Learning of bimodal versus unimodal signals in restrained bumble bees.

Similar to animal communication displays, flowers emit complex signals that attract pollinators. Signal complexity could lead to higher cognitive load for pollinators, impairing performance, or might benefit them by facilitating learning, memory and decision making. Here, we evaluated learning and memory in foragers of the bumble bee Bombus impatiens trained to simple (unimodal) versus complex (bimodal) signals under restrained conditions. Use of a proboscis extension response protocol enabled us to control the timing and duration of stimuli presented during absolute and differential learning tasks. Overall, we observed broad variation in performance under the two conditions, with bees trained to compound bimodal signals learning and remembering as well as, better than or more poorly than bees trained to unimodal signals. Interestingly, the outcome of training was affected by the specific colour-odour combination. Among unimodal stimuli, the performance with odour stimuli was higher than with colour stimuli, suggesting that olfactory signals played a more significant role in the compound bimodal condition. This was supported by the fact that after 24 h, most bimodal-treatment bees responded to odour but not visual stimuli. We did not observe differences in latency of response, suggesting that signal composition affected decision accuracy, not speed. We conclude that restrained bumble bee workers exhibit broad variation of responses to bimodal stimuli and that components of the bimodal signal may not be used equivalently. The analysis of bee performance under restrained conditions enables accurate control of the multimodal stimuli provided to individuals and to study the interaction of individual components within a compound.

Sex differences in pollinator behavior: Patterns across species and consequences for the mutualism.

Intraspecific variation in floral visitor behaviour and pollination efficiency has been much less studied than interspecific variation. Nevertheless, it is clear that large differences in these traits exist within species, and in particular between sexes within species. With the exception of a few well-studied interactions, however, the consequences of these differences in the pollinators and visited plants remain to be investigated. In this review, we document large and consistent differences in the foraging patterns of male and female pollinators that have been demonstrated to directly affect plant reproduction or that have clear potential to do so. Males and females differ in visitation frequency, type of flowers visited, and per-visit pollen transfer. Females gather more and different resources from flowers compared to males, and males generally tend to show more mobile foraging patterns than females. We argue that these sex-associated patterns have broad generality across pollinators, and that sex-associated differences can in some cases be larger than differences between species. We offer predictions about how these patterns will influence pollinator preference, specialization, and fidelity, as well as the cost, quality and quantity of pollination service to plants. In the face of increasing threats to plant-pollinator interactions, understanding their basic functioning and the variation inherent in their component parts is critical. We advocate for more attention to sex-based differences among pollinators in particular, and the consequences of intraspecific variation more broadly.

Brawls Bring Buzz: Male Size Influences Competition and Courtship in Diadasia rinconis (Hymenoptera: Apidae).

Sexual selection on male body size in species with a female-biased sexual size dimorphism is common yet often poorly understood. In particular, in the majority of bee species, the relative contribution of intrasexual competition and female choice to patterns of male body size is unknown. In this field study, we examined two possible components of male mating success with respect to body size in the solitary bee Diadasia rinconis Cockerell (Hymenoptera: Apidae): 1) ability to procure a mate and 2) the duration of copulation. We found that larger males were better able to procure mates and copulated for shorter periods of time. Although consistent with sperm competition theory, differences in copulation duration were slight; possibly, the shorter copulations of larger males instead reflect in copulo female choice. Consistent with this notion, males engaged in complex courtship while mounted, characterized for the first time in any bee in such detail via audio recordings and high-speed, high-definition video. The number of pulses in male courtship behavior was also positively associated with copulation duration and may have stimulated females to continue copulating, thereby potentially allowing smaller males to transfer a full ejaculate. Females were shown to be potentially polyandrous and although we did not observe precopulatory rejection in the field, captive females frequently rejected copulation attempts by captive males. Our work indicates that intrasexual competition selects for increased body size in a solitary bee.

Why Have Multiple Plastic Responses? Interactions between Color Change and Heat Avoidance Behavior in Battus philenor Larvae.

Having multiple plastic responses to a change in the environment, such as increased temperature, can be adaptive for two major reasons: synergy (the plastic responses perform better when expressed simultaneously) or complementarity (each plastic response provides a greater net benefit in a different environmental context). We investigated these hypotheses for two forms of temperature-induced plasticity of Battus philenor caterpillars in southern Arizona populations: color change (from black to red at high temperatures) and heat avoidance behavior (movement from host to elevated refuges at high host temperatures). Field assays using aluminum models showed that the cooling effect of the red color is greatly reduced in a refuge position relative to that on a host. Field assays with live caterpillars demonstrated that refuge seeking is much more important for survival under hot conditions than coloration; however, in those assays, red coloration reduced the need to seek refuges. Our results support the complementarity hypothesis: refuge seeking facilitates survival during daily temperature peaks, while color change reduces the need to leave the host over longer warm periods. We propose that combinations of rapid but costly short-term behavioral responses and slow but efficient long-term morphological responses may be common when coping with temperature change.

