1 Running head: warming impact on aphid-ant mutualism 2 3 Towards more intimacy: Elevated temperature enhances ant-aphid relationship 4 5 Blanchard Solène1,2, Van Offelen Julie1, Verheggen François2, Detrain Claire1* 6 7 1 Ecologie Sociale, C.P. 231, Université Libre de Bruxelles, Avenue F.D. Roosevelt, 50, 1050 8 Bruxelles, Belgique 9 10 2 Entomologie Fonctionnelle et Evolutive, Gembloux Agro-BioTech, TERRA, Université de 11 Liège, Avenue de la Faculté d’Agronomie 2B, 5030 Gembloux, Belgique 12 13 * Corresponding author: Claire Detrain 14 [email protected] 15 02/650.55.29 16 17 Key words: climate change, warming, aphid-ant interactions, behavior, ant tending level, 18 collective behaviors, honeydew collection 19 20
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21 22 Abstract 23 24 Climate change will likely affect the association between species interacting at different trophic
25 levels. However, studies focusing on the impact of an elevation of temperature on between-
26 species interactions remain scarce. In the present study we compared, in laboratory conditions,
27 the foraging behavior of the ant Lasius niger and its mutualistic interactions with the aphid
28 Aphis fabae under three conditions of temperatures (i.e. 20, 23 and 26°C), as predicted by
29 climatic scenarios. As regards the aphids, they were more mobile but as likely to release a
30 honeydew droplet at higher temperatures. As regards the ants, a moderate 3°C increase of
31 temperature positively impacted their mutualistic interaction with aphids. Such a reinforcement
32 was achieved through an increase in the walking speed of ant forager, in the flows of mobilized
33 ants as well as in the total amount of honeydew harvested by the ant colony.
34 A further elevation of temperature to 26°C reduced the benefits gained by the aphid-tending
35 ants, in terms of the lower amount of collected honeydew.
36 Based on our results, we hypothesize that, in temperate regions, a moderate increase of ambient
37 atmospheric temperature by 3°C will benefit to the L. niger - A. fabae mutuaslistic interaction
38 but that an more marked elevation of 6°C may represent a threshold thermic value above which
39 a witch of partners or a disruption of the interaction may occur under the temperatures predicted
40 by the most realistic forecast models. for the end of the century.
41 42
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43 44 Introduction
45
46 The impact of global change on terrestrial ecosystems has received growing attention from
47 scientists during the last decades. There is an increasing body of evidences that global climate
48 changes alter multi-species interactions as well as the structure of ecological communities
49 (Hughes, 2000; Kiers et al., 2010; Jamieson et al., 2012; Mackenzie et al., 2013). Increased CO2
50 concentrations or warming temperatures were repeatedly shown to directly affect plants as well
51 as their insect’s pests (Hunter, 2001; Stiling & Cornelissen, 2007; Jamieson et al., 2012; Zhou
52 et al., 2017). According to the most accurate forecasts, the mean global temperature will rise of
53 2 to 4°C by the end of the 21st century (IPCC 2007, 2013, 2019). Potential responses of insects
54 to such elevated temperatures include changes in geographic range (Pecl et al., 2017), life-
55 history traits (Robinet & Roques, 2010), population dynamics (Cammell & Knight, 1992; Porter
56 et al., 1991;) and trophic interactions (DeLucia et al., 2012). By way of consequence, the
57 economic and ecological challenges due to phytophagous insects such as aphids are likely to
58 be exacerbated by global warming, which may accelerate their individual development, favor
59 their population growth and increase the frequency of population outbreaks. Elevated
60 temperature can also induce changes in the behavioral traits of aphids such as their feeding
61 behavior or their escape response to predators (Sable & Rana, 2016; Barton & Ives, 2014; Adler
62 et al., 2007; Ma & Ma, 2012). Furthermore, global warming can have cascade effects on the
63 natural enemies and mutualistic partners of these insect pests (Hance et al., 2007; Gilman et al.,
64 2010; Barton & Ives, 2014). Since several mutualistic relationships are considered as being
65 ephemeral and unstable interactions, it is likely that rapid climatic change could promote shifts
66 from mutualism to antagonism, switches to novel partners, or even the abandonment of any
67 relationships altogether (Sachs & Simms, 2006; Kiers et al., 2010). Such alterations of the
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68 network of insect relationships can have destabilizing and disrupting effects on the whole
69 functioning of the ecosystem (Aslan et al., 2013).
70
71 Because the mutualism between pollinating insects and plants is an ecologically and
72 economically important interaction, the impact of climate change on this insect-plant mutualism
73 is one of the most documented (Brown & Paxton, 2009; Schweiger et al., 2010; Hegland et al.,
74 2009). The aphid-ant mutualism is another well-known interaction due to its frequent
75 occurrence in nature but also due to the crop pest status of several aphid species (Banks &
76 Nixon, 1958; Way, 1963; Stadler & Dixon, 1998; Detrain et al., 2010; Yao, 2012; Fischer et
77 al., 2015; Kremer et al., 2018, Van Emden & Harrington, 2017). Due to their worldwide
78 geographic distribution and their numerical dominance in different biotopes, ants play an
79 important role in the trophic network of ecosystems and are often key partners in mutualistic
80 interactions with sap-sucking insects. However, compared to the plant-pollinator system, the
81 impact of global warming on aphid-ant relationships has received little attention (Barton &
82 Ives, 2014; Marquis et al., 2014; Mooney et al., 2019).
83
84 Aphid-ant mutualism is based on reciprocal services: aphids feed ants with their honeydew and,
85 in return, receive protection against their predators and benefit from an improved hygiene in
86 their colony (Way, 1963; Holldöbler & Wilson, 1990). Ants can be attracted from a distance
87 by volatile organic compounds in the honeydew (Fischer et al., 2015) or by low-amounts of
88 aphid alarm pheromones (Verheggen et al., 2012). Once ants have come into contact with
89 aphids, the honeydew sugars are essential compounds that will cement the ant-aphid mutualistic
90 relationships (Detrain et al., 2010). When the aphid abdomen is stimulated by the antennae of
91 ant foragers, the homopteran is likely to extrude a honeydew droplet without ejecting it, thereby
92 allowing its collection and ingestion by the ants. Ants that have fed on honeydew can decide to
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93 recruit additional nestmates towards the aphid colony, through the laying of a trail pheromone.
94 The intensity of the recruitment trail as well as the level of food exploitation by ants will depend
95 on the amount and composition of carbohydrates in the aphid honeydew (Mailleux et al., 2003;
96 Detrain et al., 2010).
97
98 Because of the protection and hygienic care provided by workers, the population of ant-tended
99 aphids usually grow faster than unattended aphid colonies, thus suggesting mutual benefits for
100 both partners (El-Ziady & Kennedy, 1956; Buckley, 1987; Flatt & Weisser, 2000). However,
101 the truly mutualistic nature of this relationship can be questioned in some cases. For instance,
102 ants can occasionally switch to predation when an aphid population becomes too dense (Stadler
103 & Dixon, 2005), too mobile or more eager to disperse (Way, 1963). Likewise, in the case of
104 obligate myrmecophilous aphids, whose survival depends on the presence of ants, the energetic
105 costs of maintaining a mutualistic interaction may become quite high (Stadler & Dixon, 1998).
106 Indeed, ant-tended aphids produce tinier droplets, but in greater number, to fulfil the energetic
107 needs of ants, which may lead to a shortage of the nitrogen that is usually available for aphid
108 growth and reproduction (Yao & Akimoto, 2002). Maintaining a mutualistic relationship is thus
109 far from being taken for granted by both partners, of which the cost-benefit balance can become
110 disadvantageous. For example, any change (e.g. temperature-induced ones) in the physiological
111 and behavioral traits of aphids, that alter their relative value as a source of carbohydrates, could
112 make ants disregarding their trophobionts and may endanger the stability of the whole
113 mutualistic interaction.
