Foraging bees use visual and olfactory cues to find and select food sources and deploy innate or learned preferences to detect flowers (Lunau and Maier, 1995; Dyer et al., 2016). Primarily, a forager’s choice is biased by innate preferences for particular colors, shapes, and odors (Menzel, 1967; Giurfa et al., 1995; Lehrer et al., 1995; Lunau et al., 1996; Gumbert, 2000; Biesmeijer and Slaa, 2004; Raine and Chittka, 2007; Howard et al., 2019). These innate preferences differ among species. In several experiments, preferences for specific hues and saturation of colors could be found for honeybees and bumble bees (Lunau, 1990; Giurfa et al., 1995; Lunau et al., 1996; Papiorek et al., 2013; Rohde et al., 2013), while stingless bees sparsely show preferences for color hue or saturation (Spaethe et al., 2014; Dyer et al., 2016; Koethe et al., 2016, 2018).

With increasing foraging experience, initial individual preferences may be either consolidated or modified through associative learning (Gumbert, 2000; Sánchez et al., 2008; Roselino et al., 2016). For instance, species-specific chemical footprints deposited by bees while landing on and manipulating flowers indicate the recent presence of a forager to subsequent visitors (Hrncir et al., 2004; Jarau et al., 2004; Eltz, 2006; Saleh and Chittka, 2006; Witjes et al., 2011). An initial attraction toward the familiar scent of conspecifics (Schmidt et al., 2005) may be reinforced when individuals learn to associate the footprints with high reward levels or reversed when scent marks indicate depleted flowers (Saleh and Chittka, 2006; Roselino et al., 2016).

Learning and memory play a major role in bee foraging, enabling the repeated visit to sustainable food sources (Breed et al., 2002; Reinhard et al., 2004, 2006; Jesus et al., 2014), flower constancy (Free, 1963; Biesmeijer and Toth, 1998; Slaa et al., 1998, 2003), and the discovery of new patches of known food plants (Biesmeijer and Slaa, 2004). In addition to memorizing scent and location of resources (Reinhard et al., 2004, 2006), bees learn both color and position of landmarks, which facilitates the orientation toward food sources and the nest (Cartwright and Collett, 1983; Cheng et al., 1986, 1987; Chittka et al., 1995; Menzel et al., 2005). However, species differ concerning their learning ability (Pessotti and Lé’Sénéchal, 1981; Mc Cabe et al., 2007), which might be associated with differences in life-history and ecological traits among bee species, such as longevity of individuals (Ackerman and Montalvo, 1985), the degree of floral specialization (Cane and Snipes, 2006), and food niche-breath (Biesmeijer and Slaa, 2006).

In eusocial bees, including the stingless bees (Meliponini), bumble bees (Bombini), and honeybees (Apini), food source selection is not only based on individual foraging preferences, but relies to a large extent on social information. On their return to the nest, foragers transmit olfactory and gustatory information about the exploited food source to nestmates, which biases the subsequent food choice of the receivers (Farina et al., 2005, 2007; Mc Cabe and Farina, 2009). Moreover, returning foragers of many species announce the existence of lucrative food sources through thoracic vibrations (stingless bees: Lindauer and Kerr, 1958; Esch et al., 1965; Barth et al., 2008; Hrncir and Barth, 2014; honeybees: Esch, 1961; Waddington and Kirchner, 1992; Hrncir et al., 2011). Inactive individuals may use these mechanical signals for their decision of whether to engage in foraging or to remain in the nest. In addition, foragers of some eusocial bee species guide the recruits to the location of the exploited food patch. Honeybees (all species) use an elaborated dance language (waggle dance) communicating information about distance, direction, and quality of foraging sites (Von Frisch, 1967; Dyer, 2002). Stingless bees (few species), in contrast, lay polarized trails of species-specific pheromone marks that guide recruits with high precision toward the goal (Lindauer and Kerr, 1958; Schmidt et al., 2003; Nieh et al., 2004; Barth et al., 2008; Jarau, 2009). At the food patch, foraging choices are influenced by field-based social information, like olfactory footprints and the visual presence of con- or heterospecific foragers (Slaa et al., 2003). Depending on the composition of the foraging community at the food patch, these passively provided cues may cause local enhancement or local inhibition (Slaa and Hughes, 2009). Thus, food source selection in eusocial species is based on a complex interplay between individual preferences and social information.

