Oecologia (2003) 136:508–514 DOI 10.1007/s00442-003-1249-9
ECOPHYSIOLOGY
Jean-Luc Boev · Felix L. Wckers
Gustatory perception and metabolic utilization of sugars by Myrmica rubra ant workers Received: 12 September 2002 / Accepted: 8 March 2003 / Published online: 15 April 2003 Springer-Verlag 2003
Abstract The suitability of various nectar and honeydew sugars as a food source for the polyphagous ant species M. rubra (L.) was studied. The sugars used included monosaccharides (fructose, glucose, galactose, mannose, rhamnose), disaccharides (sucrose, maltose, trehalose, melibiose, lactose) and trisaccharides (melizitose, raffinose, erlose). Single-sugar solutions were tested on ant workers in a long-term laboratory bioassay in which acceptance of the solutions and ant survival were recorded. The acceptance of the sugars was confirmed in a second bioassay in which feeding time was established. Enzymatic hydrolysis of sucrose, maltose and melibiose was investigated through HPLC analyses of workers fed these disaccharides. Sugar acceptance and feeding time were related to ant survival. Considering the monosaccharide units of which the sugars are composed, fructose seems especially suitable as a short-term energy source, while glucose appears to be used both directly and for storage. The presence of a galactose unit appears to reduce sugar suitability. It is suggested that the workers possess invertase and maltase and to a lesser degree also galactosidase. The gustatory perception is correlated with the profitability of sugars in further metabolic processes. Keywords Formicidae · Survival · Acceptance · Enzymatic hydrolysis · Nutritive value
Introduction Ants (Hymenoptera, Formicidae) are keystone species in many terrestrial ecosystems. The biology of these social J.-L. Boev ()) Dpartement d’Entomologie, Royal Belgian Institute of Natural Sciences, IRSNB-KBIN, rue Vautier 29, 1000 Brussels, Belgium e-mail:
[email protected] Fax: +32-2-6274132 F. L. Wckers Netherlands Institute for Ecology, CTO, PO Box 40, 6666 ZG Heteren, The Netherlands
insects is extremely diversified and the worker caste can provide the colony with a range of food types, including prey organisms, sugar solutions, seeds, mycelium, etc. (Hlldobler and Wilson 1990). Ants have access to sugars in the form of honeydew, extrafloral and floral nectar, lepidopteran dorsal gland secretion, phloem and xylem sap, fruit pulp, etc. (Buckley 1982; Hlldobler and Wilson 1990; Koptur 1992; Fiedler et al. 1996; Koptur and Truong 1998). Sugars serve two main physiological functions in insects. They constitute an essential energy source, but are also required for the formation of the exoskeleton (Mullins 1985). The first function is the primary one for an ant worker since it is a long-lived adult that needs energy constantly and no longer needs to moult (Duncan and Lighton 1994). Ants often maintain mutualistic relationships with insects and plants that produce sugars in the form of honeydew and extrafloral nectar, respectively. Ants benefit from the food source, while in return they may protect the tended producers against their enemies (Bentley 1977; Buckley 1982; Hlldobler and Wilson 1990; Fiedler et al. 1996). Extrafloral nectar and honeydew are variable in their chemical composition, comprising a range of monosaccharides and oligosaccharides (Bentley 1977; Kunkel and Kloft 1977; Vlkl et al. 1999; Wckers 2001), and may contain other classes of chemical compounds such as amino acids, proteins and lipids (Baker and Baker 1973; Ziegler and Penth 1977). The monosaccharides fructose and glucose, the disaccharides sucrose, trehalose and maltose, and trisaccharides such as melizitose, raffinose and erlose have been detected in honeydew (Vlkl et al. 1999; Wckers 2000). Extrafloral nectars are commonly dominated by sucrose and its hexose components, fructose and glucose (Bentley 1977; Buckley 1982). If we want to understand to what extent the composition of sugar rewards in ant–plant or ant–insect mutualisms are adapted to the intended consumer, we need to have information on the selective advantage of these compositions for the sugar consumer.