114
115 Despite the potential consequences for the control of aphids as crop pests, it is still unclear how
116 the predicted elevation of temperature may alter the behavior of aphid-tending ants and, by way
117 of consequence, may hamper or reinforce the mutualism with their trophobionts. On the one
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118 hand, temperature it determines both the onset and the duration of ant foraging activity, along
119 with the speed of foragers (Cerda & Retana, 1998; Drees et al., 2007; Stuble et al., 2013) and
120 is thus expected to positively impact the time investment of ants in aphid-tending activities. In
121 an ant-mealybug interaction, an elevation of temperature also had a positive effect on
122 mealybugs’ honeydew excretion and ant performance, including their investment in tending
123 homopterans (Sagata & Gibb, 2016). On the other hand, elevated temperatures can alter the
124 sugar composition of aphid honeydew, with ants being less eager to tend their trophobionts
125 (Mooney et al., 2019). Furthermore, higher temperatures can decrease the aggressiveness of
126 ants towards the natural enemies of aphids, thereby leading to cascading effects on aphid
127 abundance (Barton & Ives, 2014). Since there is evidence of mixed directionality of responses,
128 it is difficult to determine the net changes in the cost/benefit balance for each partner that
129 outcome from climate warming. More in-depth studies of temperature-related changes in the
130 biological mechanisms that underlie ant-aphid interactions may improve estimates of climate-
131 warming effects on aphid crop pests.
132
133 In this study, using representative species, we raise the hypothesis that an elevation of
134 temperature will impact the facultative mutualistic interaction existing between the black
135 garden ant Lasius niger (Linnaeus, 1758) and the black bean aphid Aphis fabae (Scopoli, 1763).
136 We hypothesize that both partners, as they are poïkilothermous insects, will be more mobile
137 under elevated temperatures, thereby increasing the rate of physical contacts and interactions
138 between both partners. We will also examine whether temperature-driven changes in encounter
139 rates are related to a higher honeydew production and consumption, leading to a reinforcement
140 of the mutualistic relationship between ants and aphids.
141
142 Material and methods
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143
144 Insect rearing
145 Colonies of the garden black ant Lasius niger were collected in Brussels, Belgium (Ixelles
146 50°49'06.4"N 4°24'04.7"E and Auderghem 50°48'49.2"N 4°26'16.0"E). The colonies were
147 reared in plastic containers whose edges were covered with polytetrafluoroethylene (Fluon,
148 Withford, U.K.) to prevent ants from escaping. Aqueous sucrose solution (1 M) and water filled
149 test tubes were provided. Once a week, dead mealworms (Tenebrio molitor (Linnaeus, 1758))
150 were added as protein sources. The colonies were reared in laboratory-controlled conditions:
151 LD 16h:8h, 21 1°C, 40% relative humidity. Black bean aphids (Aphis fabae) were reared on
152 broad bean plant Vicia faba L. (var. “Major”), grown in plastic pots filled with a mix of perlite
153 and vermiculite (1:1 w/w) and placed under the same laboratory conditions as above.
154
155 Tested temperatures
156 To determine the values of the tested temperature conditions, we used the meteorological data
157 collected by the Royal Meteorological Institute of Belgium during the past seven years in
158 Brussels. At the end of spring and in summer (May to September) and during daylight (8am to
159 22pm), the average temperature was close to 20°C, while the mean maximal temperature
160 reached 26°C. Besides, the report of IPCC (Intergovernmental Panel on Climate Change)
161 (IPCC, 2013) foresees a raise of 2° to 4°C by the end of the 21th century, and up to 6°C when
162 considering the most pessimistic scenario. Based on these data, we chose to test the three
163 following temperatures: 20°C (corresponding to the mean actual summer temperature in
164 Brussels), 23°C (likely to be reached by the end of 2100) and 26°C (corresponding both to the
165 current maximal summer temperature experienced in Brussels, and to the most pessimistic
166 elevation of average temperature predicted by IPCC for 2100). These three tested temperatures
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167 nevertheless stand within the range of the development temperatures of both the host plant
168 V.faba , i.e. 18 to 23 °C (Duke, 1981), and the aphid A.fabae, i.e. 25°C (Harrington et al., 2007).
169
170 Experimental setup
171 Closed climatic chambers (120x70x60 cm) were built with polycarbonate transparent panels.
172 The ambient temperature inside each chamber was controlled using heat plates (HabiStat
173 Reptile Radiator 75 Watts HRR75), coupled to a thermostat (HabiStat Digital Temperature
174 Thermostat, 600W, HTDT). An air pump (SuperFish Koi-Flow 60, 30 L/min air flow) renewed
175 the ambient air inside the chamber, thereby helping to limit temperature fluctuations at around
176 ± 0.4°C during all the experiments.
177
178 Behavioral observations
179 We tested nine ant colonies under the three different temperatures. Three days before the
180 experiment, broad bean plants of similar age (around 16 days) and physiological stage (three
181 leaves) were selected. The chosen plants were moved into plastic pots with a standardized
182 amount of water (20 mL) and were infested with 70 adult aphids. The aphid-infested plant and
183 the ant colony were both placed in the climatic chambers, under the tested temperature, for
184 three days before the experiment. After three days the aphid populations were of about 180 ±
185 20 individuals. The plants were placed in the foraging area of the ant colony, i.e. their rearing
186 tray, and the plant pot was surrounded by plastic walls (2cm high) covered with
187 polytetrafluoroethylene (Fluon, Withford, U.K.) to prevent ants from climbing on the plant and
188 from having access to aphids. Before the experiment, the tested ant colonies could not access
189 the aphid colonies and were deprived of food for three days in order to stimulate their search
190 for food resources. The experiment was carried out on the fourth day of starvation.
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191 The experiment lasted eight hours and started when a bridge was added in the set up to connect
192 the ant colony to the aphid-infested plant. Each ant colony was tested once on each of the three
193 temperatures, meaning that nine replicates were carried out for each tested regime of
194 temperature. Between successive tests, each colony was allowed to rest for at least seven days
195 at 21°C and was given access ad libitum to water and food.
196
197 Dynamics of collective exploration and foraging
198 We quantified the foraging response of the ant colony once it had discovered the aphid colony.
199 We placed a camera (Logitech C920) on the top of the bridge connecting the ant colony and to
200 the aphid-infested plant. We video-recorded the traffic of ants crossing the bridge during the
201 first two hours. On these recordings, we counted the flows of ants that were either climbing on
202 the plant or going back to the colony. After these two first hours, the ant flows were quantified
203 every hour for 5 minutes, until the end of the experiment. Furthermore, every 15 minutes during
204 the whole experiment, we counted the number of ants exploring the foraging area, walking on
205 the bridge, and exploring the aphid-infested plant.
206
207 Ant-aphid interactions
208 We quantified the occurrence and duration of several behaviors displayed by the ants while
209 foraging over the aphid-infested plant. These observations were done by eye and by using the
210 Behavioral Observation Software BORIS (Friard & Gamba, 2016). Every hour during the
211 whole duration of the experiment, we randomly chose two ant foragers that we followed from
212 the moment they climbed on the plant until they left it. When an ant stayed on the plant more
213 than 30 minutes, we stopped recording its behavior what enabled us to observe two ants per
214 hour of experiment.
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215 First, we measured the total time spent by the ant walking over the plant and we considered a
216 worker to be inactive as long as it stopped moving without performing any other behavior for
217 at least 5 seconds. As long as the ant stayed over the plant, we quantified the number and
218 duration of the antennal contacts it performed on aphids. We also measured the number of
219 honeydew droplets emitted from the anus of aphids and collected by the ants. Finally, we noted
220 any aggressive behavior towards the aphids, food exchanges between nestmates and self-
221 grooming behavior.
