Nitrogen and amino acids in nectar modify food selection of nectarivorous bats

Authors


Summary

  1. Chiropterophilic flowers secrete sugar nectar with low-Nitrogen (N hereafter) content and small amounts of amino acids, which may function to attract animals; nevertheless, the role that micronutrients have on the foraging decisions of Neotropical nectarivorous bats is unknown.
  2. We offered the nectar specialist Leptonycteris yerbabueanae and the omnivore Glossophaga soricina pairs of experimental diets mimicking either the N content or the relative abundance of 17 amino acids found in the floral nectar from the main plant species visited by these bats in a tropical dry forest. We addressed the following research questions: (i) Do bats select N-containing or sugar-only nectar differently based on bats' N nutritional status? (ii) Does the presence of N in nectar affect the capacity of bats to discriminate and select other nectar traits such as sugar concentration? and (iii) Are bats able to distinguish among the flavours generated by the amino acid relative abundance present in the nectar from plants they typically encounter in nature?
  3. Our results showed that: (i) bats did not consider nectar N content regardless of their N nutritional condition, (ii) the nectar specialist L. yerbabuenae showed a preference for the most concentrated sugar-only nectar but changed to be indifferent when nectar contained N, and (iii) L. yerbabuenae preferred diets without amino acids and preferred the taste of the amino acids present in the nectar of Pachycereus pecten (Cactaceae) over those present in the nectar of Ceiba aesculifolia (Bombacaceae).
  4. Our results suggest that regardless of the low concentrations at which N and amino acids are present in floral nectar, their presence affects bats' food selection by interfering with the bats' ability to detect differences in sugar concentrations, and by offering particular flavours that can be perceived and selected by nectarivorous bats. We discuss the ecological implications of the presence of N and amino acids in nectar on bats' foraging decisions.

Introduction

The way in which animals obtain food in the wild is not a simple task, and foraging decisions have both physiological and ecological implications (Stephens & Krebs 1986). Animals do not simply feed on any food item they encounter, but rather they select food items higher in certain compounds and lower in others (Bozinovic, Novoa & Nespolo 1997). The way in which micronutrients affect foraging decisions in animals is an area of active research in the fields of nutrition, food science, psychology and ecology.

In general, mechanisms affecting food selection by animals have been divided into two components: (i) pre-ingestional determinants of profitability – food abundance, handling time and pursuit time (Stephens & Krebs 1986) and (ii) postingestional determinants of profitability – digestion, assimilation and metabolism (Martinez del Rio, Baker & Baker 1992). From a nutritional perspective, food items not only provide energy (carbohydrates) to fuel metabolic processes, but also provide other necessary nutrients like minerals, vitamins, lipids, sodium, water and nitrogen-containing compounds (hereafter N) in the form of proteins and/or amino acids (Bozinovic & Martinez del Rio 1996). When animals face a mismatch between nutrient availability and their minimal nutritional requirements, they either switch to compensatory feeding (Cruz-Rivera & Hay 2000), change their food preference (Mayntz et al. 2005), or change their foraging habits (Belovsky 1978). Various studies with spiders (Greenstone 1979; Mayntz et al. 2005), birds (Wheelwright 1970), opossums (Geiser, Stahl & Learmonth 1992), moose (Belovsky 1978), mice (Bozinovic, Novoa & Nespolo 1997) and chipmunks (Geiser & Kenagy 1987) have demonstrated that feeding choices may vary to satisfy the nutritional requirements depending on the animal's physiological condition.

Other features that animals utilize to choose between potential food items are sensory features such as flavour (i.e. taste, odour and texture; Sclafani 1995). Hexose and sucrose sugars are the main nutrients and phagostimulants for many leaf-eating insects (Chapman 1998). The importance of some nutrients such as amino acids and proteins on the flavour of food has been the focus of some research (Solms 1969; Baker, Opler & Baker 1978). It appears that the relative abundance of each sugar and amino acid type in food is important for providing a characteristic taste profile (Shiraishi & Kuwabara 1970; Gardener & Gillman 2001, 2002). Furthermore, animals refine their preferences as they associate the flavours of specific foods with the foods' postingestive consequences (Sclafani 1995; Morris 2001). Particular types and mixes of amino acids act as phagostimulants (Petanidou et al. 2006) in a wide range of animals like plant bugs (Strong & Kruitwagen 1970; Hatfield, Frazier & Ferreira 1982), bees (Inouye & Waller 1984; Alm et al. 1990), cockroaches (Soichi et al. 2003), crickets (Warwick et al. 2009), crabs (Robertson, Fudge & Vermeer 1981), fish (Adron & Mackie 1978), hummingbirds (Haisworth & Wolf 1976), sunbirds (Leisegneur, Verburgt & Nicolson 2007), rats (Piquard, Schaefer & Haberey 1975) and humans (Solms, Vuataz & Egli 1965).

