilic 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 interac-
tions, as corroborated by a number of recent studies (Bathori et al. 2015a, Csata et al.
2014, Haelewaters et al. 2015a, 2015b, Mark 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, se-
cretion 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 den-
sities 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 (Bathori 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 in-
dividuals died significantly faster from starvation and spent more time consuming
water than their more lightly parasitized counterparts. Starvation survival of L. neglec-
tus 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.

44 Ferenc Bathori et al. / Journal of Hymenoptera Research 58: 41-52 (2017)

Methods

Ant colony collection and laboratory conditions

We collected Myrmica scabrinodis colonies from two different regions in Hungary, in-
cluding 12 from northern Hungary which comprised six which were infected from a
habitat close to Rakaca (i.e., 48°27'N, 20°47'E, 170 m above sea level, a.s.l.) and six
that were uninfected from an area close to Aggtelek (i.e., 48°26'N, 20°30'E, 340 m
a.s.l.). We collected 12 further colonies from eastern Hungary, including six that were
infected from close to Ujléta (i.e., 47°26'N, 21°51'E, 120 m a.s.l.) and six that were
uninfected from close to Csikgat (47°25'N, 21°48'E, 110 ma.s.l.). We did not record
any sites as part of this study that comprised both infected and uninfected colonies,
so a standardisation of experiments per locality by choosing both infected and unin-
fected colonies from the same sites was unfortunately not possible. However, note that
the two paired-sites were in a similar part of Hungary and from similar habitats and
elevations, for sake to minimize the environmental effects on the populations. Thus,
our total dataset includes 24 colonies, each of which contained fertilized queens and
hundreds of workers, larvae, and pupae. To further reduce the effect of different sites,
we kept all the ants used for this study for minimum two weeks in artificial lab nests
under the same conditions: at 20 + 1°C and provided sufficient food resources (i.e.,
fed with cockroaches twice a week and with a 33% honey water solution ad libitum).
Plastic boxes treated with Fluon® on their inner walls to prevent ants from escaping
were used as formicaria (i.e., length: 16.5 cm; width: 11.5 cm; height: 6 cm); inside
these boxes, we created chambers (i.e., length: 5.5 cm; width: 4.5 cm; height: 1 cm)
with plaster floors, covered with glass plates. In advance of each experiment, colonies
were stored in the laboratory for a minimum of one month for acclimatisation (follow-
ing Bathori et al. 2015a).

Boldness test

Boldness was tested using individual workers. We assessed their boldness by measuring
how long it took for them to leave shelter (based on Gyuris et al. 2011). Prior to
the test, 18 melanised workers were randomly selected from each colony (total 2 =
432). These mini test-colonies were fed a Bhatkar diet (Bhatkar and Whitcomb 1970).
After 12 hours, single individuals were placed into different shelters in random order,
inside new sterilized thin and plugged black plastic tubes (length: 60 mm; diameter: 5
mm) for an acclimatisation period of one minute (Gyuris et al. 2011, Spicer Rice and
Silverman 2013) (Fig. 2a). After the removal of the plug, we measured the time that
passed before individual ants emerged from the tubes, allowing a maximum waiting
time of three minutes (see Bathori et al. 2015a).

The effect of Rickia wasmannii (Ascomycota, Laboulbeniales) on the aggression.... 45

0
7 R

Figure 2. Tubes used for a boldness and b aggression tests. Arrow indicates the flap which was removed
at the beginning of the test.

Aggression test

In this experiment, 120 worker pairs, including one infected and one uninfected mel-
anised worker, were randomly selected from both geographic regions. Worker pairs
from the two different geographical regions, northern and eastern Hungary were tested
separately, and two facing glass tubes (length: 53 mm; diameter: 15 mm), separated by
a thin plastic flap, formed the experimental arena (Fig. 2b). After an acclimatisation
period of 1 minute (Spicer Rice and Silverman 2013), the flap was removed so that the
infected and uninfected workers could meet. The number of different behaviour pat-
terns was registered for a period of 3 minutes (like in Bathori et al. 2015a). The follow-
ing categories were used: initiation of aggressive behaviour, antennation, mandibular
threats, biting, stinging, autogrooming and allogrooming (see Maak et al. 2014 and
references therein). At the end of the experiment, the ants were preserved in a solution
of 67.5% ethanol, and the number of fungal thalli was counted for statistical analyses
(see below) under a Leica MZ12.5 stereomicroscope at magnifications of 10-160x.

Statistical analyses

Statistical analyses were performed using the R statistical software (ver. 3.0.2, R Core
Team 2013). Data was mainly analysed using generalized linear mixed-effects models
(GLMM) of the R-package “Ime4” (Bates et al. 2014), because these types of models
offer a range of ways to handle non-Gaussian error distributions and random effects.
In GLMMs of significant correlations we acquired conditional R’ of the model using
the R-package “MuMIn” (Barton 2013).