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In insects and higher animals, ingested sugars can undergo three general metabolic steps (Weil 1978). First, any sugar polymer has to be hydrolysed into its monosaccharide units in the digestive tract before these can pass through the gut wall. The use of sugars as an energy source requires oxidation (i.e., glycolysis) as a second step. An intermediate and optional step is the storage of sugars as glycogen and, in insects, also as trehalose, which are a glucose polymer and dimer, respectively (Candy 1985). Given the extensive literature on ecological relationships between ants and sugar-producing organisms, it is surprising that etho-physiological data concerning the suitability of different sugar molecules for ants remain rather scant. Several ant species have been studied with respect to their relative sugar acceptance threshold as well as their sugar preference (Schmidt 1938; Ricks and Vinson 1970; Cornelius et al. 1996; Vlkl et al. 1999), sometimes also in combination with amino acids (Baker and Baker 1973; Lanza et al. 1993; Wada et al. 2001). A trend for Myrmica rubra, M. (=Manica) rubida and Lasius niger is that sucrose, raffinose, maltose and melizitose are perceived at lower concentrations as compared to glucose and fructose (Schmidt 1938). Galactose, lactose, rhamnose and melibiose, on the other hand, do not elicit feeding in these ant species and in Formica sanguinea (Schmidt 1938). Furthermore, L. niger prefers trisaccharides over di- and monosaccharides (Duckett 1974 in Cornelius et al. 1996; Vlkl et al. 1999). Pheidole megacephala prefers melizitose, sucrose and fructose over glucose, maltose and trehalose. Among these sugars, maltose is the lowest ranked in preference studies with Ochetellus glaber, while Paratrechina longicornis shows no preference among these six sugars (Cornelius et al. 1996). Thus, ant responses appear to be species specific; yet melizitose, sucrose and fructose are often wellaccepted sugars, whereas maltose is often poorly accepted. In the present work, we used ant workers of M. rubra, a polyphagous species feeding on insect prey as well as several sugar sources such as extrafloral nectars and honeydew (Seifert 1996). The worker caste normally reaches an age of 21 months in the laboratory (Cammaerts 1989). Thirteen mono- and oligosaccharides were selected for the experiment, based on the fact that they had been used in similar studies on other Hymenoptera (von Frisch 1934; Schmidt 1938; Wckers 1999, 2001). With the exception of lactose, all of the included sugars occur in honeydew and/or (extra)floral nectars (see literature above). We conducted laboratory bioassays as well as chemical analyses to address the gustatory perception, digestion and nutritive value. Our aim was to relate these three aspects, and to discuss whether sugar acceptance reflects the subsequent metabolic suitability of sugars.
Materials and methods Sugar acceptance and survival A large colony of M. rubra, including workers, queens and brood, was collected around Berlin (Germany) at the beginning of the autumn, and maintained in the laboratory. They received ad libitum living larvae and/or freshly killed pupae of Calliphora sp. as protein source, besides a 1 M sucrose solution and water. At the end of May, 14 groups of 45 workers each were collected (while foraging outside of the nest), isolated in Petri dishes and maintained at 20–27 C. They were provided with water only (in glass tubes plugged with cotton-wool). Two days after the transfer from the nest, the ants were offered a 50 ml droplet of a 1 M sugar solution (in addition to the water). The following 13 sugars were tested: fructose, galactose, glucose, lactose, maltose, mannose, melizitose, melibiose, raffinose, rhamnose, sucrose, erlose and trehalose. One additional group received 50 ml water and served as a control. The number of feeding ants was recorded 10 min after the 50 ml droplet had been presented. After a total of 2 h the droplets were removed. Mortality was recorded and dead ants were removed from the Petri dish. The procedure was repeated daily during 30 days, followed by three feedings at 3-day intervals, and finally another three at a 9-day interval. Thus, the experiment lasted 66 days. The decrease in sugar supply frequency was included to further challenge sugar suitability. Feeding time In a no-choice experiment, the 13 sugars listed above were tested on single workers kept in a Petri dish. Eighteen workers were tested per sugar. Before being presented with a 10 ml droplet of a 1 M sugar solution, workers were kept for 3–5 days with water only. The time spent feeding was recorded during the 10 min following sugar deposition. This observation time was extended in cases where the ant had not terminated its feeding bout at the end of the 10-min observation period. Sugar digestion Ant workers were kept individually in Petri dishes for 3 days with water only. Subsequently, ants received a droplet of a 1 M solution of either sucrose, maltose or melibiose. As a