Entomopathogenic Rickia wasmannii fungal infection generated colony-level stress effects 1 cause size decline in Myrmica ants

20 Parasitism-generated negative effects on ant societies are multifaceted, implying individual and 21 colony-level responses. Though laboratory based evidence shows that the sublethal fungus 22 Rickia wasmannii is responsible for physiological and behavioral responses that may negatively 23 affect individual workers’ resilience and life expectancy in Myrmica ant workers, colony-level 24 stress response to this parasite is largely unknown. Here, we focus on understanding of a long- 25 term, colony-level effect of Rickia infection on Myrmica scabrinodis ant populations by 26 tracking trait size-based changes. We collected worker specimens infection, and (iii) investigate shifts in body size in response to infection over time by testing 30 correlation of workers’ ages and sizes. We found that workers from infected colonies were 31 significantly smaller than their healthy congeners, but neither infection level nor the age of the 32 workers showed significant correlation with the size in infected colonies. Decreasing body sizes 33 in infected colonies can be ascribed to workers’ mediated stress toward developing larvae, 34 which are unable to attain the average body size before they pupate.


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Ants (Hymenoptera: Formicidae), the most widespread social organisms on Earth, attract an 39 amazing diversity of parasitic organisms, such as viruses [1] , bacteria [2] , fungi [3,4] , and an array 40 of uni-and multicellular animal organisms [5,6] . Many of these parasites cause lethal diseases 41 [7] , but most are sublethal, i.e. they do not necessarily pose an imminent danger, though they are 42 assumed to have detrimental effects on the quality of the hosts' lives [4,8] . 43 Entomopathogenic fungus, Rickia wasmannii Cavara, 1899 (Ascomycota: Laboulbeniales) is a 44 typical sublethal parasite of several Myrmica (Hymenoptera: Formicidae) species which has 45 long been believed to have no detrimental effect on its host individuals [9,10,11] . For a century, 46 very little was known about this parasitic organism, and only scant information was available 47 on their distribution and host specificity [4,12,13] . The effect of the fungus on the physiology of 48 its host species was largely unknown. In recent years, modern research has shed light on the 49 real nature of this widespread organism and has shown that it has negative effects on individual 50 host ants. Based on previous studies on the physiological response of infected workers of the 51 main host Myrmica scabrinodis Nylander, 1846, infected workers show higher mortality under 52 laboratory conditions [14,15] . Infected M. scabrinodis workers were also shown to exhibit 53 improved sanitary behavior [14] and a decreased level of intraspecific aggressivity [16,17] which 54 may be disadvantageous for the infected colonies in competitive interactions. 55 Although R. wasmannii is known to elicit different detrimental physiological responses in 56 individual workers, colonies seem to resist and compensate for the negative effects of the 57 infection; they contain queens, rear larvae and pupae, and all age-classes of workers are present 58 (see Csata et al. [17] ). This may be due to the fact that it is often challenging to study colony-59 level effects of an infection under in situ conditions, particularly when complex environmental 60 parameters and multifactorial relationships with a number of other organisms [8,15,18] must be 61 taken into consideration. This may be one of the reasons why a colony-level stress effect of 62 Rickia fungal parasites has never been the subject of focused research. 63 We propose that the colony-level negative effect of the infection is detectable through the 64 decline in size of colony workers. Here, we hypothesize that the infection has detrimental 65 effects on the fitness of brood caring workers, making them unable to engage in foraging 66 activity and feeding and grooming behavior toward their larvae to the necessary extent, and the 67 declining larval growth rate ultimately leads to a decrease in the size of the next generation of 68 workers. The size of the first workers infected is not affected, but R. wasmannii infection is 69 transferred over generations, causing a long-term chronic, life-long infection in a colony, which 70 is known to expand year by year in the nest with some level of seasonal fluctuation [19] . The 71 detrimental colony-level effect of the infection accumulated over time can be measured in 72 workers' sizes. We hypothesized that if R. wasmannii infection is widespread in an ant colony, 73 it will detrimentally affect the size of workers groomed by infected sisters owing to the parental 74 colony's reduced fitness. To test this hypothesis, we compared the sizes of randomly sampled 75 workers from infected and uninfected colonies.