To assess whether there was significant difference between infected and uninfected
workers in terms of their probability to leave shelter, we applied a binomial GLMM,
specifying leaving shelter as binary response (1 meaning the individual left shelter), and
infection as binary predictor factor (1 meaning the individual was infected).We also
tested if infected and uninfected workers showed significant differences in their latency
before leaving shelter by fitting a mixed-effects Cox regression model in the R-package

46 Ferenc Bathori et al. / Journal of Hymenoptera Research 58: 41-52 (2017)

‘coxme’ (Therneau 2016). In this model, time latency before leaving the shelter was
defined as follow up time, while the event of leaving was specified as the status indicator
event, and infection was a binary predictor factor variable. To test whether the number
of thalli on infected workers affected the time latency before leaving shelter, we fitted a
log-linked Gamma GLMM, because this model is able to handle Gamma-distributed
time data. When fitting this Gamma GLMM, we excluded observations from unin-
fected workers and defined the number of thalli as predictor, while using time latency
before leaving shelter as response variable. In all the models described above, the time-
block of measurements and the habitat of origin were specified as random factors.

In order to measure aggressiveness, we calculated an index based on Martin et al.
(2009), dividing the number of observed aggressive behaviours (mandible threat, bit-
ing, stinging) by the total number of interacting behaviours (mandible threat, biting,
stinging, antennae-interaction, and allogrooming). This index represent the proportion
of aggressive behaviours in the overall observed and recorded interactive behaviours.
When testing whether infection has a significant effect on the proportion of aggres-
sive behaviours, we fitted a binomial GLMM, with infection as the predictor factor,
and calculated aggression index as dependent variable. To see if infected or uninfected
workers were more likely to initiate aggressive interactions, we used a different bino-
mial GLMM that incorporated infection as a predictor, and initiation (binary variable,
1 meaning that the given ant was the first to perform aggressive behaviour in the given
trial) as a dependent variable. In the two models described above, the ID of tested
pairs and habitat of origin were specified as random factors. Furthermore, we tested
whether, the number of thalli had any effect on the proportion of aggressive behaviour
by fitting a binomial GLMM (excluding all uninfected ants) with aggression index as
the dependent and the number of thalli as the predictor variable. In this model, habitat
of origin was also specified as a random factor.

Results

Boldness

Infected ants did not differ from uninfected ones in their probability of leaving shelter
(binomial GLMM: z = -0.99; p = 0.318). However, infected workers were significantly
slower to leave shelter, i.e. showed higher latencies before leaving (mixed-effects Cox-
model: z = -2.13; p = 0.033) (Fig. 3). The number of thalli on infected workers did not
affect significantly the time latency before leaving shelter (log-linked Gamma GLMM:
z= -0.33; p = 0.744).

Aggression

Infection alone does not exert a significant effect on the proportion of aggressive be-
haviours (binomial GLMM: z = -0.41; p = 0.68). Infected workers were, however,

The effect of Rickia wasmannii (Ascomycota, Laboulbeniales) on the aggression.... 47

ME CUninfected
[] Infected

Proportion of individuals not leaving shelter

0 50 100 150
Time (sec)

Figure 3. Time latency until leaving shelter in case of uninfected (N=215) and infected (N=215) workers.

=)
Oo
C1 WNon-initiating
S © Fight-initiating
=)
ce)
=
: I
=)
at
=)

Uninfected Infected

Figure 4. Probability if initiating aggressive behaviour in cases of uninfected (N=119) and infected
(N=119) workers.

significantly less likely to initiate aggressive behaviour than uninfected ants (binomial
GLMM: z = -2.91; p = 0.004; conditional R’ = 0.23) (Fig. 4). Interestingly, the num-
ber of thalli on infected workers nevertheless seems to exert a mild, but significantly
positive effect on the proportion of aggressive behaviours (binomial GLMM: z = 2.85;
p = 0.005; conditional R’ = 0.50).

48 Ferenc Bathori et al. / Journal of Hymenoptera Research 58: 41-52 (2017)

Discussion

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 interac-
tions 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 (Mark6 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 experi-
ments, 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 locomo-
tion 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, Rk. 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: Bathori 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

The effect of Rickia wasmannii (Ascomycota, Laboulbeniales) on the aggression.... 49

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 spe-
cialization is recorded in the case of at least one ant-parasitic species [the social parasite
Microdon mutabilis (Linnaeus, 1758) hoverfly (Diptera: Syrphidae); see Schénrogge 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 in-
fecting 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 Laboulbe-
niales fungi on the behaviour of their hosts, but recent research (Bathori 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 (Bathori 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.

Acknowledgements

We would like to express our sincere thanks to Eniké Csata, Viven Marku, Anna Agnes
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 Eniké Toth 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" European Com-
munity Framework Programme. AT was supported by a “Bolyai Janos’ scholarship of
the Hungarian Academy of Sciences (MTA).

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