The effects of fungal infection and physiological condition on the locomotory behaviour of the ant Myrmica scabrinodis

Accepted Manuscript The effects of fungal infection and physiological condition on the locomotory behaviour of the ant Myrmica scabrinodis Enikő Csata, Abel Bernadou, Elena Rákosy-Tican, Jürgen Heinze, Bálint Markó PII: DOI: Reference:

S0022-1910(16)30277-3 http://dx.doi.org/10.1016/j.jinsphys.2017.01.004 IP 3594

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Journal of Insect Physiology

Received Date: Revised Date: Accepted Date:

23 August 2016 2 December 2016 8 January 2017

Please cite this article as: Csata, E., Bernadou, A., Rákosy-Tican, E., Heinze, J., Markó, B., The effects of fungal infection and physiological condition on the locomotory behaviour of the ant Myrmica scabrinodis, Journal of Insect Physiology (2017), doi: http://dx.doi.org/10.1016/j.jinsphys.2017.01.004

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Title: The effects of fungal infection and physiological condition on the locomotory behaviour of the ant Myrmica scabrinodis

Authors: Enikő CSATA1*, Abel BERNADOU2*, Elena RÁKOSY-TICAN3, Jürgen HEINZE2, Bálint MARKÓ1

Addresses: 1

Hungarian Department of Biology and Ecology, Babeş–Bolyai University, 400006 Cluj-

Napoca, Clinicilor 5-7, Romania 2

Department of Zoology and Evolutionary Biology, University of Regensburg, D-93040

Regensburg, Germany 3

Department of Molecular Biology and Biotechnology, Babeş-Bolyai University, 400006 Cluj-

Napoca, Clinicilor 5-7, Romania

* Corresponding author: Enikő CSATA Hungarian Department of Biology and Ecology, Babeş-Bolyai University, 400006 Cluj-Napoca, Clinicilor 5-7, Romania, [email protected] Abel BERNADOU Department of Zoology and Evolutionary Biology, University of Regensburg, D-93040 Regensburg, Germany, [email protected]

No. of figures: 4

Abstract

Parasite infection often results in alterations in host behaviour. These changes vary greatly in their magnitude, from slight shifts in the time spent by the host performing a given activity to the appearance of novel behaviours. The effects of parasites can differ with the age and the physiological condition of the host. Rickia wasmannii is an ectoparasitic fungal symbiont in Myrmica ants that covers the whole body surface of the host and reduces its lifespan. The fungus is present in both young and old individuals, making it an optimal subject for the study of age-related parasitic effects. We tested the effect of fungal infection on the locomotory activity of the Myrmica scabrinodis ant in different age categories. The fat content of workers was measured as a proxy for their physiological status. Based on our findings, old workers bore more thalli and were leaner than young individuals, while they tended to move at higher speeds and with a lower degree of meandering. Young individuals covered smaller distances, at slower speeds and with a higher degree of meandering. Contrary to our expectations, the infection intensity of R. wasmannii affected neither the fat content nor the locomotory activity of ant workers. However, the two age classes seem to have different strategies with regards to the relationship between fat content and distance covered. Our results suggest that characteristics of locomotory activity differ between the age classes in many respects, and are also influenced by their physiological status, but parasitism by R. wasmannii does not seem to have a straightforward effect on any of the variables studied

Key words: parasite, Laboulbeniales, Rickia wasmannii, locomotion, fat content, velocity