Nectar-eating bats cannot meet their Minimal Nitrogen Requirements (MNR) from feeding only on N found in nectar; they need to supplement their diet using other N sources like pollen, insects and/or fruit (Alvarez & González 1970; Howell 1974; Herrera et al. 2001). Because N is found in low quantities in the nectar consumed by these animals (Baker, Opler & Baker 1978; Scogin 1980; Petanidou et al. 2006), its potential effect on the foraging ecology of bats and other pollinators has been largely ignored. While the role of amino acids for food selection in pteropodid and phyllostomid bats is unknown, bats can use olfactory cues to find food sources (Rieger & Jakob 1988; Laska 1990; Thies, Kalko & Schnitzler 1998; von Helversen, Winkler & Bestmann 2000). Furthermore, they respond differently to the taste of the three most common sugar types found in nectar, indicating the existence of an ability to discriminate between subtle variation in nectar flavour (Herrera, Leblanc & Nassar 2000; Ayala-Berdon et al. 2013).

To further understand the role that N and nectar amino acids have on the foraging decisions of two Neotropical nectarivorous bats (Phyllostomidae : Glossophaginae) with different strategies of nectar use: the Saussure's long-nosed bat (Leptonycteris yerbabuenae; Martínez and Villa-R 1940; Koopman 1981) and the long-tongued bat (Glossophaga soricina; Pallas 1776), we evalaute the following research questions: (i) Do bats select N-containing or sugar-only nectar differently based on bats' N nutritional status? (ii) Does the presence of N in nectar affect the capacity of bats to discriminate and select other nectar traits such as sugar concentration? and (iii) Are bats able to distinguish among the flavours generated by the amino acid relative abundance present in the nectar from plants they typically encounter in nature?

We hypothesized that: (i) Because nectarivorous bats supplement their nectar diet in the wild using other items (e.g. insects, pollen and fruit) as an N source (Carvalho 1961; Howell 1974; Heithaus, Fleming & Opler 1975; Delorme & Thomas 1996), N-balanced bats would be unresponsive to the N-containing nectar, but N-deprived bats would prefer the N-containing nectar, (ii) As nectar feeding bats are sensitive to changes in the sugar concentration of their diets (Rodríguez-Peña et al. 2007; Ayala-Berdon et al. 2008) they would prefer the most concentrated nectar regardless of its N content, (iii) As all bat-pollinated plants produce sugar nectars with amino acids and the relative abundance of each amino acid type in food is important for providing a characteristic taste profile (Shiraishi & Kuwabara 1970; Gardener & Gillman 2001, 2002), bats would prefer the amino acid-containing nectar over the sugar-only nectar due to the flavour that amino acid relative abundance provides to nectar, (iv) Because animals refine their food preferences by associating flavours of specific foods with the foods' postingestive consequences (Sclafani 1995; Morris 2001), bats would prefer the flavour associated with the nectar source that contains more energy and (v) Because more specialized nectarivorous bats are able to discriminate smaller differences in other nectar constituents (Rodríguez-Peña et al. 2007) the more specialized nectarivore L. yerbabuenae would be able to discriminate smaller differences in the N and amino acid characteristics than G. soricina.

Materials and methods

Study site and bat collection

Bat species were collected in the Chamela region in the central Pacific coast of Mexico (c. 19°22′- 19°35′ N, 104°56′- 105°03′ W). The dominant vegetation type is tropical lowland deciduous forest (Lott 1993). Nectarivorous bats at this site consume nectar, and presumably pollinate the flowers of 22 plant species (Stoner et al. 2003). We used mist nets to capture adult nonreproductive males of Leptonycteris yerbabuenae and Glossophaga soricina. The specialized nectarivore L. yerbabuenae includes pollen, nectar, fruit and a small number of insects in its diet, but pollen constitutes the main part of the diet of this species throughout the year (Alvarez & González 1970; Gardner 1977; Fleming 1995; Valiente-Banuet et al. 1996; Stoner et al. 2003). Our second species, Glossophaga soricina, is a more generalist species that feeds on nectar, but a large part of its diet consists of fruit and insects (Alvarez et al. 1999; Nassar et al. 2003; Mirón et al. 2006). Glossophaga soricina is more insectivorous and frugivorous than L. yerbabuenae (Herrera 1999; Nassar et al. 2003; Mirón et al. 2006). In addition to dietary differences, these species differ in: (i) their digestive capacities to use the sugar in nectar, (ii) their abilities to discriminate among sugar solutions and (iii) their selection of floral resources in the field (Stoner et al. 2003; Rodríguez-Peña et al. 2007; Ayala-Berdon et al. 2008; Ayala-Berdon & Schondube 2011).