Colour learning when foraging for nectar and pollen: bees learn two colours at once.

Bees are model organisms for the study of learning and memory, yet nearly all such research to date has used a single reward, nectar. Many bees collect both nectar (carbohydrates) and pollen (protein) on a single foraging bout, sometimes from different plant species. We tested whether individual bumblebees could learn colour associations with nectar and pollen rewards simultaneously in a foraging scenario where one floral type offered only nectar and the other only pollen. We found that bees readily learned multiple reward-colour associations, and when presented with novel floral targets generalized to colours similar to those trained for each reward type. These results expand the ecological significance of work on bee learning and raise new questions regarding the cognitive ecology of pollination.

Effects of developmental change in body size on ectotherm body temperature and behavioral thermoregulation: caterpillars in a heat-stressed environment.

Ectotherms increase in size dramatically during development, and this growth should have substantial effects on their body temperature and ability to thermoregulate. To better understand how this change in size affects temperature, we examined the direct effects of body size on body temperature in Battus philenor caterpillars, and also how body size affects both the expression and effectiveness of thermal refuge-seeking, a thermoregulatory behavior. Field studies of both live caterpillars and physical operative temperature models indicated that caterpillar body temperature increases with body size. The operative temperature models also showed that thermal refuges have a greater cooling effect for larger caterpillars, while a laboratory study found that larger caterpillars seek refuges at a lower temperature. Although the details may vary, similar connections between developmental growth, temperature, and thermoregulation should be common among ectotherms and greatly affect both their development and thermal ecology.

Big maggots dig deeper: size-dependent larval dispersal in flies.

The ability of individual animals to select habitats optimal for development and survival can be constrained by the costs of moving through the environment. Animals that seek overwintering sites underground, for example, may be constrained by the energy required to burrow into the soil. We conducted field and laboratory studies to determine the relationship between individual size and overwintering site selection in the tephritid flies, Rhagoletis juglandis and Rhagoletis suavis. We also explored the effect of site selection on pupal mortality, parasitism, and the ability to emerge from overwintering sites after eclosion. In both species, and in both lab and field tests, larger pupae were found at deeper soil depths. In addition, marginally non-significant trends indicated pupae in deeper sites were 48% more likely to survive the overwintering period. Finally, larger individuals were more likely to eclose and emerge from the soil at a given depth, but flies in deep overwintering sites were less likely to emerge from those sites than flies in shallow sites. Our data indicate that overwintering site selection represents a trade-off between avoiding predators and parasites that occur at shallow sites, and the energetic and mortality costs of burrowing to, overwintering in, and emerging from, deeper sites. The size-dependent overwintering site selection demonstrated here has implications for population dynamics and pest control strategies. Some fly control measures, such as the introduction of parasites or predators, will be mitigated when the deepest and least accessible overwintering pupae represent a disproportionately large amount of the population's reproductive capacity.

Organ-specific volatiles from Sonoran desert Krameria flowers as potential signals for oil-collecting bees.

The evolution of flowers that offer oils as rewards and are pollinated by specialized bees represents a distinctive theme in plant-pollinator co-diversification. Some plants that offer acetylated glycerols as floral oils emit diacetin, a volatile by-product of oil metabolism, which is utilized by oil-collecting bees as an index signal for the presence of floral oil. However, floral oils in the genus Krameria (Krameriaceae) contain ß-acetoxy-substituted fatty acids instead of acetylated glycerols, making them unlikely to emit diacetin as an oil-bee attractant. We analyzed floral headspace composition from K. bicolor and K. erecta, native to the Sonoran Desert of southwestern North America, in search of alternative candidates for volatile index signals. Using solid-phase microextraction, combined with gas chromatography-mass spectrometry, we identified 26 and 45 floral volatiles, respectively, from whole flowers and dissected flower parts of these two Krameria species. As expected, diacetin was not detected. Instead, ß-ionone emerged as a strong candidate for an index signal, as it was uniquely present in dissected oil-producing floral tissues (elaiophores) of K. bicolor, as well as the larval cells and provisions from its oil-bee pollinator, Centris cockerelli. This finding suggests that the floral oil of K. bicolor is perfused with ß-ionone in its tissue of origin and retains the distinctive raspberry-like scent of this volatile after being harvested by C. cockerelli bees. In contrast, the elaiophores of K. erecta, which are not thought to be pollinated by C. cockerelli, produced a blend of anise-related oxygenated aromatics not found in the elaiophores of K. bicolor. Our findings suggest that ß-ionone has the potential to impact oil-foraging by C. cockerelli bees through several potential mechanisms, including larval imprinting on scented provisions or innate or learned preferences by foraging adults.