Differences among social bee species regarding ecological (habitat, food niche), physiological (learning ability, visual capacity, color vision), and behavioral features (innate preferences, foraging strategy, recruitment mechanism) may result in differences concerning the parameters used in foraging decisions. With more than 500 described species, stingless bees (Meliponini) are the most speciose group of eusocial bees with very diverse characteristics regarding body size, colony size, nesting biology, brood cell arrangement, queen production, foraging strategies, and recruitment mechanisms (Michener, 1974, 2013; Johnson, 1983; Wille, 1983; Engels and Imperatriz-Fonseca, 1990; Roubik, 2006; Barth et al., 2008). Given this biological diversity, we can expect differences concerning the mechanisms of food source selection among species. In the present study, we investigated the food source selection by two stingless bee species, Melipona subnitida and Plebeia flavocincta, and the Western honeybee, Apis mellifera. Since stingless bees show only weak preferences for colors compared to other bee species (Dyer et al., 2016; Koethe et al., 2016, 2018), alternative parameters could be of importance for foraging choices. Of interest were the roles of scent marks (olfactory footprints), the color, and the location of a food source. Melipona species are known to mark food sources with olfactory footprints (Jarau, 2009; Roselino et al., 2016). For P. flavocincta, no specific information concerning scent communication is available so far (Aguilar et al., 2005). However, given that all bee species studied to this moment deposit chemical footprints at food sources (Goulson et al., 1998; Eltz, 2006; Yokoi et al., 2007; Jarau, 2009; Witjes et al., 2011), scent cues can also be postulated for this meliponine species. A. mellifera is known for marking food sources directly (Giurfa and Nunez, 1992).

The aim was to analyze how the three investigated social bee species use the parameters color, scent marks, or location differently during the colony foraging processes. We test the hypothesis that these bees possess a hierarchy in the use of the parameters color, scent marks, and location of flowers. We expect honeybees to rely more on color cues than the two stingless bee species. For the two stingless bee species, we assume that they follow scent markings of conspecifics more reliable than honeybees. Since small stingless bee might exploit nectarrich flowers by repeated visits to the same individual flower, we assume that the location of the flower is of higher importance in the smaller bees.

This study is part of a research project on color preferences in stingless bees conducted in Australia and Brazil (Köthe, 2019).

In the first experimental series (influence of scent marks), foragers of all three bee species significantly preferred the previously visited training feeder over the clean feeder (Figure 1; M. subnitida: n = 239, z-value = −8.346, p < 0.001; P. flavocincta: n = 355, z-value = −12.15, p < 0.001; A. mellifera: n = 303, z-value = −10.46, p < 0.001).

In the second experimental series, we investigated the influence of color on the feeder choice by the three bee species. After training to a blue-colored feeder, all three species significantly preferred the blue feeder over the yellow feeder (Figure 2A; M. subnitida: n = 250, z-value = −10.24, p < 0.001; P. flavocincta: n = 230, z-value = −8.821, p < 0.001; A. mellifera: n = 538, z-value = −10.85, p < 0.001). When these workers were retrained to forage on a yellow feeder during the last training phase, the two stingless bee species significantly preferred the yellow feeder while honeybee workers visited both colors equally (Figure 2B; M. subnitida: n = 124, z-value = 2.667, p = 0.007; P. flavocincta: n = 71, z-value = 3.756, p < 0.001; A. mellifera: n = 278, z-value = 0.6, p = 0.549). When workers were initially trained to a yellow-colored feeder, both stingless bee species preferred the yellow feeder significantly over the blue feeder during the test, while A. mellifera preferred the blue feeder (Figure 2C; M. subnitida: n = 199, z-value = 3.318 p < 0.001; P. flavocincta: n = 303, z-value = 3.141, p = 0.002; A. mellifera: n = 556, z-value = 5.863, p < 0.001). Retraining to a blue feeder in the last training phase lead to a significant preference of the blue colored feeder in all three species (Figure 2D; M. subnitida: n = 52, z-value = −2.95, p = 0.003; P. flavocincta: n = 61, z-value = −2.632, p = 0.008; A. mellifera: n = 213, z-value = −6.74, p < 0.001).