Care and housing of bats

Bats were captured and handled under permission from the Oficina de Fauna Silvestre, Mexico, to JES (FAUT-0193) and met humane handling guidelines approved by the American Society of Mammalogists (Gannon & Sikes 2007). After capture, bats were transferred to a laboratory with a controlled environment of 27 °C and 50% relative humidity. Bats were maintained in groups of seven individuals in aluminium cages (100 × 100 × 100 cm) and were fed on the diet described by Mirón et al. (2006). Body mass, wing membrane elasticity and hair condition of all bats were monitored daily. All bats maintained constant body mass and were healthy during captivity. Bats lost weight under some experimental conditions; however, they regained body mass after the experiments were over. Bats were released at their capture site once the experiments were finished.

Nectar

We prepared artificial nectars to evaluate how N and amino acids affect foraging decisions of bats. Both N concentration and relative amino acid abundance were equivalent to nectar of bat-pollinated flowers consumed by nectarivorous bats in the Chamela region. In brief, nectar collections of the chiropterophilic plant species were made opportunistically depending on the flowering period (Stoner et al. 2003). Mature flower buds were covered with mesh bags 1 h before sunset. Nectar was extracted with capillary glass tubes 1 h after anthesis. To prevent possible contamination with pollen amino acids, we emasculated the flowers before nectar production started. Nectar samples were frozen before analysis.

Nitrogen concentration was measured for: Crescentia alata (Bignoniaceae, n = 3 flowers) and Pachycereus pecten (Cactaceae, n = 2 flowers; Table 1). We calculated the total concentration of amino acids in nectar by the ninhydrin method (Moore & Stein 1954; Hirs 1967), and the total concentration of soluble proteins by the Bradford method (Bradford 1976). Finally, total N concentration in nectar was calculated using a conversion factor taking into account the N contribution of both free amino acids and proteins (Table 1).

Table 1. Composition of test diets, experimental design and pairwise comparisons of diets
DietaEquivalent plant speciesCharacteristics
NN (no N) 0 μg mL−1 of N
LNa (low-N content) Crescentia alata 320 μg mL−1 of N
HNa (high-N content) Pachycereus pecten 1505 μg mL−1 of N
Cab (amino acid relative abundance) Ceiba aesculifolia arg 4·08; his 6·59; ile 7·28; leu 11·85; lys 19·37; met 0·84; phe 0·45; thr 4·31; val 3·34; asp 2·73; glu 3·49; ser 18·15; gly 2·27; ala 1·27; tyr 0·20; cys 0·19; pro 13·57.
Ppb (amino acid relative abundance) Pachycereus pecten arg 1·65; his 10·89; ile 3·39; leu 7·78; lys 7·64; met 2·32; phe 1·07; thr 3·99; val 2·88; asp 6·75; glu 6·08; ser 8·85; gly 10·78; ala 12·24; tyr 2·91; cys 1·42; pro 9·33.
NA (no amino acid)   0 μg mL−1 of amino acids
ExperimentPairwise comparison of dietsPairwise comparison of sugar concentrations (%)
  1. a

    Low-N content (LN) and high-N content (HN) diets were prepared with a soy protein–based product (PRONAT ProWinner®; www.prowinner.com.mx; Mexico City, Mexico) as an N source. This product contains a balanced mix of amino acids that will not cause a nutritional imbalance in the bats (Harper 1967; Leung, Rogers & Harper 1968).

  2. b

    Crescentia alata (Ca) and Pachycereus pecten (Pp) diets consisted of sugar solutions supplemented with 17 pure individual amino acids (100%; Sigma-Aldrich®; http://www.sigmaaldrich.com) the ratio of the different individual amino acids in solutions varied following those of the selected plant species. Solutions were prepared at a total amino acid concentration of 7 mm mimicking the amino acid concentration of Pachycereus occidentalis.