222 Concurrently to these behavioral observations of ant foragers, we placed a camera with a macro
223 lens (Kurokesu model KITC920) close to a group of 4 to 10 aphids. We video-recorded the
224 behavior of these aphids for 20 minutes every hour during the whole experiment and we
225 analyzed these data using the software BORIS. We counted the number of antennations that the
226 aphids received from the ants. We quantified the number of honeydew droplets produced by
227 aphids that were emitted either spontaneously, without any previous contact with ants, or after
228 a stimulation performed by ants. We also observed whether the emitted honeydew droplets were
229 either collected by the ants or withdrawn by the aphid and falling on the plant. We analyzed the
230 total duration of antennal contacts received by the aphids, as well as the time elapsed between
231 the emission of a honeydew droplet and its collection by ants. A short duration of antennations
232 before release of a honeyew droplet and short time ellapsed before its ingestion were used as
233 proxies for the propensity of aphids to produce honeydew and for the eagerness of ants to feed
234 on honeydew, respectively.
235
236 Statistical analyses
237 All data were analyzed with R software (v. 3.5.0) and all tests were two-tailed with a
238 significance level of α =0.05. No data met normality conditions. The number of ants present on
239 the foraging area before the beginning of the experiment was compared across temperatures by
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240 using a Kruskal-Wallis test. Other data were analyzed using Generalized Linear Mixed Models
241 (GLMMs). When necessary, transformations were applied to fit the model’s assumptions. We
242 used generalized linear mixed models (GLMMs) from the ‘lme4’ R-package, to analyze data
243 that met the models' assumption and that showed no overdispersion based on model
244 deviance/degrees of freedom values (Bates et al., 2015). Ant colonies were used as random
245 factor in all analyses. Tukey post hoc comparisons were performed using the R package lsmeans
246 (Lenth, 2016). For all count and proportion data, we used Poisson GLMMs with a Logit link
247 function. For duration data, inverse gaussian GLMMs were used with a 1/mu^2 link function.
248 For most of the observations only temperature was taken as fixed effect. For temporal changes
249 in the number of mobilized foragers or in the time they spent over the plant, we considered
250 temperature and time as fixed effects ). In this case both the ambient temperature and the time
251 were considered as fixed factors. No interaction between these two factors was ever found in
252 our data.
253
254 Results
255
256 Dynamics of collective exploration and foraging
257 Just before starting the experiment, a similar number of ants were walking over the foraging
258 area between the three temperature regimes (Kruskal-Wallis test, χ2 = 3.6325, N=24, df=2, p-
259 value = 0.545). Once the foraging area was connected to the plant, the first ant workers climbed
260 on the bridge within the first 30 minutes for all the nine tested colonies (Fig1.). We noticed an
261 effect of the time of the day on the level of ants’ foraging on the plant. In less than half an hour,
262 we observed a steep increase in the number of workers that reached the plant, with 12 to 26 ants
263 on average exploring this new resource. Then, after two hours, for all three temperature
264 regimes, the number of ants present on the plant converged to around 15 ants and stayed steady
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265 until the end of the experiment (Fig.1). We also found that temperature had a significant impact
266 on the total number of ants foraging on the plant, depending on the time of the experiment
267 (GLMM, z-value = 2.848, df = 2, N=288, p-value < 0.001). Indeed, a higher number of ants
268 foraged on the plant at 23°C compared to the other two temperatures, during the first two hours
269 of the experiment (Tukey’s post-hoc, at 10h00 p-value(20-23) = < 0.001, p-value(20-26) = 0.064, p-
270 value(23-26) = 0.122; at 12h00, p-value(20-23) = 0.018, p-value(20-26) = 0.056, p-value(23-26) = 0.393).
271 After two hours, the tested temperatures no longer had a significant effect on the number of
272 foragers. To assess whether temperature altered the ants’ investment in the exploitation of the
273 aphid-infested plant, we defined a plant occupation index for each colony. This occupation
274 index is the relative number of ants staying on the plant over the total number of ants located
275 outside the nest averaged over the 32 measures done during the whole experiment. These
276 occupation indices, did not differ between the three temperatures (GLMM, z-value = 0.555, df
277 = 2, N=9, p-value = 0.586). About 64% of the ants present on the set up were foraging on the
278 plant infested by aphid colonies at 20°C, 65% at 23°C and 59% at 26°C (Table 1).
279 By filming the bridge for the first two hours of the experiment, we measured the ascending and
280 descending flows of ants towards the plant stalk. We found that the ascending ant flows were
281 significantly impacted by the ambient temperature (GLMM, z-value= 19.470, df = 2, N=27, p-
282 value < 0.001). The cumulated number of ants in the ascending flow was the highest at 23°C
283 and 26°C compared to 20°C (Tukey’s post-hoc, p-value(20-23) = < 0.001, p-value(20-26) = < 0.001,
284 p-value(23-26) = 0.0505; Fig.2; Table 1). We did not observe any significant difference in the
285 descending ant flows, although it was correlated to the ascending flows (GLMM, z-value =
286 19.073, df =2, p-value = 0.282).
287 We also found a significant effect of temperature on the proportion of ants contacting aphids
288 with their antennae (GLMM, z-value = 2.662, df = 2, N=144, p-value = 0.008). Indeed, nearly
289 70% of the ants performed at least one antennal contact with an aphid at 26°C compared to
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290 23°C and 20°C, where less than 50% contacted aphids at least once (Tukey’s post-hoc, p-
291 value(20-23) = 0.8122, p-value(20-26) = 0.0235, p-value(23-26) = 0.004; Fig.5; Table 1). We looked
292 at the global success rate of honeydew collection , which is the total number of honeydew
293 droplets collected by all the observed ants divided by the total number of antennal contacts they
294 performed on the aphids. We found a significant effect of temperature (GLMM, z-value =
295 2.316, df = 2, N=9, p-value = 0.021) on the success rate of honeydew collection, which was the
296 highest at 23°C and significantly different from the lowest success rate observed at 26°C?.
297 (Tukey’s post-hoc, p-value(20-23) = 0.934, p-value(20-26) = 0.054, p-value(23-26) = 0.025; Fig. 7;
298 Table 1).
299 One can also extrapolate the amount of honeydew brought back to the nest after two hours of
300 food exploitation by multiplying the mean number of honeydew droplets collected per ant
301 capita with the cumulated number of ants that had returned to their nest after 2 hours of
302 foraging. We thus estimated that, on average, after two hours, a colony had retrieved a mean
303 total of 65 droplets at 20°C while 223 and 123 droplets were retrieved by at 23°C and 26°C
304 respectively. A limited 3°C increase of temperature thus favored the collection of aphid
305 honeydew while a further elevation of temperature to 6°C reduced the amount of this sugar
306 resource that was retrieved inside the ant colony.
307
308 Ant-aphid interactions
309
310 Impact of temperature on the ant behavior
311 On average, ant foragers tended to spend less time searching and foraging for food over the
312 plant with the increase of temperature, although this result was not significantly different (table
313 1; GLMM, z-value = 2.662, df = 2, N=144, p-value = 0.109).
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314 The time took by the first five ants to climb the bridge at the beginning of the experiment did
315 not differ across the three tested temperatures (GLMM, df = 2, z-value = 2.451, N=45, p-
316 value=0.090; Fig.3). However, after two hours of experiments, the ambient temperature had a
317 significant impact on the time spent by ants to cross the bridge (GLMM, df = 2, z-value = 2.742,
318 N=45, p-value < 0.001; Fig.3) which was significantly higher at 23°C and 26°C compared to
319 the ambient tested temperature (Tukey’s post-hoc, p-value(20-23) < 0.001, p-value(20-26) < 0.001,
320 p-value(23-26) = 0.2346; Table 1). Hence, a higher temperature enhanced the walking speed of
321 foragers that moving faster on the plant at 23°C and 26°C after two hours of experiment.