When analyzing the influence of the feeders’ positions on the food source choice, we observed that M. subnitida visited the feeding site closer to the nest during the scent mark trials (site 1, 15 m) significantly more often than the farther site (site 2, 17 m) (Figure 3; n = 239, z-value = −8.467, p < 0.001), while choosing randomly when tested for color differences (Figure 3; blue: n = 250, z-value = −0.045, p = 0.964; yellow: n = 199, z-value = 1.502, p = 0.133). Workers of A. mellifera significantly preferred the closer feeding site when they were trained to yellow (Figure 3; n = 556, z-value = −6.147, p < 0.001), but did not distinguish between the two sites when trained for blue (Figure 3; blue: n = 538, z-value = −0.951, p = 0.342; scent marks: n = 199, z-value = −1.502, p = 0.133). In the trial concerning scent marks workers of A. mellifera significantly preferred the farther away feeding site (Figure 3; n = 303, z-value = −10.46, p < 0.001). Workers of P. flavocincta preferred the closer feeding site when trained to blue or yellow (Figure 3; blue: n = 230, z-value = −5.036, p < 0.001; yellow: n = 71, z-value = −4.856, p < 0.001) and significantly visited the farther site when tested concerning scent marks (Figure 3; n = 355, z-value = −2.548, p = 0.011).

The results of this study show that the response to the color of feeder, scent marks, and locations differs among the tested species P. flavocincta, M. subnitida, and A. mellifera. Our results confirm previous findings about the important role of color for food plant detection in honeybees and add further findings to the diverse and sometimes less important role of color for food plant detection in stingless bees. In previous studies of color preferences in stingless bees, the results varied among species. While three species of the genus Melipona chose colors poorly, Tetragonula carbonaria chose colors according to their hue and Partamona helleri showed similar color choices as A. mellifera preferring spectrally purer colors and bluish color hues (Rohde et al., 2013; Dyer et al., 2016; Koethe et al., 2016, 2018). Particularly for the honeybee, it has been shown that besides innate preferences, absolute or differential conditioning and behavioral plasticity play important roles in how they exploit color information (Reser et al., 2012), and that strong color preferences impede learning of other features (Morawetz et al., 2013).

Workers of A. mellifera orientated most strongly according to colors. The blue-colored feeder was preferred in all tests with exception of the retraining to yellow, where A. mellifera showed no depicted choice for one of the two colors and the two stingless bee species preferred the yellow feeder. This is in accordance to previous studies showing that A. mellifera prefers blue colors over other color hues (Giurfa et al., 1995; Horridge, 2007). The two stingless bee species chose feeders according to their colors but rather preferred the feeder color of the previous training. Only when initially trained to yellow they showed weak (M. subnitida) or no preferences for the trained color (P. flavocincta). This preference for blue is in accordance with previous results of stingless bees, but also suggests that it is weaker in stingless bees than in honeybees (Dyer et al., 2016; Koethe et al., 2016). An explanation for less visually driven behavior in stingless bees could be the size differences compared to honeybees. P. flavocincta reaches a body size of 3.6–4.1 mm, M. subnitida of 7.5 mm, and A. mellifera is the largest of the three species with 13–16 mm (Hrncir and Maia-Silva, 2013; Maia-Silva et al., 2015; Imperatriz-Fonseca et al., 2017). Especially the size of the eyes, which is associated with body size, can impact the visual capacities of bees (Streinzer et al., 2016). Workers of P. flavocincta are rather small; consequently, their eyes are also small leading to poorer visual capabilities.

Both stingless bees and honeybees use scent cues to evaluate reward availability of food resources (Nunez, 1967; Butler et al., 1969; Ferguson and Free, 1979; Free and Williams, 1983; Corbet et al., 1984; Giurfa and Nunez, 1992; Giurfa, 1993; Stout et al., 1998; Williams, 1998; Stout and Goulson, 2001). In this study, all three species showed preferences for the marked feeder over the unmarked one. P. flavocincta and A. mellifera chose the marked feeder consistently (∼88% of choices), while M. subnitida preferred the marked feeder, but visited it less frequently (∼64% of choices).