1. Introduction

Animals adapt their behaviour to environmental fluctuations, to their own physiological condition, and to needs and requirements that are specific to life stage and age. Specifically, in the case of social animals age-related changes are very pronounced as the role of an individual changes with ageing; young animals may engage in completely different sets of behaviours than older animals (Wilson, 1971; Lecocq et al., 2016). Infection with parasites is also known to result in alterations in the host’s behaviour (e.g. Schmid-Hempel and Schmid-Hempel, 1993; Keller, 1995; Rosengaus and Traniello, 2001). These parasite-induced changes vary greatly in their magnitude (see Poulin, 1994) from slight shifts in the time spent performing a given activity to the appearance of entirely novel and unexpected behaviours (Poulin, 1995; Heinze and Walter, 2010; Hughes et al., 2016). The impact of parasites may change with the age of the host, e.g. older individuals with a shorter residual life expectancy might be more affected by parasitism than younger individuals (Møller and Lope, 1999; Fauchald et al., 2005). In particular cases the effect of parasites could go hand in hand with age-related modifications, thus accelerating age polyethism in social insects, e.g. in young honeybees (Natsopoulou et al., 2015; Lecocq et al., 2016). Ants are frequently targeted by parasites: bacteria, fungi, mites, fluke worms, nematodes, beetles, butterflies, and even other ants are known to exploit them (Schmid-Hempel, 1998; Csősz and Majoros, 2009; Espadaler and Santamaria, 2012; Csata et al., 2013, 2014; Otti et al., 2014; Báthori et al., 2014, 2015). One of the most diverse groups of parasites is fungi, which display a wide variety of interactions with ants from transient to obligate associations (Oi and Pereira, 1993; Roy et al., 2006; Espadaler and Santamaria, 2012; Csata et al., 2013). Fungi can affect

their hosts in many ways and behavioural, mechanical, biochemical, and even physiological changes in ants can be linked to the presence of fungal parasites (see Kaur and Mukerji, 2006, Ortiz-Urquiza and Keyhani, 2013, Hughes et al., 2016). Generally, infection by fungi causes alterations in the activity of the ant host (Oi and Pereira, 1993; Heinze and Walter, 2010; Konrad et al., 2012; Tragust et al., 2013). In extreme cases it might even cause ants to leave the colony (Heinze and Walter, 2010) or to die under conditions that would benefit the fungus by maximizing the spread of its propagules (Pontoppidan et al., 2009; Hughes et al., 2016). All these extreme behavioural syndromes are manifested by old workers performing foraging tasks, since young workers rarely if ever come into contact with infective spores. Thus, nothing is known concerning whether young workers would also be driven by parasites to change their behaviour in a similar manner. One of the most widespread Holarctic ectoparasitic fungal symbiont of ants is Rickia wasmannii (Ascomycota: Laboulbeniales) (e.g. Espadaler and Santamaria, 2012; Santamaria and Espadaler, 2015; Markó et al., 2016). It is known to reduce the lifespan of infected ants, while also causing other behavioural modifications (Csata et al., 2014; Báthori et al., 2015). The fungus does not cause the decay of the colonies, although it is present both in young and old individuals. The number of thalli can reach extreme values in infected workers (> 400 thalli per individual) (Markó et al., 2016). Due to this, the fungal infection could imply considerable physiological costs for the infected host, which could be reflected in altered locomotion, e.g., decreased mobility. However, these costs might be age-dependent, since young and old individuals differ with regard to their physiological conditions (see Bernadou et al., 2015a), while infection intensity might also differ between major age categories as suggested by previous descriptions (Lapeva-Gjonova and Santamaria, 2011).

We tested the effect of fungal infection on the locomotory activity of its main host, Myrmica scabrinodis using the fat content of the host as a proxy for its physiological condition. Since activity patterns are age-dependent in ants, due to age polyethism (Hölldobler and Wilson, 1990), we studied two main age classes (young and old) separately to identify the age-dependent effects of the parasite.

2. Material and methods 2.1. Study species and ant collection Rickia wasmannii Cavara (1899) is the most common ectoparasitic myrmecophilous Rickia species in the Holarctic (Santamaria and Espadaler, 2015), and it obligatorily exploits Myrmica ants (see Espadaler and Santamaria, 2012; Csata et al., 2013; Witek et al., 2014). The thalli attach to the outer layer of the cuticle and appear on the surface of the hosts as club-shaped setae-like structures (Tragust et al., 2016; Fig. 1.). It has been demonstrated that infection increases allogrooming and water-consumption, while reducing the lifespan of infected ants (Csata et al., 2014; Báthori et al., 2015). Its main host is Myrmica scabrinodis (Espadaler and Santamaria, 2012; Csata et al., 2013; Markó et al., 2016). Altogether 7 infected and 7 uninfected Myrmica scabrinodis nests were collected from the same area of a > 5 km radius: 8 colonies (4 infected and 4 uninfected) from Borșa Cătun (N 46.88909, E 023.70067) and 6 colonies (3 infected and 3 uninfected) from Luna de Jos (N 46.921961, E 023.734032), Romania, in September 2014. The two northern-exposed sites are both covered by meso-xeric grasslands characterized by species such as Festuca pratensis and Molinia caerulea. The prevalence of Rickia wasmannii is very high in this host population, reaching 100% in certain patches and certain colonies (Markó et al., 2016). During the process of