1HN/ NN N-balanced bats20 : 20
2HN/NN N-deprived bats20 : 20
3NN/NN18 : 27
4LN/HN18 : 27
5Ca/NA20 : 20
6Pp/NA20 : 20
7Ca/Pp20 : 20
8Ca/Pp18 : 27

The relative abundance of 17 amino acids was measured for: Ceiba aesculifolia (Malvaceae; Ca hereafter) and Pachycereus pecten (Cactaceae; Pp hereafter) using high-performance liquid chromatography in an Agilent 1100 Series equipment (Hewlett Packard), following the Agilent 1090 series method (HPLC; Table 1). We used a two-step precolumn derivatization, with ortho-phthalaldehyde (OPA) for primary amino acids and 9-fluorenylmethyl chloroformate (FMOC) for the secondary amino acid. A 0·4 N borate buffer was used with pH 10·4. Separation was performed by using a Hypersil AA-ODS 2·1 × 200 mm Agilent column. We used a solvent gradient system with two mobile phases: (A) sodium acetate, triethylamine and tetrahydrofuran water mix, and (B) sodium acetate, acetonitrile and methanol water mix buffers (pH 7·20). The gradient was started with 100% A, at 17 min 60% B, at 18·1 min flow 0·45, at 18·5 min flow 0·8, at 23·9 min flow 0·8, at 24 min 100% B and flow 0·45, at 25 min 0% B. Detection was via a Perkin Elmer (LS50B) Luminescence Spectrometer (excitation at 340 nm and emission at 450 nm for primary amino acids and excitation at 266 nm and emission at 305 nm for the secondary amino acids). Data collection was performed with FL Win Lab Perkin Elmer software. Chromatograms were compared with standards for the identification of individual amino acids. The percentage of each amino acid was calculated for each sample.

Preference tests

We offered pairs of test diets to individual bats in small flight cages (50 × 50 × 50 cm) located under laboratory conditions. Feeders were placed 50 cm apart and at a height of 30 cm. Trials lasted 10 h and were conducted from 19.00 to 05.00 h. Feeders were filled and placed at 19.00 h. The amount of food consumed per feeder was measured (g) and their position switched at the middle of the experiment at 00.00 h to control for potential positional biases (following Jackson, Nicolson & Lotz 1998). The amount of food consumed was again measured at 05.00 h. An additional feeder of each test diet was placed outside the experimental cages to control for evaporation.

Experiments

Because L. yerbabuenae is considered an endangered species by the Mexican government (listed under the old taxonomic name of L. curasoae in the Mexican law), our research permit (FAUT-0193) allowed us to have only seven individuals in captivity at any time; thus we used seven individuals per experiment. Due to the limitations of our permitted sample size our eight experiments were conducted consecutively; once each experiment was finished bats were released at their capture site and new bats were captured for the next experiment. To have a balanced design, we decided to use the same number of individuals of G. soricina. In total, we used 56 different individuals (seven in each experiment) of L. yerbabuenae and 56 different individuals of G. soricina. We performed a priori analyses (G power® software) from a pilot study and this showed that power values for statistical analyses were > 0·8 with a sample size of 4–9 bats per trial. Moreover, we conducted a posteriori analyses to determine the statistical power of each one of our experiments. A series of eight pairwise independent feeding experiments were conducted (Table 1). Treatment group is presented separately because we addressed different research questions in each experiment requiring the characteristics of the experimental diets, and in some cases, the nutritional status of bats to be different between experiments.

Experiment 1

To test whether bats demonstrated a preference for an N-containing nectar given their N nutritional status, we used N-balanced bats that under captivity were always fed a sugar and N-balanced maintenance diet (Mirón et al. 2006). We offered bats two experimental diets. The first was a sugar-only nectar (20% w/v sucrose) and contained no N (0 μg mL−1; hereafter NN diet). The second was an N-containing nectar (20% w/v sucrose; 1505 μg mL−1 N), mimicking the highest N content present in the flowers bats visit in the Chamela region (P. pecten – hereafter HN; Table 1). To see details of experimental diets see Table 1.

Experiment 2

To test whether bats demonstrated a preference for an N-containing nectar given their N nutritional status, we offered bats the same pairwise diets as in experiment one, but this time, we used N-deprived bats that were previously fed under an N-free maintenance diet for 4 days.

The remaining experiments described below were all performed with N-balanced bats that under captivity were always fed a sugar and N-balanced maintenance diet (Mirón et al. 2006).

Experiment 3

To test whether bats demonstrate a preference for sugar-only nectars (without N) at different sugar concentrations, we offered bats the natural sugar concentration found in two of the most visited plant species at our field site: 18% (w/v) simulating the total concentration of solutes in the nectar of Ca and 27% (w/v) simulating the total concentration in the nectar of Pp.