Plasticity of the worker bumblebee brain in relation to age and rearing environment.

The environment experienced during development can dramatically affect the brain, with possible implications for sensory processing, learning, and memory. Although the effects of single sensory modalities on brain development have been repeatedly explored, the additive or interactive effects of multiple modalities have been less thoroughly investigated. We asked how experience with multisensory stimuli affected brain development in the bumblebee Bombus impatiens. First, to establish the timeline of brain development during early adulthood, we estimated regional brain volumes across a range of ages. We discovered significant age-related volume changes in nearly every region of the brain. Next, to determine whether these changes were dependent upon certain environmental stimuli, we manipulated the visual and olfactory stimuli available to newly emerged bumblebee workers in a factorial manner. Newly emerged bumblebees were maintained in the presence or absence of supplemental visual and/or olfactory stimuli for 7 days, after which the volumes of several brain regions were estimated. We found that the volumes of the mushroom body lobes and calyces were larger in the absence of visual stimuli. Additionally, visual deprivation was associated with the expression of larger antennal lobes, the primary olfactory processing regions of the brain. In contrast, exposure to plant-derived olfactory stimuli did not have a significant effect on brain region volumes. This study is the first to explore the separate and interactive effects of visual and olfactory stimuli on bee brain development. Assessing the timing and sensitivity of brain development is a first step toward understanding how different rearing environments differentially affect regional brain volumes in this species. Our findings suggest that environmental factors experienced during the first week of adulthood can modify bumblebee brain development in many subtle ways.

Why study plasticity in multiple traits? New hypotheses for how phenotypically plastic traits interact during development and selection.

Organisms can often respond adaptively to a change in their environment through phenotypic plasticity in multiple traits, a phenomenon termed as multivariate plasticity. These different plastic responses could interact and affect each other's development as well as selection on each other, but the causes and consequences of these interactions have received relatively little attention. Here, we propose a new conceptual framework for understanding how different plastic responses can affect each other's development and why organisms should have multiple plastic responses. A plastic change in one trait could alter the phenotype of a second plastic trait by changing either the cue received by the organism (cue-mediated effect) or the response to that cue (response-mediated effect). Multivariate plasticity could benefit the organism either because the plastic responses work better when expressed together (synergy) or because each response is more effective under different environmental circumstances (complementarity). We illustrate these hypotheses with case studies, focusing on interactions between behavior and morphology, plastic traits that differ in their reversibility. Future empirical and theoretical research should investigate the consequences of these interactions for additional factors important for the evolution of plasticity, such as the limits and costs of plasticity.

Sex differences in the foraging behavior of a generalist hawkmoth.

Within-species variation in pollinator behavior is widely observed, but its causes have been minimally investigated. Pollinator sex is associated with large differences in behavior that may lead to predictable differences in flower foraging, but this expectation has not been explicitly tested. We investigate sex-associated differences in nectar-foraging behavior of the hawkmoth Hyles lineata, using pollen on the proboscis as a proxy for flower visitation. We tested two predictions emerging from the literature: (1) the sexes differ in the flower species they visit, (2) females are more specialized in flower choice. We also examined potential drivers underlying these predictions by performing field and laboratory experiments to test whether males (3) switch among flower species more frequently, or (4) fly farther and therefore encounter more species than females. Consistent with prediction (1), pollen load composition differed between the sexes, indicative of visitation differences. Contrary to prediction (2), females consistently carried more species-rich pollen loads than males. (3) Both sexes switched between flower species at similar rates, suggesting that differences in floral fidelity are unlikely to explain why females are less specialized than males. (4) Males flew longer distances than females; coupled with larger between-site differences in pollen composition for females, this result suggests that sex differences in mobility influence foraging, and that females may forage more frequently and in smaller areas than males. Together, our results demonstrate that sex-associated foraging differences can be large and consistent over time, and highlight the importance of sex as a driver of variation in pollinator behavior.

Long horns protect Hestina japonica butterfly larvae from their natural enemies.