collection, a hand magnifying glass (×30) was used to determine the infection status of ants, since infected ants appear unusually hairy. In addition, all collected samples were screened for fungal thalli using an Olympus SZ51 stereomiscroscope (×80) in laboratory. Ants were identified to species level based on Czekes et al. (2012) with the same stereomicroscope. All colonies were kept in artificial nests (10 × 10 cm) with a day-night cycle of 12h:12h and a constant temperature of 22°C. Ants were fed a special mixture of sugar and protein (Bhatkar and Whitcomb, 1970), which is widely used with ants, and also cockroaches every third day, with ad libitum water. In the experiments, we used individuals of different age classes, young and old, respectively. Individuals belonging to different age classes were identified based on cuticular pigmentation, which is generally used to estimate the age class of Myrmica workers (Moroń et al., 2008): young workers have yellowish-light brown colored cuticles, while old workers have a dark brown colored cuticle. According to our laboratory observations of changes in cuticular pigmentation, young individuals are ≤ 11 days old, while old individuals are ≥ 21 days old. In all cases test individuals were randomly chosen from their age classes.

2.2. Locomotory behaviour assays For the purpose of the locomotory experiment ten individuals (five old, and five young) were selected from each nest; for a total of 140 individuals. We studied the spontaneous locomotory behaviour of infected and uninfected M. scabrinodis ants by tracking their movement in a circular white plastic arena (28 cm diameter) with Fluon®-coated walls in constant laboratory conditions. Only one individual was tested at a time. The ant was gently placed into a small plastic cylinder (diameter: 2 cm) in the centre of the observation arena. It was allowed to acclimatize for 60 s and then the cylinder was removed and the individual could

freely move in the arena. The movement of the ant was recorded for ten minutes with a Canon® Legria HF M56 camera (1280×720 pixels resolution) placed 70 cm above the arena. The arena was surrounded by a white cardboard frame and was lit from above with white neon lights in order to homogenize the light and mask any visual cues that might have influenced the ant’s trajectory. After each assay, the arena was cleaned with alcohol and left to dry before subsequent use. The ants’ locomotory behaviour was analysed using automatic tracking software (EthoVision® XT 7.0; Noldus Information Technology, 2007). A threshold movement of 0.05 cm was used as an input filter to eliminate system noise or slight body movements that were not associated with locomotion (Bernadou et al., 2015b). Ants were tracked using the image subtraction and only objects that are darker than the background methods. For the trajectory analysis, the circular arena was divided into two zones: a central zone (26 cm diameter) and a border zone (1 cm width). To avoid edge effects on locomotory parameters (velocity and meander), only parts of the trajectories in the central zone were analysed. Five behavioural parameters were calculated on the basis of each digitized path, one related to general activity (1), two path characteristics (2-3), two related to spatial location (4-5). 1) total distance travelled by an individual in the course of the ten minutes during which it was under observation (cm); (2) velocity (cm/s); (3) degree of meandering (°/cm): mean absolute change in direction of movement of an ant relative to the distance moved; (4) time in the central zone and the border zone (s): total time spent in these zones during the period of observation; (5) distance to point (cm), as the shortest distance between the ant individual and the centre of the arena (Bernadou et al., 2015b).

2.3. Fat content analyses The workers used for the locomotory experiment were placed individually in labeled vials, killed by freezing, and stored at –20 °C. Individuals were dried at 60°C for five days and weighed individually to the nearest 0.0001 mg with a Sartorius SC2 ultra-microbalance to measure their dry mass. Subsequently, the fat was extracted by soaking each worker for two days in 2 ml petroleum ether (boiling range 40–60 °C, Merck, Darmstadt, Germany) at room temperature. After two days, the workers were transferred into new vials and the petroleum ether was renewed, and each worker was kept in petroleum ether again for two days. Afterwards the ants were dried at 60 °C for six days and weighed again to determine their lean mass. The percentage of fat was calculated according to the standard equation: (dry mass – lean mass) × 100 / dry mass (e.g. Bernadou et al., 2015a).