Experiment 4

To test whether bats demonstrate a preference for N-containing nectars at different sugar concentrations, we offered bats diets that simulated the natural sugar and N concentrations found in these two plant species in the field (Rodríguez-Peña et al. 2007). The low-N diet (hereafter LN) with 320 μg mL−1 simulated the natural N nectar concentration of Ca and also contained its natural nectar sugar concentration of 18% (w/v), whereas the HN diet with 1505 μg mL−1 simulated the natural N nectar concentration of Pp and contained its natural nectar sugar concentration of 27% (w/v).

Experiment 5

To determine whether bats were able to differentiate between a sugar-only nectar and an amino acid-containing nectar (20% w/v of sucrose), we offered bats an amino acid-free sugar solution versus an artificial nectar mimicking the amino acid relative abundance of Ca (amino acid-containing nectar).

Experiment 6

To determine whether bats species were able to differentiate between a sugar-only nectar and another amino acid-containing nectar we offered bats again an amino acid-free sugar solution but this time, we offered an artificial nectar mimicking the amino acid relative abundance of Pp (amino acid-containing nectar; 20% w/v of sucrose). Thus, in experiments 5 and 6, we examined whether bats were able to detect the amino acids present in two of their natural diets. This time the experimental diets mimicked the relative abundance of 17 specific free amino acids in the nectar of Ca and Pp. To see details of experimental diet preparations see Table 1.

Experiment 7

To examine whether bats demonstrate a preference for a particular amino acid-containing nectar, we offered bats two sucrose diets at 20% of sugar concentration that had the same 17 amino acids types but differed in their proportion. In other words, diets differed in their relative abundance of the 17 amino acids mimicking those found in the field for Ca and Pp.

Experiment 8

To examine the effect of sugar concentration on bat preferences for amino acid composition, we offered the two diets that differed in their relative abundance of 17 amino acids, but this time at the natural total concentration present in the nectar of each of these plant species, thus Ca (18% w/v of sucrose) and Pp (27% w/v of sucrose).

Data analysis

We used the Box and Cox method to transform and normalize the intake data. We performed a two-way Analysis of Variance with diet, bat species, and their interaction to assess preference for experimental diets. A Tukey's test was performed to assess differences in the total nightly intake of N-mantained and N-deprived bats. All analyses were performed in Minitab 16 (Minitab, State College, PA, USA).

Results

Bats showed no preferences for the sugar-only nectar, nor for the N-containing nectar when bats where N-balanced and N-deprived (Fig. 1 Experiments 1 and 2; Table 2). No differences in intake were found within N-balanced and N-deprived bats (Tukey > 0·05; d.f. = 24). Furthermore, no differences were found in total nightly intake by bats' nutritional state (F = 0·79; P = 0·383; d.f. = 1) or bats' nutritional state × bat species interaction (F = 0·05; P = 0·823; d.f. = 1). Leptonycteris yerbabuenae showed a preference for the most concentrated sugar-only nectar when NN/NN was 18 : 27, whereas G. soricina showed no preferences. Significant differences were found between diet, species and the diet × species interaction (Fig. 1 Experiment 3). However, neither bat species exhibited preferences for the most concentrated N-containing nectar (Fig. 1 Experiment 4, Table 2).

Table 2. Results from two-way anovas for foraging experiments on nectarivorous bats
ExperimentDiet (F; P)Bat species (F; P)Diet × Bat species (F; P)
  1. Ca, Crescentia alata; HN, high-N content; LN, low-N content; NA, no amino acid; NN, no N; Pp, Pachycereus pecten. Significant values are highlighted in bold.

HN/NN 20 : 20 N-balanced bats0·46; 0·5043·76; 0·0640·98; 0·333
HN/NN 20 : 20 N-deprived bats0·01; 0·9980·54; 0·4691·29; 0·322
NN/NN 18 : 2720·40; 0·0014·04; 0·0569·43; 0·005
LN/HN 18 : 271·88; 0·1833·77; 0·0640·01; 0·915
Ca/NA 20 : 209·43; 0·00516·01; 0·0017·29; 0·012
Pp/NA 20 : 2015·94; 0·00138·05; 0·0013·07; 0·093
Ca/Pp 20 : 203·35; 0·08014·04; 0·0012·50; 0·127
Ca/Pp 18 : 279·93; 0·0047·56; 0·01110·53; 0·003
Figure 1.

