For the first time, this study investigates the effect of temperature and carbon dioxide levels, alone and in combination, on the production of aphid honeydew and on its sugar content.
We found that a combined elevation of temperature and carbon dioxide concentration led to an increase in the amount of honeydew released by A. fabae aphids, in the total content of carbohydrates, as well as in the concentration of the main honeydew sugars, i.e. melezitose and fructose. Excepting for the fructose concentration, these impacts of abiotic conditions on aphid honeydew were however not statistically significant most probably due to the limited sample size.
Phloem-feeding aphids can respond differently to changes in the sap of plants reared under conditions of elevated CO2. In a non-myrmecophilous aphid species, Acyrthosiphon pisum, an elevation of CO2 impacted negatively the production of honeydew (Boullis et al. 2018). Conversely, other studies showed that higher amounts of honeydew were produced by Aphis craccivora reared on lucerne (Kremer et al. 2018) and by Aphis gossypii reared on cotton (Sun et al. 2009), under higher CO2 levels. These results, based on aphids and plants raised under daily temperatures of 26/18°C (Day-Night) (Kremer et al. 2018), and 28°C (Sun et al. 2009) are in accordance with our own findings, where an elevation of CO2 at 23°C also enhanced the production of honeydew by A. fabae aphids. Thus, aphids seem to adapt their feeding behavior to the changing nutritional value of plants reared under elevated CO2 conditions (Newman et al. 2003; Wang et al. 2004; Prichard et al. 2007; Sun and Ge 2011).
Noticeably, the enhancing effect of elevated CO2 on honeydew production by A. fabae was not observed at 20°C and took place only at 23°C. This could be explained by some synergistic effects of elevated temperature and CO2 levels on the ingestion of phloem sap by aphids. Indeed, higher temperatures induce higher rates of sap-feeding that go along with a generalized increase of aphid metabolism and that enable aphids to recover from water loss. On the other hand, elevated CO2 conditions reduce the nitrogen to carbon ratio in the phloem sap (Awmack and Leather 2002; Stiling and Cornelissen 2007; Sudderth et al. 2005) what leads aphids to ingest more phloem sap in order to meet their needs in amino acids.
Besides, the release of honeydew droplets is obviously linked to the feeding efficiency of aphids. When aphids drill into plant tissues with their stylet, their capacity to find sieve tubes is linked to the sensing of sucrose and pH in the microenvironment around the stylet tip (Tjallingii 2006, Hewer et al. 2010). Further studies should focus on how the feeding behavior of aphids also depends on temperature and/or CO2-induced changes in the phloem sap of their host plant.
Sugar composition of honeydew
In this study, melezitose as well as fructose were the major sugars found in A. fabae honeydew, which together accounted for more than 70% of the total amount of identified sugars. A. fabae also contained smaller amounts of monosaccharides such as glucose, xylose and maltose, disaccharides such as sucrose and trehalose, and the raffinose trisaccharide. The honeydew analyzed in this study included several sugars that are attractive to the ants (Detrain et al. 2010) and for which temperature and/or CO2-induced changes in concentration are expected to influence the interactions between ants and aphids. Previous studies showed that honeydew composition can be highly variable since it depends on the host plant of aphids (Hendrix et al. 1992; Völkl et al. 1999; Yao and Akimoto 2001; Pringle et al. 2014), on the aphid species (Wäckers 2000, Wäckers 2001; Fischer and Shingleton 2001; Hogervorst 2007; Woodring et al. 2004; Monticelli et al. 2020) and even on the aphid clone (Vantaux et al. 2011). Here, we found that the temperature and CO2 levels can also influence the carbohydrate composition of aphid honeydew, albeit to different degrees depending on the sugar.
Several honeydew sugars, such as fructose, glucose, sucrose, or xylose are not synthesized by aphids, but come directly from the phloem sap of the plant (Hendrix et al. 1992). One can thus assume that the relative amount of these sugars in honeydew to be -at least partially- related to those in the phloem sap. However, in this study we found that the relative amount of these sugars in the honeydew of A. fabae aphids did not significantly change when we tested separately either the impact of an elevated temperature or the impact of a CO2 enrichment.