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How can we rule out the possibility that the size decline is caused not by direct larval infection 77 but by mediated colony-level stress among workers? Rickia wasmannii fungus is found to grow 78 on imago [14,20] and is not known to infect ant larvae [11,21] but in order to make sure that a 79 possible size decline is ascribed to stress mediated by nursing workers and is not caused by 80 direct larval infections, trait scaling patterns were also observed. The background of this 81 approach is that infections in the larval stage often cause scrambled trait combinations in adults 82 [22] via altered static trait allometries. If significant shifts in trait scaling were detected in 83 infected colonies, parasitism generated stress might not only be mediated by infected workers. 84 We also tested whether the extent of infection (i.e., number of thalli on the cuticular surface of 85 the ants) and the workers' ages affect the detected changes in imaginal size. Therefore, we 86 registered both infection level and estimated age of the workers in the colony. socially parasitic organisms which live together with their colonies [8,23] . This network is very sensitive, and every single component of this very complex system might have a regulatory role 93 of its own.     Earlier, laboratory based findings and field studies have shown that this fungal infection does 144 not attack Myrmica larvae [11,21,24,25] , but is known to cause an array of detrimental changes in 145 physiology in individual ant workers, such as a decrease in life expectancy, particularly in cases 146 of water and food deprivation [15] , increased sanitary behavior [14] , and a reduced ability to fight 147 against competitors [16,17] . Yet, despite the number of negative effects detected under laboratory 148 conditions, infected colonies in the field have been thought to function relatively well, as they 149 contain queens, larvae and pupae, and all age-classes of workers (see Csata et al. [17] ). not penetrate the cuticle of the host, so the most likely hypothesis concerning the feeding of the 161 parasitic fungus is that it absorbs the necessary nutrients from the workers' cuticle surface or 162 directly from the environment [25] . Ants have numerous exocrine glands, the secretions of which 163 are spread on the cuticle surface by self-grooming and allogrooming. This may be confirmed 164 by the fact that infected M. scabrinodis workers show increased sanitary behavior [14] . This 165 behavior has been observed in the invasive garden ant (Lasius neglectus van Loon boomsma et 166 Andrásfalvy, 1990) infected by Laboulbenia formicarum Thaxt. [26] , where fungus also was not 167 found to penetrate the cuticle of its hosts [25] . This increased sanitary behavior could mean that individuals' cuticular surface. We also know that the intensity of infection varies throughout 175 the year, and the peak number of thalli takes place in late autumn [19] . This fluctuation, combined 176 with the emergence of smaller workers from the overwintering larvae of Myrmica ants [27] , 177 makes it difficult to map the exact processes.

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Although only negative effects of the fungus are currently known, we speculate that the 179 increased sanitary behavior may be advantageous against other parasites, and R. wasmannii 180 may also have unknown positive effects on its hosts, similar to the L. formicarum [14,26] . 181 We also speculate that the decline in size among workers in infected Myrmica ant colonies is   [19] . screened (by FB) for fungal thalli (Fig. 6). All fungal thalli on the whole ant body were counted 216 using a Leica MZ125 stereomicroscope at ×10-160 magnification as described by Báthori et al.
217 [20] . After the fungal thalli had been counted, colony infection level was calculated based on 218 arithmetic mean thalli number of 15 randomly sampled workers from each colony.  As was done by Báthori et al. [20] , all infected M. scabrinodis workers screened for fungal thalli 226 were separated into different age groups. Based on the degree of cuticular pigmentation, five 227 different age categories were described by Cammaerts-Tricot [28] . The infected individuals were 228 classified into categories according to cuticle coloration from younger to older (1-5). The highly 229 pigmented oldest workers were given the highest numbers. The measured morphometric characters are defined as in [22] . The measurements of altogether 233 300 M. scabrinodis workers (10 from each colony) were made with an ocular micrometer using 234 a Leica MZ125 stereomicroscope at a magnification of ×50 for CL, CW and FR, ×100 for FL,

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SL and PPW (all measurements were recorded in μm). All measurements were made by FB.

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Measured characters are defined in Table 1 All measurements are subject to error, therefore repeatability, i.e., the degree of agreement 240 between pairs of observations made on the same measurand under the same conditions, i.e. 241 made by the same observer, using the same microscope, following the same measurement 242 protocol as defined in Csősz et al. [29] , was tested before the statistic framework was created.

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The repeatability of the recorded size parameters was assessed via Intraclass Correlation the only axis with an eigen-value higher than 1), which corresponded to 77% of the total 264 variation in the six body measurement variables and was positively correlated with all variables.

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To test whether there is a significant difference in body size between infected and uninfected 266 colonies, we used a mixed-effects linear regression model (LMM) with Gaussian error 267 distribution using the R-packages "lme4" and "lmerTest" [32,33] , specifying the abovementioned 268 PCA axis representing body size as the dependent variable and infection as independent factor.

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To test how the severity of fungal infection and age affected body size, we fitted another LMM

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(naturally, only using data from infected colonies) with the body size PCA axis as a dependent 271 variable and the log-transformed number of thalli and age as independent variables. Because 272 previously it had been shown that age and thalli number can be correlated, we estimated 273 variance inflation factors to see whether there is substantial multicollinearity between the 274 independent variables used and the "car" package [34] . In addition, we tested the association