Food selection for nectar-eating bats between experimental nectars (mean ± SE) mimicking either the N content (experiments 1–4) or the relative abundance of 17 amino acids (experiments 5–8) in the nectar of bat-pollinated plant species bats consume in the field. Values for Glossophaga soricina are represented by dark circles and those for Leptonycteris yerbabuenae are represented by white circles.

Leptonycteris yerbabuenae was able to differentiate between the sugar-only and the amino acid-containing nectar preferring the sugar-only solutions. Significant differenes were found between diet, bat species and the diet × bat species intereaction when comparing Ca/NA 20 : 20 (Fig. 1 Experiment 5, Table 2) and between diet and bat species when comparing Pp/NA 20 : 20 (Fig. 1 Experiment 6, Table 2). Leptonycteris yerbabuenae preferred the Pp amino acid-containing nectar over the Ca amino acid-containing nectar, when tested at equal sugar concentrations and at field sugar concentrations (Fig. 1 Experiments 7 and 8, Table 2). Significant differences were found between bat species when comparing Ca/Pp 20 : 20 (Experiment 7), and diet, bat species, and the diet x bat species interaction when comparing Ca/Pp 18 : 27; (Experiment 8,Table 2). However, G. soricina showed no preferences (Fig. 1 Experiments 5–8, Table 2).

Discussion

Because of the low concentration of N and amino acids in the nectar of bat-pollinated flowers, their role in the foraging ecology of nectar-eating bats has been ignored in the past. Although our results suggested that bats did not consider nectar N content within a nutritional basis in making foraging decisions, we found that N presence and the flavours provided by the relative abundance of amino acids in nectar influenced the specialist nectar feeding bat L. yerbabuenae food selection in two ways. First, the presence of N and free amino acids in nectar causes an incapacity to discriminate between sugar concentrations; and secondly, the flavour provided by the relative abundance of amino acids in nectar can be perceived and preferred. We focus this discussion on the ecological implications of the influence of N and amino acids in nectar on bats' foraging decisions.

Do bats select N-containing or sugar-only nectar differently based on their N nutritional status?

Our results showed that regardlessof N status, neither bat species preferred N-containing nectar over sugar-only nectar.(Fig. 1). To date, several studies have demonstrated that nectar-eating bats cannot meet their MNR while feeding only on floral nectar. To experimentally test this idea, we calculated how much N the bats ingested under our experimental conditions and compared this data with their MNR from the literature. While there is information on the MNR for L. yerbabuenae (13–17 mg N day−1; Howell 1974; Voigt & Matt 2004), the only report for G. soricina (Herrera, Ramírez & Mirón 2006) used a different methodology and the data reported for this species is not comparable, and thus, we present the analysis only for L. yerbabuenae. Considering that this species ingested 16·5 mL day−1 from the nectar of P. pecten (Ayala-Berdon et al. 2008), it only can obtain between 30·3% of its MNR when feeding exclusively on P. pecten nectar (4 mg N day−1). While we cannot calculate these values for G. soricina, we expect a similar result for this species.

Keeping bats under N-deprived conditions (feeding on a sugar-only diet during four nights) caused bats to lose hair, reduced their flying abilities and generally weakened the bats. We considered that keeping bats under this N-depleted condition for a longer period of time would result in bats being unable to fly for feeding within the experiments and their inminent death. Unexpectedly, our results did not coincide with the response found in other animals that vary their feeding choices to satisfy the requirements of nutrients when they face an unbalanced nutrient availability (Belovsky 1978; Greenstone 1979; Geiser & Kenagy 1987; Wheelwright 1970; Geiser, Stahl & Learmonth 1992; Torres-Contreras & Bozinovic 1997; Maynts et al. 2009). We believe that the indifference that both bat species exhibited for our N-containing nectar is the result of its lack of nutritional value for the bats. Bats percieve the highest N content found in a floral nectar at our study site as low, and therefore, they do not discriminate between diets, even when they are N-deprived, because it is inconsequential in obtaining their N daily requirements. However, this interpretation requires further exploration. As a result, nectarivorous bats in the field need to supplement their diet using other items as an N source (Carvalho 1961; Alvarez & González 1970; Howell 1974; Heithaus, Fleming & Opler 1975; Baker & Baker 1982; Lemke 1984; von Helversen 1993; Delorme & Thomas 1996; Herrera et al. 2001; Winter & von Helversen 2001). At our study site, both bat species supplement their nectar diet with other sources. Glossophaga soricina includes fruits, pollen and insects in its diet and uses primarily insects to satisfy its nitrogen requirements (c. 70%, Herrera et al. 2001). It has been reported that Leptonycteris yerbabuenae meets its N requirements mostly from ingesting pollen (Howell 1974), and at Chamela, both bat species have been recorded actively foraging on the anthers of the flowers of several plant species (Quesada et al. 2003).