However, we observed additive effects of elevated temperature and CO2, which both tend to increase the total amount of honeydew sugars as well as the concentration of phloem-derivate sugars like fructose. According to the existing literature, an elevation of CO2 levels was usually found to increase photosynthetic rates in plants, what can lead to the accumulation of carbohydrates in leaves (Moore et al. 1999; Ainsworth and Long 2005; Leakey et al. 2009), where an elevation of temperature commonly results in a decrease of carbohydrates’ concentration in plants (Sami et al. 2016; Yelle et al. 1989; Olszyk et al. 2003; Farrar and William 1991). One would therefore predict that effects resulting from an increase of CO2 (increased sugar content) would be levelled off by those expected from an increased temperature (decreased sugar content). This is not the case in the present study, in particular for the concentration of fructose that significantly increased under a combined elevation of temperature of CO2 and temperature. This increase may result from a higher accumulation of this sugar in plant sap, under elevated CO2 concentration but also from an enhanced metabolism of aphids at higher temperatures, which speeds up the enzymatic hydrolysis of sap sucrose into a fructose and a glucose unit. This confirms the need to consider all the processes involved in the production of honeydew, whose composition results not only from the characteristics of plant sap but also from the sugar metabolism of homopterans. Climate-induced changes in the composition of the phloem sap do not imply similar effects on the honeydew released by sap-feeding homopterans. Differences across sugars in their rate of transports through the gut walls of the aphids, in their level of degradation by hydrolysis or conversely, in their need to be biosynthesized by the aphids themselves, have to be investigated in order to understand how climate change would alter the honeydew-mediated mutualism between aphids and ants.
Some honeydew sugars are directly synthesized by aphids. This is the case of melezitose, a trisaccharide which is synthesized in the aphid’s gut by the coupling of two glucose units and one fructose unit (Bacon and Dickinson 1957; Ashford et al. 2000, Fisher and Shingleton 2001). We found that melezitose concentration increases under the combined elevation of temperature and CO2 concentration. The biosynthesis of this trisaccharide was potentially facilitated by a higher sucrose content in the phloem sap under elevated CO2 (Krumbein et al. 2010), which after being hydrolyzed in the gut of aphids, makes a glucose and a fructose unit available for the melezitose biosynthesis. Noticeably, since melezitose is made of two glucose units for only one fructose unit, the synthesis of this trisaccharide may leave fructose units in excess. This may explain why, in this study, the increase of fructose in honeydew went along with that of melezitose when A. fabae aphids were reared under a combined elevation of temperature and carbon dioxide.
Potential consequences for ant-aphid mutualism
The volume of honeydew excreted is a good indicator of phloem intake in sap-sucking insects, as well as a predictor of the potential strength of the ant–aphid mutualisms (Stadler and Dixon 1998).
Honeydew “richness” can be defined as the emission rate/volume of honeydew droplets coupled to their total concentration in sugars (Woodring et al. 2004). The total amount as well as the concentration of carbohydrates in honeydew is crucial for the initiation and maintenance of mutualistic relationships between ants and aphids (Beattie 1985; Blüthgen and Fiedler 2004; Buckley 1987; Engel et al. 2001; Stadler and Dixon 2005; Way 1963). In this study, the combined elevation of CO2 and temperature had a positive impact on A. fabae honeydew richness as it tended to increase both the amount of released droplets and their total concentration in sugars. Ants increase their feeding behavior as well as the intensity of collective recruitment to food according to the energetic value of carbohydrates sources, i.e their volume and/or their sugar content (e.g. Detrain et al. 1999, Detrain et al. 2010, Dussutour and Simpson 2008, Mailleux et al. 2000, Mailleux et al. 2003, Völkl et al. 1999). Under a scenario of climate change, one may thus expect that a higher honeydew richness will reinforce the aphid-ant mutualism.