Animals sometimes have prominent projections on or near their heads serving diverse functions such as male combat, mate attraction, digging, capturing prey, sensing or defence against predators. Some butterfly larvae possess a pair of long frontal projections; however, the function of those projections is not well known. Hestina japonica butterfly larvae have a pair of long hard projections on their heads (i.e., horns). Here we hypothesized that they use these horns to protect themselves from natural enemies (i.e., predators and parasitoids). Field surveys revealed that the primary natural enemies of H. japonica larvae were Polistes wasps. Cage experiments revealed that larvae with horns intact and larvae with horns removed and fitted with horns of other individuals succeeded in defending themselves against attacks of Polistes wasps significantly more often than larvae with horns removed. We discuss that the horns counter the paper wasps' hunting strategy of first biting the larvae's 'necks' and note that horns evolved repeatedly only within the Nymphalidae in a phylogeny of the Lepidoptera. This is the first demonstration that arthropods use head projections for physical defence against predators.

Flowers help bees cope with uncertainty: signal detection and the function of floral complexity.

Plants often attract pollinators with floral displays composed of visual, olfactory, tactile and gustatory stimuli. Since pollinators' responses to each of these stimuli are usually studied independently, the question of why plants produce multi-component floral displays remains relatively unexplored. Here we used signal detection theory to test the hypothesis that complex displays reduce a pollinator's uncertainty about the floral signal. Specifically, we asked whether one component of the floral display, scent, improved a bee's certainty about the value of another component, color hue. We first trained two groups of bumble bees (Bombus impatiens Cresson) to discriminate between rewarding and unrewarding artificial flowers of slightly different hues in the presence vs absence of scent. In a test phase, we presented these bees with a gradient of floral hues and assessed their ability to identify the hue rewarded during training. We interpreted the extent to which bees' preferences were biased away from the unrewarding hue ('peak shift') as an indicator of uncertainty in color discrimination. Our data show that the presence of an olfactory signal reduces uncertainty regarding color: not only was color learning facilitated on scented flowers but also bees showed a lower amount of peak shift in the presence of scent. We explore potential mechanisms by which scent might reduce uncertainty about color, and discuss the broader significance of our results for our understanding of signal evolution.

Sensory bias and signal detection trade-offs maintain intersexual floral mimicry.

Mimicry is common in interspecies interactions, yet conditions maintaining Batesian mimicry have been primarily tested in predator-prey interactions. In pollination mutualisms, floral mimetic signals thought to dupe animals into pollinating unrewarding flowers are widespread (greater than 32 plant families). Yet whether animals learn to both correctly identify floral models and reject floral mimics and whether these responses are frequency-dependent is not well understood. We tested how learning affected the effectiveness and frequency-dependence of imperfect Batesian mimicry among flowers using the generalist bumblebee, Bombus impatiens, visiting Begonia odorata, a plant species exhibiting intersexual floral mimicry. Unrewarding female flowers are mimics of pollen-rewarding male flowers (models), though mimicry to the human eye is imperfect. Flower-naive bees exhibited a perceptual bias for mimics over models, but rapidly learned to avoid mimics. Surprisingly, altering the frequency of models and mimics only marginally shaped responses by naive bees and by bees experienced with the distribution and frequency of models and mimics. Our results provide evidence both of exploitation by the plant of signal detection trade-offs in bees and of resistance by the bees, via learning, to this exploitation. Critically, we provide experimental evidence that imperfect Batesian mimicry can be adaptive and, in contrast with expectations of signal detection theory, functions largely independently of the model and mimic frequency. This article is part of the theme issue 'Signal detection theory in recognition systems: from evolving models to experimental tests'.

Patterns of phenotypic plasticity in common and rare environments: a study of host use and color learning in the cabbage white butterfly Pieris rapae.

Phenotypic plasticity is adaptive in variable environments but, given its costs, may be disfavored if only one environment is commonly encountered. Yet species in relatively constant environments often adjust phenotypes successfully in rare or novel environments. Developmental biases may reduce the costs of plasticity in common environments, favoring the maintenance of plasticity. We explored this proposition by studying the flexibility of visually guided host-selection behavior in cabbage white butterflies (Pieris rapae), wherein common and rare environments consisted of green and red host types, respectively. We demonstrated in greenhouse assays that adult females display an innate bias toward green color during host search but alter that bias through learning in red-host assemblages such that, after several hours of experience, red hosts are located as efficiently as green hosts. Full-sib analyses suggested there was genetic variation in host and color choice that was more pronounced in the red-host environment. We found no evidence of genetic correlations in behavior across host environments or of fitness costs of plasticity in color choice. Our results support the idea that learning may persist in less variable environments through the evolution of innate biases that reduce operating costs in common environments.

Brain size: a global or induced cost of learning?