Does the presence of N in nectar affect the capacity of bats to discriminate and select other nectar traits such as sugar concentration?

Leptonycteris yerbabuenae preferred the most concentrated diet when confronted with sugar-only nectars, while it became indifferent to the sugar concentration when confronted with the N-containing nectars. The effect of nectar sugar concentration on bats′ sugar preferences has been studied in the past (Roces, Winter & Helversen 1993; Rodríguez-Peña et al. 2007). Roces, Winter & Helversen (1993) found that G. soricina always preferred the higher of two nectar concentrations, when the differences in sugar concentration between the two solutions were > 10%. Rodríguez-Peña et al. (2007) found that L. yerbabuenae and G. soricina preferred the more concentrated sugar solution even when the difference in concentration between the two solutions were small (3% for L. yerbabuenae and 9% for G. soricina). Here, our results showed that bats had no preferences for diets with different sugar concentrations when N was added to the artificial nectar solutions. While the two bat species have the ability to detect small differences in sugar concentration, they were indifferent when facing two sugar solutions differing in 9% when N was added. This was a surprising result and suggests that the presence of N-containing compounds in nectar causes a confounding effect that modifies bats' capacity to discriminate among sugar concentrations. A similar situation is presented in the nuptial gifts of Gryllodes sigillatus and Blatella germanica (Orthoptera; Warwick et al. 2009 and Dictyoptera; Kugimiya et al. 2003). In these species, males offer a nuptial gift to the female that brings the female to the precopulatory position and ensures his reproductive success. However, males increase the attractiveness of the feeding gift by ‘swittenning’ (candymaker hypothesis), a relatively low-value food item, by incorporating free amino acids that act as a phagostimulant. Thus, producing a sensory trap for the female and not necessarily providing her with a nutritional benefit.

In this case, if the N-containing compounds present in nectar reduce the ability of specialized bats to discriminate for sugar concentrations, as our results indicate, the presence of these substances in floral nectar could be the result of a plant strategy to confound floral visitors. Plants could produce nectars with lower sugar concentrations, and by adding small amounts of N-containing compounds, they could trick their visitors to perceive different sugar solutions as energetically similar, saving sugars and maintaining visit rates (See Gilbert, Haines & Dickson 1991; Kunze & Gumbert 2001; Schiestl, Huber & Gomez 2011; Bronstein, Alarcón & Geber 2006; Anderson & Johnson 2006; Kessler, Gase & Baldwin 2008; Wright, Choudhary & Bentley 2009 for strategies of plants cheating pollinators). However, the relative cost of using nitrogen in nectar to reduce the ability of pollinators to detect sugar concentration is unknown and needs to be explored.

Are bats able to distinguish among the flavours generated by the amino acid relative abundance present in the nectar from plants they typically encounter in nature?

Leptonycteris yerbabuenae had preferences for sugar-only diets (NA) when offered an amino acid-containing counterpart. Amino acid rejection has been reported for other pollinators like bees (Inouye & Waller 1984), hummingbirds (Haisworth & Wolf 1976) and sunbirds (Leisegneur, Verburgt & Nicolson 2007). An amino acid imbalance is defined as adverse effects caused by the ingestion of a surplus of an individual, or group of amino acids (Krehl et al. 1945; Harper, Benevenga & Wohlheuter 1970). Adverse effects of amino acid imbalance have been well documented and go from moderate depression of food intake and reduced growth, to the development of pathological lesions and low survival rates (Harper 1967; Harper, Benevenga & Wohlheuter 1970). Usually, this rejection is associated with the presence of amino acid concentrations higher than those found naturally in the nectar ingested by these animals in the field. Rodents and other mammals have been found to select protein-free diets, or a diet with a balanced amino acid pattern, over those presenting amino acid imbalance (Harper 1967; Leung, Rogers & Harper 1968). In this study, L. yerbabuenae first showed no preferences between sugar-only and N nectar diets when these diets where balanced in their amino acid composition (experiments 1–4). However, they preferred the sugar-only solutions when they were offered a specific amino acid composition as their other option (experiments 5 and 6). As we offered the bats the specific amino acid composition found in the plants they eat in the field, this ‘rejection’ is surprising. Nevertheless, as they are unable to find amino acid-free nectars in the field they must feed on nectars containing amino acids to survive, but our data show that they do not prefer them. We speculate that L. yerbabuenae rejection of amino acid-containing solutions is due to the flavour that amino acid relative abundance provides to the experimental solutions. As documented in rats (Sclafani 1995), the selection–rejection process involves the sensory characteristics of the foodstuff, in particular its flavour (i.e. taste, odour and texture). Given the extremely low concentration of amino acids in the nectar, it is hard to imagine that the amino acid imbalance would be a factor affecting the foraging decesions of this species. Because the capacity of this bat species to discriminate between sugar solutions diminished from a capacity to separate solutions that differed in only 3% to being unable to discriminate between solutions differing in 9% when N was added to the nectar, bats could prefer the N-free solutions to avoid this bias; however, this interpretation requires further exploration.