2.4. Infection status After fat content analysis, individuals from infected colonies (N = 70) were mounted, and the number of fungal thalli on their right side was counted, separately for each body part (head, clypeus, mandible, scape, thorax, 1st, 2nd, and 3rd leg, petiole, postpetiole, gaster) (see Markó et al., 2016). Only mature fungal thalli were considered. The screening was carried out by the same person with an Olympus SZ51 stereomicroscope at ×80 magnification. An ocular micrometer was used to set the axial line through the ant’s body to separate the right and left sides.

2.5. Statistical analyses We tested all data for normality (Kolmogorov-Smirnov test) and performed log2transformations when necessary. To identify potential background factors with regards to the

variability in fat content of the individuals a linear mixed-effect model approach (LMM, N = 140) was applied with fat content as a response variable. Age (young or old) of the individuals, thalli number (infection intensity), infection status of the colony (infected vs uninfected), and the dry mass of the individuals were introduced as explanatory variables, and colony ID was a random factor in all three cases. The infection status of the colonies was used as an explanatory variable, since even in a few cases individuals from infected nests did not have mature thalli, spores might have resided on their cuticle and their activity might have been altered due to exposure to infection risk. In a second analysis, we examined whether infection intensity differed between the two age groups in infected colonies, and also in relationship to their dry mass. A generalized linear mixed model (GLMM, N = 140) was applied with colony ID as random factor. Because of overdispersion, the standard errors were corrected using a quasi-GLM model (Zuur et al., 2009). We used the LMM approach (N = 140) to investigate the effect of age, fat content, thalli number, and infection status of the colony on the four locomotory variables measured: total distance travelled (cm) by individuals in the whole arena in the course of ten minutes, velocity (cm/s), and degree of meandering (°/cm), and the distance to point (cm). All two-way interaction terms between these four factors were also included in the model (except the interaction between thalli number and infection status of the colony). We obtained a minimal model by successively removing the non-significant variables by stepwise backward elimination procedure and by comparing the nested models by likelihood ratio tests. In each model, colony ID was entered as random variable. Data on a single old uninfected individual was excluded from the statistical analysis as an extreme value, because the total distance travelled by it was 3.45 cm on the whole arena.

The time spent by individuals in the two different zones (the central zone and the border zone) was also analysed using the LMM approach (N = 140), according to which the age, fat content, total number of fungal thalli, and infection status of the colony were introduced as variables, and the individual ID nested in colony ID was included as a random factor. All statistical analyses were carried out using the R 3.2.3 statistical environment (R Development Core Team 2015). LMMs were performed using the lme function of the nlme R package (Pinheiro et al., 2016). GLMMs were performed using glmer function in lme4 R package (Bates et al., 2015) and glmmPQL function of the MASS R package (Ripley et al., 2014). The exact significance levels of input variables were retrieved with the use of Anova function in car R package (Fox and Weisberg, 2015). The graphs were carried out using the ggplot2 R package (Wickham, 2009).

3. Results 3.1. General condition of individuals Old workers contained less fat than young workers (old: 10.38% ±5.02; young: 15.38% ±5.87; LMM χ² = 64.46, p < 0.0001), and fat content increased with dry mass of the individuals (χ² = 25.58, p < 0.0001). Infection intensity (χ² = 1.05, p = 0.30, N = 140) and the infection status of the colony (χ² = 0.005, p = 0.93, N = 140) did not have any significant effect on the fat content of workers. Infection intensity increased with age: old individuals had more thalli than young workers (GLMM t = -6.73, p < 0.0001; Fig. 2). In the case of more corpulent individuals, the infection was usually more advanced (t = 16.52, p < 0.0001).