control, individuals were kept with water only. To assess the digestion rate we analysed the sugar levels at three different time intervals (t=15 min, 18 h and 40 h), counted from the time at which individuals started to feed. At the indicated time, ants were killed and their gaster extracted in 0.5 ml of 70% ethanol. In the case of the control and t=15 min, the gasters of three workers were pooled for analysis, whereas single gasters were analysed for both t=18 and 40 h. The ethanol extract was filtered on a RP-18 column (Merck), before injection on a Dionex DX 500 HPLC system (Dionex Corp., Sunnyvale, Calif.), equipped with a GP 40 gradient pump, a Carbopac PA1 guard (450 mm) and analytical column (4250 mm), and an ED 40 electrochemical detector for pulsed amperimetric detection (PAD). The eluent flow at 1 ml/min was isochratic with 90% water and 10% NaOH 1 M. Based on daily calibrations of the equipment, the amount of the sugars present in a sample was calculated with the software PeakNet version 5.1. We were not able to distinguish glucose and galactose due to their similar retention times. Field versus laboratory ants Control experiments were performed to estimate whether sucrose feeding of the laboratory colony had resulted in possible physiological adaptations. We collected 360 workers of M. rubra at the beginning of June from a single colony found in the neighbourhood of Brussels (Belgium). In the laboratory, the individuals from this field stock were equally divided among six Petri dishes. In addition,
510 three colonies of M. rubra had been collected from the same area, 20 months before. These colonies were kept on a sucrose solution, a protein source and water (see above). From each colony of this laboratory stock, two groups of 60 workers were isolated in Petri dishes. Thus, the experiment started with 12 replicates of 60 workers each, half of the replicates directly from the field and half from the laboratory. The ants received water only, and their mortality was recorded daily. At the day when the cumulative mortality, averaged separately over field and laboratory individuals, reached 50% the ants were allowed to feed once on a 1 M solution of either glucose, fructose or galactose. Each monosaccharide was tested at random on two groups of ants of each stock (i.e., from the laboratory versus from the field). The amount of solution provided per Petri dish was an equivalent of 1 ml per ant, related to the number of surviving individuals. This amount was totally ingested. Daily mortality counts were continued until all ants had died.
Results Acceptance and survival The cumulative ant mortality curves (Fig. 1) show that 50% mortality was reached first with rhamnose and water (19 days), followed by lactose (28), galactose, mannose, melibiose and raffinose (48), erlose (57), and fructose and maltose (66). With the remaining sugars (i.e. glucose, melizitose, sucrose and trehalose) mortality had not reached 50% by day 66 (i.e., the end of the experiment). Thus, the majority of ants, with the exception of those fed lactose and rhamnose, survived the first 30 days in which they were exposed to the sugar solution daily (Fig. 2). This is interesting, given that clear differences in sugar acceptance were observed during the same period (Fig. 2). Feeding time In general, feeding by the ants occurred in discontinuous bouts within the 10 min of observation. For each worker, the total time spent feeding was measured and its mean value for each sugar is given in Fig. 3. In the case of galactose, mannose, rhamnose, melibiose and lactose, the ants fed less than 100 s. On fructose, glucose, maltose, trehalose and raffinose feeding times ranged between 100 and 300 s, while the highest times (>300 s) were recorded on sucrose, erlose and melizitose. A short feeding time (10 ants,
Table 1 Ant mortality (M) and survival (S), out of an initial 45 individuals,during 30 days of daily sugar supply (D30); the data refer to Fig. 1. The sugars are listed according tothe number of monosaccharide units: (n Fru) fructose, (n Glu) glucose, (n Gal) galactose
Fru D30 M 22 S 20 n Fru 1 n Glu n Gal
Fig. 1 Cumulative ant mortality, out of an initial 45 individuals, during 30 days of daily sugar supply, and a subsequent period of less frequent sugar supply. Each sugar supply consisted of offering a 50 ml droplet of a 1 M sugar solution during 2 h. Otherwise, all ants were provided continuously and ad libitum with water only
as during the first 30 days in the survival study). Sugars that yielded a longer feeding time showed lower mortalities in the survival study (P = 0.005, Fisher exact probability test, N=13 sugars, see Fig. 3 and Table 1). The observation time needed to be extended beyond the 10-min observation period in 20% of the total of 234
Glu
Gal
Man
Rha
Suc
Mal
Tre
Meb
Lac
Erl
Mez
Raf
2 43
11 34
11 34
43 2
8 37
11 34
5 40
13 32
27 18
4 41
4 41
7 38
12 31
21 13
34 0
2 0
5 32 1 1
14 20
11 29
30 2
16 2
2
2
1 1
1 1
35 6 1 2
13 28 1 2
30 8 1 1 1
1 1
511
test, G=64.51 and 125.00, first and second period, respectively, df=10 and P