322 As regards the ant-aphid interactions, the elevation of temperature did not impact the average
323 number of antennations made by each ant that contacted aphids (GLMM, z-value = 1.967, df =
324 2, N=57, 52 and 76 respectively for 20°C, 23°C and 26°C, p-value = 0.884; Fig.6; Table 1).
325 However, the total duration of the antennations made by each ant significantly differed under
326 the tested temperatures (GLMM, z-value = 0.159, df = 2, N=57, 52 and 76 respectively for 20,
327 23 and 26°C, p-value < 0.001; Table 1). In total, the antennal contacts made by each ant lasted
328 les time at 23°C and 26°C compared to 20°C (Tukey’s post-hoc, p-value(20-23) = < 0.001, p-
329 value(20-26) = < 0.001, p-value(23-26) = 0.785). Furthermore, the mean number of honeydew
330 droplets that were collected per contacting ant was significantly impacted by the temperature
331 (GLMM, z-value = 2.145, df = 2, N=57, 52 and 76 respectively for 20, 23 and 26°C, p-value =
332 0.032). Each ant collected a higher amount of honeydew at 23°C compared to 20°C and 26°C
333 (Tukey’s post-hoc, p-value(20-23) = 0.041, p-value(20-26) = 0.99 p-value(23-26) = 0.039; Table 1).
334 We looked at the individual success rate of honeydew collection, which is the number of
335 antennal contacts performed per ants on aphids which were followed by the collection of a
336 honeydew droplet by the same ant, and found no significant effect of temperature (GLMM, t-
337 value = 0.840, df = 2, N = 28, 17 and 25 respectively for 20, 23 and 26°C, p-value = 0.404;
338 Table 1).
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339 We never observed any agonistic behavior from the ants towards their aphid partners, whatever
340 the temperature studied.
341
342
343
Behavioral observation 20°C 23°C 26°C Cumulated ant flows; 71.42 ± 29.84 a 172.15 ± 48.47 b 152.37 ± 49.78 ab Mean + SE; N=9 Occupation index of the plant; 0.64 ± 0.05 a 0.65 ± 0.04 a 0.59 ± 0.05 a Mean + SE; N=9
Proportion of ants contacting a a b 0.39 ± 0.04 0.40 ± 0.04 0.53 ± 0.04 aphids; Mean + SE; N=9 Global success rate of honeydew
collection triggered by an ab a b Ant colony Ant 0.23 ± 0.04 0.26 ± 0.05 0.16 ± 0.04 antennal contact Mean + SE; N=9 Estimated number of droplets retrieved per colony after 2h 64.72 ± 11.67 223.16 ± 57.65 122.97 ± 29.16 foraging; Mean + SE; N=9 Time spent by each forager on the 296.44 ± 30.47 a 249.58 ± 28.92 a 225.36 ± 25.21 a plant Mean + SE; N=144 Time spent by ants to cross the bridge at the beginning of the 31.58 ± 2.50 a 31.87 ± 2.35 a 31.40 ± 2.41 a experiment (s); Mean + SE; N=45 Time spent by nants to cross the
bridge after 2h of foraging (s); 30.68 ± 1.68 a 22.37 ± 1.67 b 21.99 ± 1.68 b Mean + SE; N=45 Number of antennations made per 4.79 ± 0.62 a 4.88 ± 0.79 a 5.66 ± 0.79 a dual Forager dual ants having contacted aphids; N=57 N=52 N=76 Mean + SE
Indivi Total duration of antennations 10.44 ± 1.31 4.90 ± 0.32 5.27 ± 0.33 made per ant having contacted a b b N=57 N=52 N=76 aphids; Mean + SE Number of droplets collected per 0.93 ± 0.20 1.42 ± 0.35 0.93 ± 0.20 ant having contacted aphids; a b a N=57 N=52 N=76 Mean + SE
15
Individual success rate of 0.17 ± 0.03 0.15 ± 0.03 0.13 ± 0.02 honeydew collection after a a a N = 57 N = 52 N = 76 contacting aphids; Mean + SE 344 Table 1: Summary of the behavioral observations made by the ants during the experiment. Collective behaviors and individual 345 behaviors are separated here. N values are the number of observations made per temperature. Tukey’s post-hoc were performed 346 for all observations. Experimental conditions that shared a common letter were not significantly different. 347 348 Impact of temperature on the aphid behavior
349 The impact of an elevation of temperature on aphid’s mobility was analyzed on video
350 recordings focused on a section of the plant stem, where aphids were present in patches of 5 to
351 15 adult individuals. We found that the proportion of mobile aphids changed according to the
352 temperature (GLMM, z-value = 4.882, df = 2, N=64, p-value < 0.001) by being slightly higher
353 at 26°C than at the other two temperatures (Tukey’s post-hoc, p-value(20-23) = 0.9564, p-value(20-
354 26) = < 0.001, p-value(23-26) = < 0.001; Fig. 4; Table 2).
355 The elevation of temperature did not impact the number of antennations that were received per
356 aphid (GLMM, z-value = 1.844, df = 2, N=64, p-value = 0.385; Table 2). Only a few (at most
357 11 %) of these antennations triggered the emission of a honeydew droplet regardless of the
358 tested temperature conditions (GLMM, z-value = 2.563, df = 2, N = 59, 61 and 64 respectively
359 for 20°C, 23°C and 26°C, p-value = 0.659 Table 2). Furthermore, we found no impact of
360 temperature on the total number of honeydew droplets emitted per aphid over 120 minutes of
361 observation (GLMM, z-value = 6.132, df = 2, N= 64, p-value = 0.090; Table 2). Also, similar
362 amount of honeydew was collected from the aphids by the ants after stimulation, indifferently
363 of temperature (GLMM, z-value = 2.632, df = 2, N = 64, p-value = 0.085; Table 2). We also
364 found no impact of temperature both on the motivation of ants to trigger the release of aphid
365 honeydew and on the aphid responsiveness to these ants’ stimulations. Indeed, the duration of
366 the antennation that preceded the release of a honeydew droplet by the simulated aphid did not
367 differ between the tested temperature conditions (GLMM, z-value= 1.564, df = 2, N = 32, 39
368 and 33 respectively for 20°C, 23°C and 26°C, p-value = 0.486; Table 2). Furthermore, aphids
16
369 were as reactive to the stimulations provided by the ant tenders since the time elapsed between
370 a stimulating antennation and the emission of a honeydew droplet was similar across
371 experimental conditions (GLMM, z-value= 0.124, df = 2, N = 32, 39 and 33 respectively for
372 20°C, 23°C and 26°C, p-value = 0.461; Table 2).
373 To sum up, at the aphid scale, an elevation of temperature increased the number of contacts
374 they received from the ants, although this higher level of stimulation did not concurrently lead
375 to an increase in the number of honeydew droplets they released. The honeydew produced by
376 the aphids under the three tested temperatures seemed to be as attractive for the ants that were
377 similarly quick to ingest the emitted droplet.
378 Behavioral observation 20°C 23°C 26°C Proportion of mobile aphids 0.45 ± 0.03 a 0.46 ± 0.03 a 0.51± 0.03 b Mean + SE; N=64 Number of contacts received per observed aphid 3.06 ± 0.43 a 3.27 ± 0.40 a 3.48 ± 0.29 a Mean + SE; N=64 Proportion of antennations received by aphids that 0.11 ± 0.02 0.08 ± 0.01 0.07 ± 0.01 a a a triggered honeydew emission N=59 N=61 N=64 Mean + SE; Total number of honeydew droplets emitted per aphid 0.25 ± 0.03 a 0.25 ± 0.03 a 0.27 ± 0.03 a Mean + SE; N=64 Proportion of honeydew droplets collected per observed aphid over the 0.73 ± 0.08 a 0.72 ± 0.08 a 0.73 ± 0.08 a antennations they received Mean + SE; N=64 Duration of antennation 22.44 ± 5.94 19.48 ± 3.41 15.30 ± 2.80 triggering honeydew emission a a a N=32 N=39 N=33 (s) Mean + SE Time elapsed between 10.67 ± 2.60 8.73 ± 3.24 11.19 ± 3.38 honeydew emission and a a a N=32 N=39 N=33 ingestion by an ant (s)
17
Mean + SE 379 Table 2: Summary of the behavioral observations made on aphids during eight hours. N values are the number of observations 380 made per temperature. Tukey’s post-hoc were performed for all observations. Experimental conditions that share a common 381 letter were not significantly different. 382
383 Discussion
384
385
386 The present study suggests that an elevation of temperature – corresponding to the predicted
387 scenarios of climate change by the end of the 21th century – has reinforcing effects on aphid-
388 ant mutualism (Table 3). Elevated temperature had a direct impact on the activity, the foraging
389 and the exploitation of food by the ant colony. Individually, the foragers moved faster and
390 collected higher amount of resources, especially at 23°C. Aphids did not show any changes in
391 their behaviors, except for a higher mobility directly due to an increase in temperature.