3.2. General locomotory activity

Old workers covered significantly longer distances (474.24 cm ±186.28) than young workers (346.68 cm ±184.85; LMM χ² = 18.04, p < 0.001). Fat content, infection intensity, and infection status of the colony did not significantly influence the total distance moved (χ² = 1.38, p ≥ 0.23). However, the interaction of fat content and age had a significant effect on the distance moved (χ² = 5.64, p < 0.01). In old individuals, the fat content and distance were negatively correlated, while in young individuals they were positively correlated (Fig. 3).

3.3. Path characteristics and location of individuals The velocity of young individuals was significantly lower (0.85 cm/s ±0.32) than that of old individuals (0.95 cm/s ±0.3; LMM χ² = 9.60, p < 0.001), and it increased significantly with fat content (χ² = 7.54, p < 0.01). Other parameters, such as infection intensity (χ² = 1.67, p < 0.19) and infection status of the colonies (χ² = 0.67, p < 0.41), did not have a significant effect. The degree of meandering was related significantly negatively to fat content (LMM χ² = 6.58, p = 0.01). In young individuals, meandering increased in comparison to old workers (187.94°/cm ±97.30 versus 175.69°/cm ±110.8; LMM χ² = 3.68, p = 0.05) (Fig. 4). None of the other parameters, such as the number of fungal thalli (χ² = 0.03, p = 0.85) or the infection status of the colony (χ² = 0.17, p = 0.67), had a significant effect on this feature. Finally, none of the variables measured had any significant effect on the time an individual spent in the different zones of the arena (cumulative zone: 281.38 s ±137.37; border zone: 318.61 s ±137.37): GLMM age χ² = 1.71, p = 0.19; fat content χ² = 0.21, p = 0.64; no. of fungal thalli χ² = 0.66, p = 0.41; infection status of the colony χ² = 2.18, p = 0.13. The distance to point was also not influenced by any of the variables tested: GLMM age χ² = 3.51, p = 0.06; fat

content χ² = 0.22, p = 0.63; no. of fungal thalli χ² = 1.64, p = 0.19; infection status of the colony χ² = 1.47, p = 0.22.

4. Discussion The fungus Rickia wasmannii can reach extreme abundance on its Myrmica host ants (see Markó et al., 2016), causing earlier death of the host (Csata et al., 2014; Báthori et al., 2015). The results of our research indicate that old individuals have more thalli than young individuals, indicating that infection advances with age. Laboulbeniales fungi usually need at least two weeks (but sometimes even more) to attain full maturity (Richards and Smith, 1955; Kaur and Mukerji, 2006), and after that they need considerably more time to reach a high abundance on the surface of their host. We expected that the negative effects of the fungus would increase with the age of their host. Several studies have shown that infected individuals in general may be more active, may leave their nests, may show altered patterns of ground level activity, and may become more exploratory because the parasites change their behaviour in order to increase chances of parasite transmission to the next host (Libersat et al., 2009; Wilson-Rich et al., 2009; Gyuris et al., 2016; Lecocq et al., 2016). Moribund, infected ant workers are also known to loosen their social ties and move away from their nest mates (Heinze and Walter, 2010). Thus, there are two opposing effects acting on the infected individual: maximization of the amount of work due to reduced residual life expectancy and minimization of social interactions (Wilson-Rich et al., 2009; Heinze and Walter, 2010; Moroń et al., 2012). Altogether, this might act in the same direction: increasing the time spent outside the nest and also the distance travelled. Behavioural development of infected young workers might be accelerated, as in the case of honeybees infected by Noseam ceranae (Lecocq et al., 2016). Usually, diseased young individuals could