Our results showed that L. yerbabuenae preferred the flavour of the amino acids of P. pecten (Cactaceae) over those found in C. aesculifolia (Malvaceae) when total nectar concentration was equal in both experimental solutions, or when the sugar concentration resembled the natural sugar concentrations found in the field in these two plant species. The fact that L. yerbabuenae preferred the flavour provided by the specific composition and relative abundance of the amino acids present in the nectar of P. pecten over those present in C. aesculifolia is evidence that this species perceives and recognizes the flavour that amino acid relative abundance provides to the nectar of each plant species. Gardener & Gillman (2001) suggested that amino acid composition has more influence than amino acid concentration on the taste profiles of nectar.

Our results indicate that the relative abundance of the different amino acids in nectar also contributes to provide a specific taste to nectar and that bats can use this information to select food resources in the field (Baker & Baker 1973; Baker 1977; Baker, Baker & Hodges 1998; Gardener & Gillman 2002). Kubota et al. (1998) found two natural amino acids isolated from the fruit of Scorodocarpus borneensis to play an important role in developing the main odorous components of the characteristic garlic odour found in some fruits and most bat-pollinated flowers. The influence of amino acids on smell and how bats can use it to select food resources in the field is a topic that remains to be studied.

The amino acid related flavour preferred by L. yerbabuenae is associated with the nectar with the natural highest sugar concentration (27%; P. pecten). We speculate that L. yerbabuenae is associating the flavour caused by the particular amino acid relative abundance in the nectar of this cactus over other nectar attributes like sugar concentration. Leptonycteris yerbabuenae preferred the nectar imitation of P. pecten over that of C. aesculifolia. This is not surprising if we consider that P. pecten has more sugar in its nectar in the wild than C. aesculifolia.

Leptonycteris yerbabuenae and G. soricina differed in their ability to discriminate between solutions with N and nectar amino acids. Our results suggest that ecological specialization has led the nectar specialists L. yerbabuenae to evolve a fine-scale flavour discrimination capacity that allows it to identify high-energy nectar. Because of its large body size (15–25 g), L. yerbabuenae needs more concentrated nectars to maintain a positive energy balance (Ayala-Berdon et al. 2008), compared with the smaller bodied G. soricina (8–12 g). Additionally, L. yerbabuenae has a better capacity to discriminate between solutions with different sugar concentrations (Rodríguez-Peña et al. 2007). Although both bat species consume nectar, fruits and insects in the field, our results suggest that among bats, ecologically more specialized nectarivores will be better at using amino acid flavour cues to select the highest reward nectars available to them in the field; however, this hypothesis remains to be tested with other species of nectar specialists.

In conclusion, our study shows that N content and free nectar amino acids could affect the foraging decisions of nectar-eating bats, regardless of the low concentrations at which they are present in floral nectar. The fact that N presence in nectar confounds bats′ capacity to distinguish among sugar concentrations and that some bats have preferences for the taste that the relative abundance of free amino acids provides to nectar of specific plant species, suggests that plants can use variation in this trait to attract bat pollinators. Relating nectar concentration with amino acid flavour could allow bats to use the presence of certain amino acids to influence foraging decisions under natural conditions in the field. Future studies should further evaluate how N content and free nectar amino acids affect bat foraging decisions by using a greater variety of nectar and bat species to test some of the ideas we present here.

Acknowledgements

The authors acknowledge support by a grant from the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica PAPITT (IN226007and IN226710-3), Universidad Nacional Autónoma de México to KES and JES. Scholarship support to Nelly Rodríguez-Peña was provided by Consejo Nacional de Ciencia y Tecnología CONACYT. We thank J. Gilbert and one anonymous reviewer for making valuable suggestions to improve our manuscript. We thank J.M. Lobato for assistance in the field, L. Barbo for help in the laboratory, and the Estación de Biología Chamela for logistical support.

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