Interaction Ants Aphids Ant flows (+)23 Ant’s and aphid’s mobility (+)26 and 23 (+)26 Contact rate given or received (+)26 (0) Proportion of ants contacting aphids (+)26 Honeydew emission by aphids (0) Ant eagerness for honeydew (0) Reactivity towards antennal contacts (0) Honeydew collection (+)23 (-)26 392 Table 3: Impact of the elevation of temperature on the behaviours of ants and aphids. (+) means significant increase of the 393 behavior performed by an ant or an aphid, (-) meansa significant negative impact . (0) when no impact of temperature was 394 found. Squares left in blank mean that the selected behavior was not applicable for one of thespecies. 23° and 26° represents 395 the temperature at which the significant impact was found.
396 397 Impact of elevated temperature on the ants’behaviour
398 Food iscovery and foraging flows
18
399 Ants are poïkilothermous organisms, whose foraging activityclosely depends on the thermal
400 conditions (Azcarate et al., 2007; Hurlbert et al., 2008; Jayatilaka et al., 2011), as long as the
401 temperature value stays beneath critical thermic zones (Cerda et al., 1998). As regards the
402 search for food resources, the aphid tending ant species, Lasius niger explore the nest
403 surroundings either on an individual basis or collectively by laying an exploratory trail
404 (Devigne & Detrain, 2002; Detrain et al., 2019).In the 20°-26°C range of tested temperatures,
405 we found no impact of temperature on the global exploratory activity displayed by ant colonies
406 at the beginning of the experiment.
407 Once ants hand discovered the aphid-infested plants, at the beginning of food recruitment,
408 foragers walked at a similar speed regardless of the ambient temperature but the ant flows
409 towards the infested plant doubled at 23°C and 26°C compared to 20°C with the highest number
410 of workers being mobilized at 23°C. Then, after two hours, the flows of recruited foragers
411 slightly decreased and stabilized at similar values for all the tested temperatures while t ant
412 workers walked faster on the bridge at 23°C and 26°C than at 20°C. As a result, throughout the
413 experiment, the total flow of foragers ascending the bridge to the plant, or going back to their
414 nest, was the lowest at 20°C. A moderate 3°C increase of temperature triggered a higher level
415 of foragers’ mobilization. An elevation of temperature of 6°C did not further enhance the
416 mobilization of foragers whose ascending flows on the plant became even lower than at 23°C.
417 Similar results were shown in another ant-homopteran mutualistic interaction, but at a higher
418 temperature, 29°C, compared to 23°C and 25°C (Sagata & Gibb, 2016), probably due to the
419 fact that the ant species studied is known to face higher extreme temperatures (Walter &
420 Mackay, 2004).
421 The direct effect of temperature on the metabolism of foragers (Jayatilaka et al., 2011) explains
422 the higher speed of foragers, resulting in more intense flows of ants heading towards the aphid-
423 infested plant at higher temperature conditions. Temperature-induced changes in ant flows
19
424 could also indirectly result from changes in the quantity and/or quality of aphid honeydew,
425 which in turn, lead to differences in the intensity of recruitment trails laid by ant foragers (Völkl
426 et al., 1999; Detrain et al., 2010, Detrain & Prieur, 2014). Further studies are still needed to
427 assess whether the recruitment signals transmitted by successful scouts having discovered food
428 actually change with a warming of the ambient temperature. At most, one can say that a
429 potentially higher evaporation rate of trail pheromone at 23°C and 26°C compared to 20°C did
430 not hamper the dynamics of collective exploitation of aphid honeydew by L.niger foragers.
431 Furthermore, ant species such as L.niger, whose trail pheromone compounds have a relatively
432 long half-lifetime (around 40 minutes, Beckers et al., 1992), would show a recruitment
433 dynamics more resilient to elevated temperatures than other mass-recruiting ant species relying
434 on highly volatile trails (Van Oudenhove et al., 2011; Boullis et al., 2016).
435 Aphid-Tending behavior
436 Once they get over the plant, foragers spent a slightly lower, although not significantly different,
437 amount of time on the aphid-infested plant at warmer temperatures, probably in relation with
438 their concurrent increase of locomotory activity. As a correlate, each ant spent less time at
439 contacting and tending the aphids with the increase of ambient temperature. On the other hand,
440 the increased locomotory activity at higher temperatures made ant workers more likely to
441 encounter trophobionts per unit of time, thereby leading to a higher proportion of aphid-tending
442 foragers at 26°C compared to the other two temperature conditions. Similarly, in the case of
443 an ant-mealybug mutualism (Zhou et al., 2017); the ant tending level (i.e. the number of
444 interacting ants per mealybug) increased with temperature (26°C compared to 23°C). On the
445 contrary, such tending level was found to decrease concurrently to an elevation of temperature
446 in another study on ant-aphid mutualism (Mooney et al., 2019). Such differences could be due
447 to the fact that the field study done by Mooney et al., 2019 was focused on different ant and
448 aphid’s species and was carried out under temperatures which daily and seasonally varied. Such
20
449 variations may have included extremely high temperatures, above 29°C during summer, which
450 might have negatively affected the tending of aphid colonies by ants.
451 Honeydew collection
452 Through antennal contacts, ants typically encourage their mutualistic partners to increase their
453 honeydew production (Larsen et al., 1992, Degen et al., 1986; Sagata & Gibb, 2016). The higher
454 the proportion of tending ants at warmer temperatures contributed to reinforce the mutualisitic
455 interaction between the two partners. However, in terms of food income for the ant colony, the
456 total amount of collected honeydew was estimated to be the highest at 23°C. Indeed, this
457 moderate increase of temperature increased both the cumulated ant flows and the global success
458 of foragers at getting a honeydew droplet. At the colony level, a further increase of temperature
459 to 26°C reduce the total amount of honeydew that was brought back to the nest, mainly due to
460 to a lower success of ants to trigger the release of honeydew by contacted aphids.
461 When considering the global impact of warming on ant colonies, a moderate increase of
462 temperature to 23°C seemed to be optimal in temrs of honeydew reward but a further elevation
463 to 26°C made the benefits brought along less pronounced. On may even assume that, in the
464 long term, at 26°C, foragers may become less and less successful at bringing honeydew
465 resources to their nest thereby destabilizing the cost-benefit balance of this mutualistic
466 relationship.
467
468 Impact of temperature on the aphids
469 As for the ants, due to their enhanced metabolic activity, aphids were more mobile over the
470 plant at 26°C compared to the two tested temperatures. In some aphid species, an increased
471 mobility can trigger aggressiveness from their tending ants, but we never observed such
472 agonistic behavior for the tested range of temperature . Furthermore, in the case of a low density
473 of aphids as in our experiment, predation rarely occurrs because the homopterans are primarily
21
474 used by the ant as honeydew producers (Sakata, 1995). Although higher flows of L.niger
475 foragers were mobilized towards the aphid infested plants at higher temperatures, each aphid
476 received, at its local scale, the same number of antennations and potentially the same level of
477 care from the ants, regardless of the ambient temperature. Likewise, warming temperatures did
478 not alter the amount of honeydew droplets emitted by the aphids nor the number of antennal
479 contacts required to trigger the emission of a honeydew droplet. This suggests that aphids were
480 as likely to produce honeydew within the tested range of thermal conditions. Finally, we
481 examined whether the honeydew quality could change according to the ambient temperature.