Not only the amount but also the nature of honeydew sugars matters for triggering ant attendance. On the one hand, some sugars including maltose, xylose or melibiose elicit only a weak feeding response from ant foragers (Boevé and Wäckers 2003, Detrain et al. 2010). In contrast, melezitose, sucrose, fructose and glucose are regularly consumed by the majority of ants, even though species-specific sugar preferences may occur (Blüthgen and Fiedler 2004, Völkl et al. 1999). In a climate change perspective, based on our chemical analyses, we expect an enhanced feeding of ants due the increased amount of fructose and melezitose in A. fabae honeydew. As for sucrose, which makes up 80% of the organic components of the phloem sap in most C3 plants (including bean species), it is an effective phagostimulant for herbivorous and sap-feeding insects (Moon 1967; Hawker 1985, Weibull 1990). However, unlike a previous study (Detrain et al. 2010), we found a surprisingly little amount of this disaccharide in A. fabae honeydew in each environmental condition, what makes sucrose playing a marginal role in the ant-aphid interaction.
Finally, a special attention should be paid to melezitose sugar, which was the main sugar produced in A. fabae honeydew and whose concentration increased under a combined elevation of temperature and carbon dioxide. Melezitose is known to attract aphid-tending ants such as Lasius niger (Fischer et al. 2005; Völkl, et al. 1999; Woodring et al. 2004), whose foragers prefer to exploit aphid clones that secrete a melezitose-rich honeydew (Kiss 1981; Völkl et al. 1999; Fischer et al. 2001; Vantaux et al. 2011). In addition to stimulating ants’ feeding, melezitose triggers the laying of a recruitment trail by ant foragers (Detrain et al. 2010; Detrain and Prieur 2014), thereby playing a key-role in the initiation and the maintenance of high flows of ants travelling towards the aphid colonies. When considering all these effects of melezitose on ant behavior, one may assume that the increased production of this trisaccharide by the A. fabae aphids would lead to the reinforcement of aphid attendance and honeydew exploitation by ants under conditions of climate change.
The benefits acquired by aphids from these enhanced interactions could however be mitigated in the long term since the increased production of a sugar-enriched honeydew may incur higher physiological costs and may impair homopterans’ development (Stadler and Dixon 2005). Beside the ant mutualists, one should consider how honeydew changes can impact the survival and reproduction of aphid predators and parasitoids (Wäckers 2000). For instance, some species (e.g. Cotesia glomerata L., a parasitoid wasp) achieve the longest lifespan when feeding on sucrose, fructose and glucose and the shortest longevity in the presence of melezitose (Wäckers 2001). Attention should also be paid to the honeydew volatiles that enable aphid predators such as hoverflies to find suitable oviposition sites (Leroy et al. 2011) or mutualists such as ants to find aphid partners at a distance (Fisher et al. 2015). Finally, the bacterial community in the aphid gut (Fischer et al. 2015) can shape the profile of honeydew volatiles and potentially alters the predator/mutualist ratio of aphids. Some bacteria also enhance the resistance of aphids to parasitoids and to thermal stress (Renoz et al. 2019). Future research should investigate how climate change could indirectly alter aphid fitness by modifying their bacterial community and whether bacteria conferring thermal resistance could be naturally selected by making aphids more tolerant to heat waves.
To conclude, aphids and their ant partners take part to a multitrophic network involving amplifying processes, negative feed-backs as well as cascading effects. Like any other complex systems, it is difficult to predict how slight changes in local interactions between agents would influence the global functioning and the stability of the whole multitrophic system. Based on our results, one may assume that, in a scenario of global warming, the changes induced in A. fabae honeydew will enhance their mutualism with ant partners. In addition to testing the generic value of this prediction for other plant-aphid-ant combinations, much work is however needed to see whether the predicted impact of global warming on aphid-ant relationship could be further enhanced or counteracted by changes in plant phloem sap, aphid metabolism, parasitoid/predator behavior as well as in the bacteria community of sap-feeding insects.