The role of brain size as a cost of learning remains enigmatic; the nature and timing of such costs is particularly uncertain. On one hand, comparative studies suggest that congenitally large brains promote better learning and memory. In that case, brain size exacts a global cost that accrues even if learning does not take place; on the other hand, some developmental studies suggest that brains grow with experience, indicating a cost that is induced when learning occurs. The issue of how costs are incurred is an important one, because global costs are expected to constrain the evolution of learning more than would induced costs. We tested whether brain size represented a global and/or an induced cost of learning in the cabbage white butterfly, Pieris rapae. We assayed the ability of full sibling families to learn to locate either green hosts, for which butterflies have an innate search bias, or red hosts, which are more difficult to learn to locate. Naïve butterflies were sacrificed at emergence and congenital brain volume estimated as a measure of global costs; experienced butterflies were sacrificed after learning and change in brain volume estimated as a measure of induced costs. Only for the mushroom body, a brain region involved in learning and memory in other insects, was volume at emergence related to learning or host-finding. Butterfly families that emerged with relatively larger mushroom bodies showed a greater tendency to improve their ability to find red hosts across the two days of host-search. The volume of most brain regions increased with time in a manner suggesting host experience itself was important: first, total number of landings during host-search was positively related to mushroom body calyx volume, and, second, experience with the red host was positively related to mushroom body lobe volume. At the family level, the relative volume of the mushroom body calyx and antennal lobes following learning was positively related to overall success in finding red hosts. Overall, our results suggest that within species, brain size might act as a small global cost of learning, but that environment-specific changes in brain size might reduce the overall costs of neural tissue in the evolution of learning.

Sonicating bees demonstrate flexible pollen extraction without instrumental learning.

Pollen collection is necessary for bee survival and important for flowering plant reproduction, yet if and how pollen extraction motor routines are modified with experience is largely unknown. Here, we used an automated reward and monitoring system to evaluate modification in a common pollen-extraction routine, floral sonication. Through a series of laboratory experiments with the bumblebee, Bombus impatiens, we examined whether variation in sonication frequency and acceleration is due to instrumental learning based on rewards, a fixed behavioral response to rewards, and/or a mechanical constraint. We first investigated whether bees could learn to adjust their sonication frequency in response to pollen rewards given only for specified frequency ranges and found no evidence of instrumental learning. However, we found that absence versus receipt of a pollen reward did lead to a predictable behavioral response, which depended on bee size. Finally, we found some evidence of mechanical constraints, in that flower mass affected sonication acceleration (but not frequency) through an interaction with bee size. In general, larger bees showed more flexibility in sonication frequency and acceleration, potentially reflecting a size-based constraint on the range over which smaller bees can modify frequency and acceleration. Overall, our results show that although bees did not display instrumental learning of sonication frequency, their sonication motor routine is nevertheless flexible.

Mimicry in viceroy butterflies is dependent on abundance of the model queen butterfly.

Mimics should not exist without their models, yet often they do. In the system involving queen and viceroy butterflies, the viceroy is both mimic and co-model depending on the local abundance of the model, the queen. Here, we integrate population surveys, chemical analyses, and predator behavior assays to demonstrate how mimics may persist in locations with low-model abundance. As the queen becomes less locally abundant, the viceroy becomes more chemically defended and unpalatable to predators. However, the observed changes in viceroy chemical defense and palatability are not attributable to differing host plant chemical defense profiles. Our results suggest that mimetic viceroy populations are maintained at localities of low-model abundance through an increase in their toxicity. Sharing the burden of predator education in some places but not others may also lower the fitness cost of warning signals thereby supporting the origin and maintenance of aposematism.

Multimodal signals enhance decision making in foraging bumble-bees.

Multimodal signals are common in nature and have recently attracted considerable attention. Despite this interest, their function is not well understood. We test the hypothesis that multimodal signals improve decision making in receivers by influencing the speed and the accuracy of their decisions. We trained bumble-bees (Bombus impatiens) to discriminate between artificial flowers that differed either in one modality, visual (specifically, shape) or olfactory, or in two modalities, visual plus olfactory. Bees trained on multimodal flowers learned the rewarding flowers faster than those trained on flowers that differed only in the visual modality and, in extinction trials, visited the previously rewarded flowers at a higher rate than bees trained on unimodal flowers. Overall, bees showed a speed-accuracy trade-off; bees that made slower decisions achieved higher accuracy levels. Foraging on multimodal flowers did not affect the slope of the speed-accuracy relationship, but resulted in a higher intercept, indicating that multimodal signals were associated with consistently higher accuracy across range of decision speeds. Our results suggest that bees make more effective decisions when flowers signal in more than one modality, and confirm the importance of studying signal components together rather than separately.