482 Indeed, high temperatures (41°C) can alter the nutritional content of honeydew (Salvucci et al.,
483 1999), affecting the nutritional requirements and ant preference for sap-sucking insects (Kiss,
484 1981). The composition of honeydew was not studied here, but some hints about honeydew
485 quality are provided by the ant responses toward emitted honeydew droplets, which might
486 hesitate before taking this droplet, or just leave it if not interested. In our experiments, the
487 honeydew droplets were immediately collected by the ants at the same rate, suggesting an equal
488 attractivity of honeydew regardless of the tested temperature. In addition, a similar number of
489 honeydew droplets were left uncollected by the ants for all tested conditions.
490
491 To sum up, a moderate warming of 3°C seems to impact positively the ants which, at the colony
492 level, mobilize larger flows of foragers and that retrieve a higher total amount of honeydew,
493 with potential cascading effects for aphids, which might also get direct benefits from the
494 increase of interactions with ants. It has been suggested that benefits to the homopterans from
495 ant tending are strongly related to the ant tending level (Breton & Addicott, 1992; Zhou et al.,
496 2015). The more foragers tend aphids and get in return high amount of food, the more likely
497 they protect the aphid colony against predators and keep their environment clean thereby
498 preventing the development of fungi, or sooty molds on the plant (Way, 1963; Dixon, 1998;
22
499 Stadler & Dixon, 2005). An average temperature of 23°C during summer days and/or an 3°C
500 increase of atmospheric temperature as predicted by climatic scenarios may therefore benefit
501 for both partners and reinforce the mutualism between Lasius niger and Aphis fabae in
502 temperate regions. On the other hand, a further increase of temperature to 26°C may represent
503 a threshold thermic value, below which the L.niger-A.fabae mutualism is the strongest, and
504 above which a witch of partners, or disruption of the interaction may be observed. Under natural
505 conditions, some episodes of extremely high warming are expected in temperate regions, with
506 temperatures frequently reaching 26°C and above (Meehl & Tebaldi, 2004; IPCC, 2013) as
507 well as long-term exposure to elevated temperature. In such cases, the increased ant flows and
508 the higher proportion of ants interacting with aphids may lead to an overstimulation of the
509 trophobionts, a resulting lower ability to respond to ant solicitation due to a limited renewal rate
510 of honeydew droplets (Stadler & Dixon, 1998) and potentially higher physiological costs and
511 impaired development of the homopterans. Aphids may also experience long-term changes of
512 their honeydew quality, through the impact of warming on their host plant (Walther, 2003;
513 Adler et al., 2007). It is well established that changes in plant chemistry can alter honeydew
514 quality (Fischer et al., 2005; Katayama et al., 2013; Pringle et al., 2014) and modulates the
515 aphid-ant mutualism (Breton & Addicott, 1992). The high cost of producing high quantity and
516 high-quality honeydew for aphids in these conditions might become a limiting factor in their
517 interaction with ants and could lead in time to a disruption of this aphid-ant mutualism (Mooney
518 et al., 2019) and to a switch of partners towards other aphid species better adapted to warmer
519 climates (Mooney et al., 2019, Offenberg, 2001).
520 Besides, in natural conditions, climate warming is associated with higher CO2, which is also
521 likely to affect nutritional value of the sap and aphid honeydew (Roderick & Berry, 2001;
522 Thomas et al., 2004; Kremer et al., 2018). Studying the effect of multiple abiotic stressors on
523 aphid-ant mutualism may provide new understanding on the evolution of this interaction in the
23
524 next decades. Because of the species-specificity of aphids and ants, and the difficulty to
525 investigate multitrophic interactions under multiple climatic factors, providing clear
526 conclusions are still challenging.
527
528 Acknowledgements
529 This work was supported by a research grant from the Belgian National Fund for Scientific
530 Research (FRS-FNRS) grant number n° T.0202.16.
531 We also acknowledge the Institut Royal de Météorologie de Belgique (IRM Belgium) for
532 providing the temperature data.
533 534 References 535 536 Adler, L. S., De Valpine, P., Harte, J., & Call, J. (2007). Effects of long-term experimental
537 warming on aphid density in the field. Journal of the Kansas Entomological Society, 80(2),
538 156-168.
539
540 Aslan, C. E., Zavaleta, E. S., Tershy, B., & Croll, D. (2013). Mutualism disruption threatens
541 global plant biodiversity: a systematic review. PLoS one, 8(6), e66993.
542
543 Banks, C. J. (1962). Effects of the ant Lasius niger (L.) on insects preying on small populations
544 of Aphis fabae Scop. on bean plants. Annals of Applied Biology, 50(4), 669-679.
545
546 Banks, C., & Nixon, H. L. (1958). Effects of the ant, Lasius niger L., on the feeding and
547 excretion of the bean aphid, Aphis fabae Scop. Journal of Experimental Biology, 35(4), 703-
548 711.
549
24
550 Bates, D., Maechler, M., Bolker, B., Walker, S., Christensen, R. H. B., Singmann, H., ... &
551 Bolker, M. B. (2015). Package ‘lme4’. Convergence, 12(1), 2.
552
553 Barton, B. T., & Ives, A. R. (2014). Direct and indirect effects of warming on aphids, their
554 predators, and ant mutualists. Ecology, 95(6), 1479-1484.
555
556 Beckers, R., Deneubourg, J.L., Goss, S. (1992a). Trails and U-turns in the selection of a path
557 by the ant Lasius niger. J. Theor. Biol. 159, 397–415.
558
559 Boullis A., Detrain C., Francis F., Verheggen F. (2016). Will climate change affect insect
560 pheromonal communication? Current opinion in insect science, 17: 87-91.
561
562 Breton, L. M., & Addicott, J. F. (1992). Does host-plant quality mediate aphid-ant mutualism?.
563 Oikos, 253-259.
564
565 Breton, L. M., & Addicott, J. F. (1992). Density‐dependent mutualism in an aphid‐ant
566 interaction. Ecology, 73(6), 2175-2180.
567
568 Brown, M. J., & Paxton, R. J. (2009). The conservation of bees: a global perspective.
569 Apidologie, 40(3), 410-416.
570
571 Buckley, R. C. (1987). Interactions involving plants, Homoptera, and ants. Annual review of
572 Ecology and Systematics, 18(1), 111-135.
573
25
574 Cammell, M. E., & Knight, J. D. (1992). Effects of climatic change on the population dynamics
575 of crop pests. In Advances in Ecological Research (Vol. 22, pp. 117-162). Academic Press.
576 Cerdá, X., Retana, J., & Cros, S. (1998). Critical thermal limits in Mediterranean ant species:
577 trade‐off between mortality risk and foraging performance. Functional Ecology, 12(1), 45-55.
578
579 Degen, A. A., Gersani, M., Avivi, Y., & Weisbrot, N. (1986). Honeydew intake of the weaver
580 antPolyrhachis simplex (Hymenoptera: Formicidae) attending the aphidChaitophorous
581 populialbae (Homoptera: Aphididae). Insectes Sociaux, 33(2), 211-215.
582
583 DeLucia, E. H., Nabity, P. D., Zavala, J. A., & Berenbaum, M. R. (2012). Climate change:
584 resetting plant-insect interactions. Plant physiology, 160(4), 1677-1685.