become foragers more rapidly, and in consequence, they were able to spend more time outside the nest and cover longer distances than their uninfected counterparts. However, surprisingly, we did not find any such effect of R. wasmannii on its host, and none of the studied parameters seemed to have been influenced by infection intensity or generally by the health status of the colony. According to previous studies, however, the fungus causes other behavioural modifications in the host ants, such as increases in allo-and autogrooming frequency (Csata et al., 2014) and water consumption (Báthori et al., 2015). Our findings confirm previous results concerning the age-related nature of fat content in social insects and in particular in ants (Schulz et al., 1998; Toth and Robinson, 2005; Smith et al., 2011; Robinson et al., 2012; Mersch et al., 2013; Bernadou et al., 2015a; Dussutour et al., 2016). Old workers are the leanest individuals, while young individuals have the largest fat reserves. Although in the case of infected colonies young individuals were clearly less infected, the abundance of R. wasmannii did not seem to be reflected in the level of fat reserve. Nalepa and Weir (2007) found that infection with the fungus Hesperomyces virescens, from the order Laboulbeniales, did not influence the wet weight of the host ladybird, Harmonia axiridis. The activity patterns of the two age classes of Myrmica workers clearly differed from each other. Old workers tended to travel longer distances at a higher speed and with a lower degree of meandering. Young individuals covered smaller distances at a slower speed and with an increased degree of meandering. Fat content and age were also correlated with the velocity of the ants and the degree of meandering. The path characteristics of workers – velocity and the degree of meandering – are important factors during foraging activities in old individuals, e.g. when exploring new environments or escaping from predators or rivals (Hölldobler and Wilson, 1990; Ślipiński and Żmihorski, 2016). These findings are consistent with the general pattern of

age polyethism in social insects. Usually, young workers perform inner-nest tasks, such as brood care and nest maintenance, while old individuals spend more time outside the nest performing foraging-related tasks (Hölldobler and Wilson, 1990). In other words, the riskier extranidal tasks, such as foraging over long distances, are more readily undertaken by workers with a shorter life expectancy (Tofilski, 2002). Studies have already shown that fat content is negatively correlated with the propensity to forage in ants (Porter and Jorgensen, 1981; MacKay, 1983) and other social insects (O’Donnell and Jeanne, 1995). Based on our findings, the relationship between the distance travelled and fat content could be age-dependent. Specifically, in Myrmica scabrinodis fat content and distance travelled were negatively correlated, but only in old individuals. In the case of young workers fat content and distance travelled were positively correlated. This relationship could suggest that young individuals make decisions about the level of their activity in relation to available fat reserves. Young individuals in Myrmica are known to contribute to male production (e.g. Leppänen et al., 2014), and physiological condition could be a good proxy for the reproductive status of the individual. Reproductively more active individuals, which might have a higher fat content, could be more engaged in policing activities. This would then give them a similarly higher activity level, as in the case of old foragers (Barth et al., 2010). Alternatively, freshly hatched young individuals, could still have reduced fat reserves and could move less.

4.1. Conclusions In conclusion, our findings show that age and fat content influence the locomotory behaviour of M. scabrinodis workers differentially, while an infection with R. wasmannii does

not display any significant effects on the locomotory behaviour of the host. The fact that the fungus does not seem to modify the locomotory activity of its host might actually show that the major interests of both parties coincide in ensuring the functioning of the colony, since this secures the survival of both organisms, host and parasite.

Acknowledgements We would like to thank two anonymous reviewers for their useful comments on the previous versions of this manuscript, and Thomas Cooper for thorough linguistic revision. E.Cs. and B.M were supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS–UEFISCDI, project number PN-II-RU-TE-2014-4-1930.

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Figures

Fig. 1. Gaster of a M. scabrinodis worker infected with R. wasmannii (SEM photo taken with JSM-5510 by Ciprian Mihali).

Fig. 2. Fungal infection intensity of ant workers belonging to different age classes in infected M. scabrinodis colonies (median, quartiles, min-max values).

Fig. 3. Relationship between total distance travelled (cm) in the whole arena and fat proportion (relative to dry mass, %) for individuals of different age categories. Confidence intervals of 95percent are drawn around the estimated regression lines.

Fig. 4. Relationship between degree of meandering (°/cm) and fat proportion (%) of the individuals in the central zone (A), and the difference between individuals of different age categories with respect to degree of meandering (B) (median, quartiles, min-max values).

Highlights 1. We study the effect of fungal infection, age and fat content on ant activity. 2. Old ant workers bore more thalli of Rickia wasmannii than young ones. 3. Fungal infection does not affect the locomotory behavior of workers. 4. Old workers are leaner and travel longer distances than young ones. 5. Young workers decrease their activity with decreasing fat content, old ones do not.