Plebeia flavocincta was the only species that significantly preferred the closer feeding site when tested concerning colors and the farther feeding site when tested regarding scent marks. One interpretation is that P. flavocincta does not differentiate between colors and choses the closer feeding site, while the preference during the scent mark trial could be based on the fact that the scent marked feeder was positioned twice at the farther site and only once at the closer site. In contrast, M. subnitida was the only species in the scent mark trials that visited the food site with shorter distance to the hive more frequently. It seems likely that M. subnitida orientates on location rather than on scent marks. Previous studies showed that species of the genus Melipona mark food sites directly and do not lay scent trails (Hrncir et al., 2004). In order to recruit new foragers, it seems possible that M. subnitida relies strongly on piloting—leading new foragers from hive to food site during flight (Nieh et al., 2003). Foragers of M. subnitida could be observed to frequently arrive in small groups, while A. mellifera and P. flavocincta workers seemed more independent from each other. Scent marks play an important role for the communication of reward availability, but their impact on recruitment seems dependent on the specific strategy used by species (Free and Williams, 1983; Corbet et al., 1984; Giurfa and Nunez, 1992; Giurfa, 1993; Stout et al., 1998; Stout and Goulson, 2001; Schmidt et al., 2003). The attractiveness of scent marks, whether or not they were used for recruitment purposes, appears to be strong because scent-marked feeders were preferred by all three tested bee species. During the experiments workers foraged in groups and could be influenced by the presence of other individuals. An influence by social facilitation (Wilson, 1971) could not be excluded during the experiments, but when comparing the results for choices of blue and yellow feeders, after the respective training, an influence solely by the presence of conspecifics seems unlikely.

Another aspect that can explain the diverse results for the three tested bee species could be their natural habitat. M. subnitida originates from the Caatinga, which is an open habitat, while P. flavocincta inhabits a spacious habitat that extends from the Caatinga to the Atlantic Rainforest, which is a densely vegetated forest (Imperatriz-Fonseca et al., 2017). Because of its domestication, the honeybee is widespread all over the world. It originates from diverse habitats of Europe, the Middle East, and Africa. Open habitats are brightly illuminated, while forest habitats are characterized by dim-light conditions (Endler, 1992). Based on the light conditions of their respective habitat, it appears to be possible that M. subnitida and A. mellifera could rely to a greater extent on visual signals than P. flavocincta that encounters dim-light conditions and a less visually structured vegetation. In a densely vegetated habitat, scent marks could be a more reliable signal to guide foragers to a food source. Furthermore, temperate and sub-tropical regions experience more distinct seasons concerning weather conditions and the rhythm of flowering plants is directly influenced, while tropical and semi-arid regions have more steady weather conditions but are challenging for their inhabitants because of high temperatures (Prado, 2003; Zanella and Martins, 2003; Machado and Lopes, 2004; Maia-Silva et al., 2012, 2015; Hrncir et al., 2019).

Social bee species that face seasonal variations mass-collect floral resources for provision of the hive (Ramalho, 2004). These variations in floral resource availability could be another explanation for more distinct preferences for visual signals in honeybees when compared to tropical species, like M. subnitida and P. flavocincta, because only honeybees face strong seasonal variations (Michener, 1974; Kleinert-Giovannini, 1982; Roubik, 1982a; Seeley, 1985). Nonetheless, this would not explain the differences between M. subnitida and P. flavocincta.

The three tested bee species reacted vaguely similar to color, scent marks, and location of food sources, but their main focus varies: While A. mellifera choose food sites according to both color and scent marks, M. subnitida orientates on location and color of food sites, and P. flavocincta relies mainly on scent marks. These variations are possibly based on different recruitment mechanisms (e.g., waggle dance of honeybees vs. piloting, excited movements, vibration, and scent mark deposition by stingless bees) or they could be the result of adaptations to the bees’ respective habitat and obliged morphological constraints. Although highly eusocial stingless and honeybees do not communicate the color of flowers to nestmates (Michelsen, 2014), flower color has a large impact on foraging decisions. This impact is demonstrated by the results of this study, that bees exhibit a spontaneous response to color cues and that they memorize color cues following experience; spontaneous response of bees and discrimination after conditioning might rely on different color parameters, such as color saturation and color hue (Rohde et al., 2013). Flower color has also been identified as a floral filter excluding bees from visiting the less preferred flower colors, i.e., red, UV-absorbing and white, UV-reflecting hummingbird-pollinated flowers (Lunau et al., 2011). Stingless bees are known as nectar robbers of hummingbird-pollinated flowers (Roubik, 1982b); it remains to be tested if the less pronounced color preferences in stingless bees are helpful for finding food on flowers displaying colors that are not adapted to bee-color vision and color preferences.

All datasets generated for this study are included in the article/Supplementary Material.

KL conceived and developed the research concept and supervised the study. SK designed the experiments. MH and KL supported the design of the experiments. SK, VF, LR, and SB conducted the experiments and collected and analyzed the data. SK wrote the manuscript. SB, VF, LR, SB, MH, and KL supported the writing of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.