585
586 Detrain, C., Verheghen, F., Diez, L., Wathelet, B. & Haubruge, E. (2010). Aphid-ant
587 mutualism: how honeydew sugars influence the behaviour of ant scouts. Physiological
588 Entomology, 35, 168–174.
589
590 Detrain, C., & Prieur, J. (2014). Sensitivity and feeding efficiency of the black garden ant Lasius
591 niger to sugar resources. Journal of insect physiology, 64, 74-80.
592
593 Detrain, C., Pereira, H., & Fourcassié, V. (2019). Differential responses to chemical cues
594 correlate with task performance in ant foragers. Behavioral ecology and sociobiology, 73(8),
595 107.
596
597 Devigne, C., & Detrain, C. (2002). Collective exploration and area marking in the ant Lasius
598 niger. Insectes sociaux, 49(4), 357-362.
26
599
600 Drees, B. M., Summerlin, B., & Vinson, S. B. (2007). Foraging activity and temperature
601 relationship for the red imported fire ant. Southwestern Entomologist, 32(3).
602
603 Duke, J. A. (1981). Caesalpinia spinosa. Handbook of Legumes of World Economic
604 Importance. Plenum Press, New York, 32-33.
605
606 El-Ziady, S., & Kennedy, J. S. (1956). Beneficial effects of the common garden ant, Lasius
607 niger L., on the black bean aphid, Aphis fabae Scopoli. In Proceedings of the Royal
608 Entomological Society of London. Series A, General Entomology (Vol. 31, No. 4‐6, pp. 61-
609 65). Oxford, UK: Blackwell Publishing Ltd.
610
611 Fischer, M. K., & Shingleton, A. W. (2001). Host plant and ants influence the honeydew sugar
612 composition of aphids. Functional Ecology, 15(4), 544-550.
613
614 Fischer, M. K., Voelkl, W., & Hoffmann, K. H. (2005). Honeydew production and honeydew
615 sugar composition of polyphagous black bean aphid, Aphis fabae (Hemiptera: Aphididae) on
616 various host plants and implications for ant-attendance. European Journal of Entomology,
617 102(2), 155-160.
618
619 Fischer, C. Y., Lognay, G. C., Detrain, C., Heil, M., Grigorescu, A., Sabri, A., Thonart, P.,
620 Haubruge, E. & Verheggen, F. J. (2015). Bacteria may enhance species association in an ant–
621 aphid mutualistic relationship. Chemoecology, 25(5), 223-232.
622
27
623 Flatt, T., & Weisser, W. W. (2000). The effects of mutualistic ants on aphid life history traits.
624 Ecology, 81(12), 3522-3529.
625
626 Friard, O., & Gamba, M. (2016). BORIS: a free, versatile open‐source event‐logging software
627 for video/audio coding and live observations. Methods in Ecology and Evolution, 7(11), 1325-
628 1330.
629
630 Gilman, R. T., Fabina, N. S., Abbott, K. C., & Rafferty, N. E. (2012). Evolution of plant–
631 pollinator mutualisms in response to climate change. Evolutionary Applications, 5(1), 2-16.
632
633 Hance, T., van Baaren, J., Vernon, P., & Boivin, G. (2007). Impact of extreme temperatures on
634 parasitoids in a climate change perspective. Annu. Rev. Entomol., 52, 107-126.
635
636 Harrington, R., Clark, S. J., Welham, S. J., Verrier, P. J., Denholm, C. H., Hulle, M., ... &
637 European Union Examine Consortium. (2007). Environmental change and the phenology of
638 European aphids. Global change biology, 13(8), 1550-1564.
639
640 Hegland, S. J., Nielsen, A., Lázaro, A., Bjerknes, A. L., & Totland, Ø. (2009). How does climate
641 warming affect plant‐pollinator interactions?. Ecology letters, 12(2), 184-195.
642
643 Holldöbler, B. & Wilson, E.O. (1990). The Ants. Harvard University Press, Cambridge,
644 Massachusetts
645
646 Hughes, L. (2000). Biological consequences of global warming: is the signal already apparent?.
647 Trends in ecology & evolution, 15(2), 56-61.
28
648
649 Hunter, M. D. (2001). Effects of elevated atmospheric carbon dioxide on insect–plant
650 interactions. Agricultural and Forest Entomology, 3(3), 153-159.
651
652 Hurlbert, A. H., Ballantyne, F., & Powell, S. (2008). Shaking a leg and hot to trot: the effects
653 of body size and temperature on running speed in ants. Ecological Entomology, 33(1), 144-154.
654
655 IPCC, C. C. (2007). The physical science basis. Contribution of working group I to the fourth
656 assessment report of the Intergovernmental Panel on Climate Change. Cambridge University
657 Press, Cambridge, United Kingdom and New York, NY, USA, 996, 2007.
658
659 IPCC Working Group I. (2013). Climate Change 2013-The Physical Science Basis: Summary
660 for Policymakers. Intergovernmental Panel on Climate Change.
661
662 IPCC, Intergovernmental Panel On Climate Change (2019). Special report on global warming
663 of 1.5 C (SR15).
664
665 Jamieson, M. A., Trowbridge, A. M., Raffa, K. F., & Lindroth, R. L. (2012). Consequences of
666 climate warming and altered precipitation patterns for plant-insect and multitrophic
667 interactions. Plant physiology, 160(4), 1719-1727.
668
669 Jayatilaka, P., Narendra, A., Reid, S. F., Cooper, P., & Zeil, J. (2011). Different effects of
670 temperature on foraging activity schedules in sympatric Myrmecia ants. Journal of
671 Experimental Biology, 214(16), 2730-2738.
672
29
673 Kiers, T. E., Palmer, T. M., Ives, A. R., Bruno, J. F., & Bronstein, J. L. (2010). Mutualisms in
674 a changing world: an evolutionary perspective. Ecology letters, 13(12), 1459-1474.
675
676 Kiss A. (1981). Melezitose, aphids and ants. Oikos. 37:382.
677
678 Kremer, J. M., Nooten, S. S., Cook, J. M., Ryalls, J. M., Barton, C. V., & Johnson, S. N. (2018).
679 Elevated atmospheric carbon dioxide concentrations promote ant tending of aphids. Journal of
680 Animal Ecology, 87(5), 1475-1483.
681
682 Larsen, K. J., Heady, S. E., & Nault, L. R. (1992). Influence of ants (Hymenoptera: Formicidae)
683 on honeydew excretion and escape behaviors in a myrmecophile, Dalbulus quinquenotatus
684 (Homoptera: Cicadellidae), and its congeners. Journal of Insect Behavior, 5(1), 109-122.
685
686 Lenth, R.V. (2016) Least-squares means: the R package lsmeans. J Stat Softw, 69, 1–33.
687
688 Ma, G., & Ma, C. S. (2012). Climate warming may increase aphids’ dropping probabilities in
689 response to high temperatures. Journal of Insect Physiology, 58(11), 1456-1462.
690
691 Mailleux, A. C., Deneubourg, J. L., & Detrain, C. (2003). Regulation of ants' foraging to
692 resource productivity. Proceedings of the Royal Society of London. Series B: Biological
693 Sciences, 270(1524), 1609-1616.
694
695 Marquis, M., Del Toro, I., & Pelini, S. L. (2014). Insect mutualisms buffer warming effects on
696 multiple trophic levels. Ecology, 95(1), 9-13.
697
30
698 Meehl, G. A., & Tebaldi, C. (2004). More intense, more frequent, and longer lasting heat waves
699 in the 21st century. Science, 305(5686), 994-997.
700
701 Mooney, E., Davidson, B., Den Uyl, J., Mullins, M., Medina, E., Nguyen, P., & Owens, J.
702 (2019). Elevated temperatures alter an ant‐aphid mutualism. Entomologia Experimentalis et
703 Applicata, 167(10), 891-905.
704
705 Offenberg, J. (2001). Balancing between mutualism and exploitation: the symbiotic interaction
706 between Lasius ants and aphids. Behavioral Ecology and Sociobiology, 49(4), 304-310.
707
708 Pecl, G. T., Araújo, M. B., Bell, J. D., Blanchard, J., Bonebrake, T. C., Chen, I. C., ... Williams
709 S.E. (2017). Biodiversity redistribution under climate change: Impacts on ecosystems and
710 human well-being. Science, 355(6332), eaai9214.
711
712 Porter, J. H., Parry, M. L., & Carter, T. R. (1991). The potential effects of climatic change on
713 agricultural insect pests. Agricultural and Forest Meteorology, 57(1-3), 221-240.
714
715 Robinet, C., & Roques, A. (2010). Direct impacts of recent climate warming on insect
716 populations. Integrative Zoology, 5(2), 132-142.
717
718 Roderick, M. L., & Berry, S. L. (2001). Linking wood density with tree growth and
719 environment: a theoretical analysis based on the motion of water. New Phytologist, 149(3),
720 473-485.
721
31
722 Sable, M. G., & Rana, D. K. (2016). Impact of global warming on insect behavior-A review.
723 Agricultural Reviews, 37(1), 81-84.
724
725 Sachs, J. L., & Simms, E. L. (2006). Pathways to mutualism breakdown. Trends in ecology &
726 evolution, 21(10), 585-592.
727
728 Sagata, K., & Gibb, H. (2016). The effect of temperature increases on an ant-hemiptera-plant
729 interaction. PloS one, 11(7).
730
731 Salvucci, M. E., Hendrix, D. L., & Wolfe, G. R. (1999). Effect of high temperature on the
732 metabolic processes affecting sorbitol synthesis in the silverleaf whitefly, Bemisia argentifolii.
733 Journal of Insect Physiology, 45(1), 21-27.
734
735 Sakata, H. (1995). Density-dependent predation of the ant Lasius niger (Hymenoptera:
736 Formicidae) on two attended aphids Lachnus tropicalis and Myzocallis kuricola (Homoptera:
737 Aphididae). Researches on Population Ecology, 37(2), 159-164.
738
739 Schweiger, O., Biesmeijer, J. C., Bommarco, R., Hickler, T., Hulme, P. E., Klotz, S., Ingolf
740 Kühn Mari Moora Anders Nielsen Ralf Ohlemüller Theodora Petanidou Simon G. Potts Petr
741 Pyšek Jane C. Stout Martin T. Sykes Thomas Tscheulin Montserrat Vilà Gian‐Reto Walther
742 Catrin Westphal Marten Winter Martin Zobel... & Petanidou, T. (2010). Multiple stressors on
743 biotic interactions: how climate change and alien species interact to affect pollination.
744 Biological Reviews, 85(4), 777-795.
745
32
746 Stadler, B., & Dixon, A. F. G. (1998). Costs of ant attendance for aphids. Journal of Animal
747 Ecology, 454-459.
748
749 Stadler, B., & Dixon, A. F. (2005). Ecology and evolution of aphid-ant interactions. Annu. Rev.
750 Ecol. Evol. Syst., 36, 345-372.
751
752 Stiling, P., & Cornelissen, T. (2007). How does elevated carbon dioxide (CO2) affect plant–
753 herbivore interactions? A field experiment and meta‐analysis of CO2‐mediated changes on
754 plant chemistry and herbivore performance. Global Change Biology, 13(9), 1823-1842.
755
756 Stuble, K. L., Pelini, S. L., Diamond, S. E., Fowler, D. A., Dunn, R. R., & Sanders, N. J. (2013).
757 Foraging by forest ants under experimental climatic warming: a test at two sites. Ecology and
758 evolution, 3(3), 482-491.
759
760 Thomas, D. S., Montagu, K. D., & Conroy, J. P. (2004). Changes in wood density of Eucalyptus
761 camaldulensis due to temperature - the physiological link between water viscosity and wood
762 anatomy. Forest Ecology and Management, 193(1-2), 157-165.
763
764 Van Emden, H. F., & Harrington, R. (2007). Aphids as crop pests. CAB International.
765
766 Van Oudenhove, L., Billoir, E., Boulay, R., Bernstein, C., Cerda, X. (2011). Temperature limits
767 trail following behavior through pheromone decay in ants. Naturwissenschaften, 98:1009-1017.
768
769 Verheggen, F. J., Diez, L., Sablon, L., Fischer, C., Bartram, S., Haubruge, E., & Detrain, C.
770 (2012). Aphid alarm pheromone as a cue for ants to locate aphid partners. PloS one, 7(8).
33
771
772 Villalpando, S. N., Williams, R. S., & Norby, R. J. (2009). Elevated air temperature alters an
773 old‐field insect community in a multifactor climate change experiment. Global Change
774 Biology, 15(4), 930-942.
775
776 Völkl, W., Woodring, J., Fischer, M., Lorenz, M. W., & Hoffmann, K. H. (1999). Ant-aphid
777 mutualisms: the impact of honeydew production and honeydew sugar composition on ant
778 preferences. Oecologia, 118(4), 483-491.
779
780 Walters, A. C., & Mackay, D. A. (2004). Comparisons of upper thermal tolerances between the
781 invasive Argentine ant (Hymenoptera: Formicidae) and two native Australian ant species.
782 Annals of the Entomological Society of America, 97(5), 971-975.
783
784 Walther, G. R. (2003). Plants in a warmer world. Perspectives in plant ecology, evolution and
785 systematics, 6(3), 169-185.
786
787 Way, M. J. (1963). Mutualism between ants and honeydew-producing Homoptera. Annual
788 review of entomology, 8(1), 307-344.
789
790 Yao, I., & Akimoto, S. I. (2002). Flexibility in the composition and concentration of amino
791 acids in honeydew of the drepanosiphid aphid Tuberculatus quercicola. Ecological
792 Entomology, 27(6), 745-752.
793
794 Yao, I. (2012). Ant attendance reduces flight muscle and wing size in the aphid Tuberculatus
795 quercicola. Biology letters, 8(4), 624-627.
34
796
797 Yoo, H. J. S., & Holway, D. A. (2011). Context‐dependence in an ant–aphid mutualism: direct
798 effects of tending intensity on aphid performance. Ecological Entomology, 36(4), 450-458.
799
800 Zhou, A., Kuang, B., Gao, Y., & Liang, G. (2015). Density-dependent benefits in ant-
801 hemipteran mutualism? The case of the ghost ant Tapinoma melanocephalum (Hymenoptera:
802 Formicidae) and the invasive mealybug Phenacoccus solenopsis (Hemiptera: Pseudococcidae).
803 PloS one, 10(4).
804
805 Zhou, A., Qu, X., Shan, L., & Wang, X. (2017). Temperature warming strengthens the
806 mutualism between ghost ants and invasive mealybugs. Scientific reports, 7(1), 1-10.
807 808 809 810
Figure 1: Number of ants present on the plant as a function of time. The number of ants staying over leaves and stem were counted every fifteen minutes for the whole duration of the experiment, at three different ambient temperatures. N=288
35
811 812 813 814
Figure 2: cumulated flow of ants leaving the aphid-infested plant as a function of time for the three tested temperatures. N=9 for each condition. For each time and each condition, the mean value and the standard errors are represented.
36
815 816 Figure 3: Comparison of the time spent by the 5 first ants and the five last ants to cross the bridge in function of the 817 temperature. N=45
818
819 820 Figure 4: Percentage of mobile aphids during the observations under the three tested temperatures. N=64 for each 821 temperature.
822 823
37
824
Figure 5: proportion of ants antennating at least one aphid as a function of temperature. N=9 for each condition. 825
826 827 Figure 6: Mean number of antennations received by one aphid for all the tested temperatures. N=64
828 829 830
38
831
832 833 Figure 7: Success of collection of a honeydew droplet making an antennation for all observed ants during the whole 834 experiment, under the three tested temperatures. N=9
835 836 837 838 839
39